Abasic site–peptide cross-links are blocking lesions repaired by AP endonucleases

Anna V Yudkina, Nikita A Bulgakov, Daria V Kim, Svetlana V Baranova, Alexander A Ishchenko, Murat K Saparbaev, Vladimir V Koval, Dmitry O Zharkov

► To cite this version:

Anna V Yudkina, Nikita A Bulgakov, Daria V Kim, Svetlana V Baranova, Alexander A Ishchenko, et al. Abasic site–peptide cross-links are blocking lesions repaired by AP endonucleases. Nucleic Acids Research, 2023, 51 (12), pp.6321-6336. 10.1093/nar/gkad423. hal-04289451

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Abasic site-peptide cross-links are blocking lesions repaired by AP endonucleases

<table><tr><td>Journal:</td><td>Nucleic Acids Research</td></tr><tr><td>Manuscript ID</td><td>NAR-03976-Q-2022.R1</td></tr><tr><td>Manuscript Type:</td><td>1 Standard Manuscript</td></tr><tr><td>Key Words:</td><td>DNA-peptide cross-links, AP sites, DNA polymerases, AP endonucleases, base excision repair</td></tr></table>

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All data are provided in the paper and the supplementary materials.

KEY POINTS

(3 bullet points summarizing the manuscript's contribution to the field)

â€¢ Abasic (AP) siteâ€“peptide cross-links arise in the proteolytic repair of AP siteâ€“protein cross-links
â€¢ AP siteâ€”peptide cross-links are blocking and follow the A-rule or induce primer misalignment

â€¢ AP siteâ€”peptide cross-links can be repaired by the TIM barrel superfamily AP endonucleases
# Abasic site–peptide cross-links are blocking lesions repaired by AP endonucleases

Anna V. Yudkina<fnref n="1" />,<fnref n="2" />*, Nikita A. Bulgakov<fnref n="1" />, Daria V. Kim<fnref n="1" />,<fnref n="2" />, Svetlana V. Baranova<fnref n="1" />, Alexander A. Ishchenko<fnref n="3" />, Murat K. Saparbaev<fnref n="3" />, Vladimir V. Koval<fnref n="1" /> and Dmitry O. Zharkov<fnref n="1" />,<fnref n="2" />,*

## KEY POINTS

* Abasic (AP) site–peptide cross-links arise in the proteolytic repair of AP site–protein cross-links

* AP site–peptide cross-links are blocking and follow the A-rule or induce primer misalignment

* AP site–peptide cross-links can be repaired by the TIM barrel superfamily AP endonucleases

## ABSTRACT

Apurinic/apyrimidinic (AP) sites are abundant DNA lesions arising from spontaneous hydrolysis of the N-glycosidic bond and as base excision repair (BER) intermediates. AP sites and their derivatives readily trap DNA-bound proteins, resulting in DNA–protein cross-links. Those are subject to proteolysis but the fate of the resulting AP–peptide cross-links (APPXLs) is unclear. Here we report two *in vitro* models of APPXLs synthesized by cross-linking of DNA glycosylases Fpg and OGG1 to DNA followed by trypsinolysis. The reaction with Fpg produces a 10-mer peptide cross-linked through its N-terminus, while OGG1 yields a 23-mer peptide attached through an internal lysine. Both adducts strongly blocked Klenow fragment, phage RB69 polymerase, *Saccharolobus solfataricus* Dpo4, and African swine fever virus PolX. In the residual lesion bypass, mostly dAMP and dGMP were incorporated by Klenow and RB69 polymerases, while Dpo4 and PolX used primer/template misalignment. Of AP endonucleases involved in BER, *E. coli* endonuclease IV and its yeast homolog Apn1p efficiently hydrolyzed both adducts. In contrast, *E. coli* exonuclease III and human APE1 showed little activity on APPXL substrates. Our data suggest that APPXLs produced by proteolysis of AP site-trapped proteins may be removed by the BER pathway, at least in bacterial and yeast cells.

<footnote n="1">¹SB RAS Institute of Chemical Biology and Fundamental Medicine, Novosibirsk, 630090, Russia</footnote>

<footnote n="2">²Department of Natural Sciences, Novosibirsk State University, Novosibirsk, 630090, Russia</footnote>

<footnote n="3">³Groupe "Mechanisms of DNA Repair and Carcinogenesis", Equipe Labellisée LIGUE 2016, CNRS UMR9019, Université Paris-Saclay, Gustave Roussy Cancer Campus, F-94805 Villejuif, France</footnote>

<footnote>* To whom correspondence should be addressed. Tel: +7 383 363 5187; Fax: +7 383 363 5128; Email: dzharkov@niboch.nsc.ru. Correspondence may also be addressed to Anna Yudkina. Tel: +7 383 363 5188; Fax: +7 383 363 5128; Email: ayudkina@niboch.nsc.ru.</footnote>
## INTRODUCTION

Apurinic/apyrimidinic (AP) sites occur by hydrolysis of the N-glycosidic bond in deoxyribonucleotides and are among the most abundant spontaneous and induced DNA lesions in the cell (1,2). They are also formed as intermediates in the base excision repair pathway, often being more genotoxic and mutagenic than the original lesion and thus requiring tight control over their processing (3,4). Chemically, AP sites are a family of lesions, which includes the much-studied aldehydic AP site as well as its derivatives oxidized at C1' (deoxyribolactone, DRL) (5-7), C2' (C2-AP) (8), C4' (C4-AP) or C5' position (5'-(2-phosphoryl-1,4-dioxobutane), DOB) (9,10).

Normally, AP sites are quickly removed from the genome by the base excision DNA repair (BER) system. However, there are important differences in the processing of different types of AP sites. The repair of aldehydic AP sites follows the classical short-patch BER branch, which, in human cells, involves the AP endonuclease (APE1) followed by polymerase and deoxyribophosphate lyase (dRPase) activities of DNA polymerase β (POLβ) (11). At the same time, C2-AP is not a substrate for the dRPase activity of POLβ and is removed through the long-patch branch of BER: the DNA flap containing the lesion at the 5'-end is displaced during the repair DNA synthesis and hydrolyzed by FEN1 nuclease (8,12). DRL repair is even more problematic: this lesion forms DNA-protein cross-links (DPXLs) with Lys residues in the active sites of DNA glycosylase Nth and POLβ (13,14), and the β-elimination product of DRL can similarly trap DNA glycosylases Fpg and NEIL1 (14). The presence of a 1,4-dicarbonyl moiety in C4-AP and DOB also promotes covalent capture of POLβ and DNA polymerase λ (15,16). The irreversible inhibition of the main DNA repair enzymes leads to significant cytotoxicity of modified AP sites (7).

Both aldehydic and oxidized AP sites readily form DPXLs. The covalent capture of many DNA-binding proteins by AP sites has been reported, including histones (6,17,18), integrase (19), DNA ligase (20), Ku antigen (21), as well as RNA-binding proteins (22,23), chaperones (22) and enzymes of carbohydrate metabolism (22,24). Recently, a unique mechanism for the repair of AP sites in single-stranded DNA was discovered (25-27), based on the formation of cross-links between the AP site and the HMCES protein (in human cells) or YedK protein (in E. coli), followed by switching to the proteasome-dependent pathway of DPXL repair. Thus, DPXL with AP sites are highly relevant adducts *in vivo*.

DPXLs are not limited to cross-links with AP sites and appear through a multitude of mechanisms including reactions with aldehydes and heavy metals, UV and ionizing radiation, and failed action of enzymes such as topoisomerases or C5-methyltransferases (28,29). Thus, a special pathway dedicated to DPXL repair has evolved. It is tightly coupled to replication and involves proteolytic degradation of the protein part of the cross-link (30-33). However, at the moment it remains poorly understood how the last peptide remnant conjugated with DNA is finally removed. It was suggested that this role can be played by nucleotide excision repair (NER), which normally protects the genome from bulky adducts (34-38). Also, Fpg/Nei-like DNA glycosylases are able to excise oxidized purine residues cross-linked to peptides (38). Recombination repair does not appear to be involved in the removal of peptide conjugates from DNA, although it may play a role in tolerance to larger DPXLs (31,36). It should be noted that all these data were obtained on cross-links chemically different from
those formed by AP sites, and peptide adducts of this type remain uncharacterized with respect to their repair and their effect on replication. While the studied peptide adducts with nucleobases usually retain some base-pairing ability, the bulky nature and non-instructing properties of AP site-peptide cross-links make these lesions more problematic for the cell. Here we report a model of stable, chemically determined AP site-peptide cross-links (APPXLs), conjugated through either a terminal or an internal position in the peptide, and show that they block DNA polymerases and can be repaired by AP endonucleases.

## MATERIAL AND METHODS

### Oligonucleotides and enzymes

E. coli formamidopyrimidine-DNA glycosylase (Fpg) (39), endonuclease VIII (Nei) (40) and endonuclease IV (Nfo) (41), human AP endonuclease (APE1) (42), 8-oxoguanine-DNA glycosylase (OGG1) (43), endonuclease VIII-like protein 1 (NEIL1) (44) and endonuclease VIII-like protein 2 (NEIL2) (45), yeast AP endonuclease (Apn1p) (41), 3'→5'-exonuclease-deficient (exo⁻) Klenow fragment of E. coli DNA polymerase I (KF) (46) and exo⁻ bacteriophage RB69 DNA polymerase (RBpol) (47) were overexpressed and purified essentially as described. Cloning and purification of DNA polymerase X from African swine fever virus (ASFV PolX) is described below. E. coli exonuclease III (Xth) was purchased from Roche (Basel, Switzerland), E. coli uracil-DNA glycosylase (Ung) and exonuclease-proficient KF, from SibEnzyme (Novosibirsk, Russia), full-length E. coli DNA polymerase I (Pol I) and Saccharolobus (Sulfolobus) solfataricus DNA polymerase IV (Dpo4), from New England Biolabs (Ipswich, MA) and trypsin, from Samson-Med (St. Petersburg, Russia). Purity of the DNA polymerases and AP endonucleases, except for those obtained commercially, is illustrated in Supplementary Figure S1. Oligonucleotides (Supplementary Table S1) were synthesized in-house from commercially available phosphoramidites (Glen Research, Sterling, VA).

### ASFV PolX cloning and purification

The coding frame of PolX optimized for expression in E. coli was synthesized by Gene Universal (Newark, DE, USA) and confirmed by Sanger sequencing. The insert was subcloned into the pET-24b vector (Merck, Darmstadt, Germany) at Ndel/XhoI sites. The plasmid was subsequently introduced into the E. coli Rossetta 2(DE3) strain (Merck). One liter of LB medium supplemented with 100 µg/ml of kanamycin was inoculated with 5 ml of an overnight culture. The cells were grown with vigorous shaking at 37°C to A₆₀₀ = 0.6, isopropyl-β-D-thiogalactopyranoside was added to 1 mM, and the growth was continued for 4 h at 37°C. The cells were harvested by centrifugation at 12,000×g at 4°C for 20 min and stored at -72°C. Before the purification, the pellet was thawed on ice in 40 ml of Buffer A consisting of 20 mM sodium phosphate (pH 7.5), 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM dithiothreitol (DTT) and supplemented with 500 mM NaCl and 1 mM phenylmethylsulfonyl fluoride. The cells were sonicated, and the lysate was clarified by centrifugation at 12,000×g at 4°C for 30 min. The supernatant was filtered through a 0.45-µm polyvinylidene difluoride membrane, diluted with four volumes of Buffer A and loaded onto a 5-ml SP Sepharose
HiTrap column (GE Healthcare, Chicago, IL) equilibrated in Buffer A plus 100 mM NaCl. The column was washed with Buffer A supplemented with 100 mM NaCl, and PolX was eluted with a NaCl gradient at ~650 mM. The fractions containing PolX were pooled, diluted with six volumes of Buffer A and loaded onto a 5-ml heparine Sepharose HiTrap column (GE Healthcare) equilibrated in Buffer A plus 100 mM NaCl. The column was washed with the same buffer, and PolX was eluted with a NaCl gradient at ~730 mM. The fractions containing PolX were pooled, diluted with six volumes of Buffer A and additionally purified on a 1-ml MonoS column (GE Healthcare) previously equilibrated in Buffer A plus 100 mM NaCl. PolX was eluted with a NaCl gradient at ~600 mM. The fractions containing >90% homogeneous protein were pooled, dialyzed against the buffer containing 20 mM sodium phosphate (pH 7.5), 400 mM NaCl, 1 mM EDTA, 1 mM DTT, and 50% glycerol, and stored at -20°C.

## Synthesis of model peptide conjugates with AP sites

DNA cross-links to Fpg or OGG1 proteins were obtained as described (48-50). Briefly, ~2 nmol of the oligonucleotide duplex (40oG//28down or 28oG//21comp; Supplementary Table S1) was treated with 3-6 nmol of the enzyme in 50 mM sodium phosphate (pH 6.8), 1 mM EDTA, 1 mM DTT and 100 mM NaBH₄ in a total volume of 40 µl. The reaction was allowed to proceed at 37°C for 2 h. The resulting DPXL was denatured at 95°C for 10 min, cooled, and pH of the solution was adjusted to ~8.0 by adding Tris base. The mixture was supplemented with 2 µg of trypsin and incubated for 4 h at 37°C. The oligonucleotide-peptide conjugate was separated by electrophoresis in 20% polyacrylamide/7 M urea, excised under UV illumination, desalted by reverse-phase chromatography on Isolute C18 sorbent (Biotage, Uppsala, Sweden) and characterized by MALDI MS. If necessary, the oligonucleotides for the following experiments were 5'-labeled using γ[³²P]ATP and annealed to a 1.5-fold molar excess of the complementary strand.

## DNA polymerase assays

For the standing-start assay, the substrates were assembled from the 40-mer template strand containing an APPXL and the ³²P-labeled primer (13pri; Supplementary Table S1) or the 28-mer template strand containing an APPXL and the ³²P-labeled primer (16pri; Supplementary Table S1). The reaction mixture (10 µl) contained 20 nM pre-annealed substrate, DNA polymerase (100 nM KF or RBpol, 400 nM PolX, 0.1 U/µl Dpo4), dNTPs (either 200 µM individual dATP, dCTP, dGTP, or dTTP, or a mixture of all four dNTPs each at 200 µM) in the reaction buffer (50 mM Tris-HCl (pH 7.5), 5 mM MgCl₂ and 1 mM DTT for KF, RBpol and PolX; 20 mM Tris-HCl (pH 8.8), 10 mM (NH₄)₂SO₄, 10 mM KCl, 2 mM MgSO₄, 0.1% Triton X-100 for Dpo4). The reaction was allowed to proceed at 37°C for 30 min, quenched with an equal volume of the loading solution (80% formamide, 20 mM Na-EDTA, 0.1% xylene cyanol, 0.1% bromophenol blue), and heated for 1 min at 95°C. The reaction products were resolved by 20% denaturing PAGE and visualized on a Typhoon FLA 9500 imager (GE Healthcare). The bands were quantified using Quantity One v4.6.8 (Bio-Rad Laboratories, Hercules, CA). For the running-start assay, the substrates were assembled from the 40-mer template strand containing an APPXL and the ³²P-labeled primer (11pri; Supplementary Table S1). The substrates
were taken at 50 nM, and the enzymes, at 50 nM (KF or RBpol), 200 nM (PolX) or 0.1 U/µl (Dpo4);
aliquots were taken at 2, 5, and 30 min; otherwise the reactions were run and processed as above.

## AP endonuclease assay

The substrates were assembled from the ³²P-labeled 40-mer strand containing an APPXL and the complementary 40-mer strand (40compC; Supplementary Table S1). The reaction mixture (10 µl) contained 50 nM substrate prepared as above and 10–1000 nM Xth, APE1, Apn1p or Nfo in the respective buffer optimal for BER or NIR (Supplementary Table S2). As a control, instead of the APPXL, we used the same oligonucleotide construct with an aldehydic AP site, freshly prepared by treatment of the U-containing substrate with Ung at 37°C for 20 min. The endonuclease reaction was allowed to proceed at 37°C for 30 min, treated and analyzed as above.

## AP endonuclease kinetics

The reaction mixture (10 µl) contained increasing concentrations of the ³²P-labeled 40-mer APPXL substrates or control substrates 23OHU:23comp or 20THF:20comp (1–500 nM) in the BER or NIR buffer. Concentrations of AP endonucleases were optimized for each substrate and buffer to produce ≤20% cleavage and were in the 5 pM – 0.5 nM range. The reaction was allowed to proceed for 10 min at 37°C, treated and analyzed as above. The data were fitted by nonlinear regression to the Michaelis–Menten equation using Sigmaplot v11.0 (Systat Software, Chicago, IL). The reported values are averages of 3–5 independent experiments.

## AP endonuclease / DNA polymerase assay

The reaction mixture (10 µl) contained 200 nM ³²P-labeled duplex 40-mer APPXL substrate and 50–150 nM Nfo in the BER buffer (Supplementary Table S2). After 10 min, the reaction mixture was supplemented with dNTPs (200 µM each), 5 mM MgCl₂, and 50–250 nM KF. The reaction was allowed to proceed at 37°C for 2, 5, or 30 min, treated and analyzed as above.

## DNA glycosylase assay

The reaction mixture (10 µl) contained 10 nM labeled duplex substrate (see above), 1 µM DNA glycosylase (*E. coli* Fpg or Nei, human OGG1, NEIL1, or NEIL2), 50 mM Tris–HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA and 1 mM DTT. The reaction was allowed to proceed at 37°C for 30 min, treated and analyzed as above.

## RESULTS

### Synthesis and characterization of DNA–peptide cross-links with AP site

Since AP sites can form cross-links to a variety of proteins, which can be further processed to APPXLs, it is usually assumed that the repair of such adducts does not depend on the exact nature of the cross-linked peptide. Hence, to obtain model APPXLs, we made use of two enzymes, *E. coli* Fpg and human OGG1 DNA glycosylases, that form transient covalent intermediates with AP sites during
the catalysis. To excise the damaged base (8-oxoguanine; 8-oxoG) from DNA, Fpg and OGG1 carry out a nucleophilic attack by an amino group at the C1' atom of the target nucleotide forming a labile Schiff base, which may be nearly quantitatively reduced by NaBH₄ or NaBH₃CN to a stable conjugate (Figure 1). This reaction was successfully used to crystallize covalent Fpg–DNA and OGG1–DNA complexes (39,51) and to study the interaction of full-length DPXLs with the replication and repair machinery (36,48-50). Thus, we first prepared DPXLs by treatment of 8-oxoG-containing duplexes with Fpg or OGG1 in the presence of NaBH₄ and subjected them to trypsinolysis (Supplementary Figures S2A, S2B). Fpg and OGG1 belong to different structural superfamilies and use different nucleophilic residues for catalysis: N-terminal Pro1 in Fpg and Lys249 in OGG1. Hence, after trypsinolysis, the resulting peptide conjugates are expected to have different structures (Figure 1). The Fpg–DNA conjugate gives an adduct with the decapeptide (P)ELPEVETSR (the residue that forms the cross-link is in parentheses), in which the peptide forms a bond with DNA through its N-terminus. On the other hand, trypsinolysis of the OGG1 conjugate should give an adduct with the 23-mer peptide ALCILPGVGT(K)VADCICLMALDK, cross-linked through an internal position of the peptide. Based on the visual resemblance (Figure 1), we will further call these AP site–peptide cross-links APPXL-I (terminally adducted, linear peptide) and APPXL-Y (an adduct through an internal residue with two peptide branches).

APPXL-I and APPXL-Y were purified by denaturing polyacrylamide gel electrophoresis followed by reverse-phase chromatography and characterized by MALDI-TOF mass spectrometry (Supplementary Figures S2C, S2D). The yield of the homogeneous stable APPXLs was ~5–15% of the initial 8-oxoG-containing oligonucleotide. In addition to the cross-link with the full-length modified strand, we have observed some products that likely corresponded to products of strand cleavage by Fpg/OGG1, which were easily separated from the major adduct by electrophoresis (Supplementary Figures S2A, S2B). Despite the long oligonucleotide parts, of the cross-links, their masses were better resolved by MALDI mass spectrometry in a positive mode and were within a few Da of the expected values. No peaks of the starting material (expected [M+H⁺], 12138.9 Da) was evident, indicating good separation of the APPXLs by electrophoresis. In the APPXL-I mass spectrum, we observed two peaks corresponding to the expected molecular weight of the adduct of the 40-mer oligonucleotide with the (P)ELPEVETSR peptide (observed [M+H⁺], 13131.0 Da, expected, 13127.9 Da) and (P)ELPEVETSRR peptide (observed [M+H⁺], 13285.8 Da, expected, 13284.0 Da), most likely resulting from incomplete trypsin cleavage at two adjacent Arg residues (Supplementary Figure S2C). As heterogeneity of cross-linked peptides may be naturally expected from proteolysis of DPXLs in the cell, we have proceeded to characterize the biochemical properties of our adducts.

## AP site–peptide cross-links block DNA polymerases

To investigate the ability of DNA polymerases to bypass APPXLs, we annealed the cross-linked template oligonucleotide with a labeled primer with its 3'-terminus placed 12 bases upstream of the cross-link site ("running start" conditions, see the substrate schemes in Figures 2A, 2B, 3A). This distance exceeds the footprints of the full-length proteins from which the cross-linked peptides were derived, and thus the peptide was not expected to prevent DNA polymerase binding. We also tested
the ability of DNA polymerases to carry out synthesis through the APPXL in the context of DNA duplex when the polymerase has to displace the downstream strand (Figures 2C, 2D, 3B). As APPXLs are non-instructive, both APPXL-I and APPXL-Y are expected to be strongly blocking. Therefore, we used a natural freshly prepared AP site as a control to determine which events would result from DNA polymerases blocking due to the lack of a base or due to the nature of cross-linked peptide. Non-damaged primer–template or primer–downstream strand–template constructs were used as polymerase activity controls.

Four DNA polymerases, representing a range of structural and functional features, were investigated. As we were interested in the general ability of DNA polymerases to bypass APPXLs, we opted to use easily accessible representative members of various structural families originating from bacteria, archaea, and viruses, rather than human enzymes. The Klenow fragment of *E. coli* DNA polymerase I (KF) belongs to Family A of DNA polymerases, acting in DNA repair and replication. The homologous human enzymes are DNA polymerases γ, θ, and v. Bacteriophage RB69 DNA polymerase (RBpol) is a high-fidelity replicative polymerase from Family B and is often taken as a convenient model of structurally related human replicative DNA polymerases α, δ, and ε, and a translesion DNA polymerase ζ. In both cases, we have used 3'→5'-exonuclease-deficient versions of the polymerases to prevent primer degradation; exonuclease-proficient KF was also brought in for comparison. DNA polymerase X (PolX) from African swine fever virus (ASFV) belongs to structural Family X, mostly engaged in DNA repair and encompassing human DNA polymerases β, λ, and μ. Finally, DNA polymerase IV from *Saccharolobus* (formerly *Sulfolobus solfataricus* (Dpo4) is a representative of Family Y translesion polymerases, of which human cells possess DNA polymerases η, ι, and κ.

Under the running start conditions, the blocking properties of APPXL-I were comparable with the properties of the natural AP site: KF demonstrated slightly better bypass of APPXL-I compared to AP site in comparison with RBpol, which was slightly more efficient on AP site-containing substrates. After 30 min, KF was able to fully elongate 6.8% of the primer if APPXL-I was in the template, and only 4.6% if an AP site was in the template whereas RB69 demonstrated 6.6% bypass of APPXL-I and 11% of the AP site (Figure 2A). Moreover, two strong pause points were evident one nucleotide before the cross-linking site and directly opposite the cross-link (23- and 24-nt long products; marked by grey arrows in Figures 2 and 3), indicating low ability of DNA polymerases both to incorporate a dNMP opposite APPXL-I and to extend the primer after such incorporation. The absence of any additional pause points suggests that the bulky peptide does not hamper moving DNA polymerase access to the cross-link site. The aldehyde AP site produced a single pause point one nucleotide before the lesion. Exonuclease-proficient KF demonstrated the same behavior as KF exo⁻ except for noticeable primer degradation at longer reaction times (Supplementary Figure S3). Thus, APPXL-I, while strongly blocking, still allows better incorporation of a dNMP than the smaller non-instructive AP site. PolX, which is known to be nearly fully blocked by AP sites (52), demonstrated no bypass of the APPXL-I as well as the natural AP site, showing a single pause point corresponding to the 23-mer synthesis product (Figure 3A). Blocking of PolX may be due to its distributive mode of synthesis, which facilitates DNA release and complicates polymerase binding near the blocking lesion.
Translesion DNA polymerase Dpo4, as expected, demonstrated the strongest ability to bypass the AP site (Dpo4 was able to fully elongate ~28% of the primer) and APPXL-I (~30% of the bypass), producing the same two pause points in the latter case (Figure 3A). The presence of a downstream strand complicated the bypass of the blocking lesion, yet the synthesis pause points and the relative efficiency of lesion bypass seen with the primer–template system were preserved (Figure 2C, Figure 3B).

Strong polymerase inhibition and two pronounced pause points for KF, RBpol and Dpo4 were also evident for substrates containing the branched APPXL-Y adduct (Figures 2B, 2D, 3, Supplementary Figure S3). The bulkier APPXL-Y turned out to be even more blocking than APPXL-I. The pause point corresponding to the 24-nt product for KF and RBpol was weaker than the corresponding pause for APPXL-I, and the full-length product decreased significantly for KF and Dpo4.

We have also addressed polymerase-blocking ability of AP site–peptide cross-links present in an internal position of the non-template downstream strand. However, only for PolX was a minor pause point observed, while KF and RBpol displaced the cross-linked strand without effort, and Dpo4, while having lower strand displacement activity, did not pause at the cross-link (Supplementary Figure S4).

Interestingly, when different substrates were compared, we systematically observed primer utilization efficiency decreasing in the order AP site > APPXL-I > APPXL-Y. Possibly, the peptide part may partially interfere with DNA binding by the polymerase even when the primer end is 12 nt away from the cross-link site, for example, by obstructing non-specific DNA binding and facilitated diffusion used by polymerases to locate the primer end (53). The only exception was Dpo4 with APPXL-Y in the downstream strand, in which case the bulky peptide adduct might destabilize the duplex and improve the poor strand displacement ability of Dpo4.

## AP site–peptide cross-links mostly direct A and G incorporation and can induce primer–template misalignment

Since we observed some dNMP insertion opposite APPXLs, we sought to explore the spectra of the incorporated nucleotides. To do this, we moved the primer to place its 3'-end immediately next to the site of the lesion and presented the polymerases with the individual dNMPs as well as with their mixture ("standing start" conditions, Figure 4). Here, KF utilized the APPXL-I adducted template much better than the APPXL-Y adduct, whereas for RBpol the efficiency was somewhat better for the APPXL-Y. Notably, in all cases the primer elongation was significantly lower than under the running start conditions, most likely because the bulky peptide interferes with DNA polymerases' binding to the primer/template junction. Nevertheless, the polymerase preferences for dNMP incorporation opposite APPXLs were clear. Both KF and RBpol predominantly incorporated dAMP, and, to a lesser extent, dGMP (Figures 4A, 4B). We observed the preference of dNMP incorporation in the order A > G > C > T, which is consistent with the "A rule", the preference pattern for better dAMP incorporation opposite the AP site (54-56). dGMP incorporation could possibly be explained by template/primer slippage since the next base after the lesion in the template is C. To address this possibility, we repeated the experiment with the substrate of another sequence featuring G as the next base (Supplementary Figure S5), for which template/primer slippage would guide the
incorporation of dCMP. However, the same general dNMP incorporation pattern A > G > C > T for KF and RBpol was observed (Supplementary Figures S5A, S5B), confirming that these preferences characterize the miscoding properties of the peptide adducts rather than the tendency for template misalignment. The order of preference of dNTP incorporation by the Family Y Dpo4 was G >> A > C, T (Figure 4C); however, when the substrate was switched, the preference for dNTP usage completely to C >> A > G, T (Supplementary Figure S5C), indicating that Dpo4 use +1 nucleotide template misalignment mechanism rather than the “A rule” during the synthesis on the AP-peptide APPXL-containing substrates. Dpo4 tended to misincorporate several nucleotides, in agreement with its reported low fidelity characteristic of Family Y DNA polymerases (57). PolX also bypassed the peptide adduct by +1 slippage mechanism, albeit with a low efficiency: the enzyme preferred G > A, T, C on the substrate presenting C as the +1 template nucleotide while changing the specificity to C > A >> T, G on the substrate presenting G at this position (Figure 4D, Supplementary Figure 5D).

## AP site–peptide cross-links can be processed by AP endonucleases

Aldehydic AP sites and many of their natural and synthetic modifications are removed from DNA through the BER pathway initiated by AP endonucleases. These enzymes belong to two major unrelated structural groups: the exonuclease–endonuclease–phosphatase (EEP) superfamily and the triosephosphate isomerase (TIM) barrel superfamily. Therefore, we have tested representative members of each group – *E. coli* exonuclease III (Xth) and the main human AP endo/exonuclease 1 (APE1) from the EEP superfamily and *E. coli* endonuclease IV (Nfo) and yeast endonuclease Apn1p from the TIM barrel superfamily – for their ability to cleave oligonucleotide substrates containing APPXL-I or APPXL-Y. Notably, all these enzymes but Xth, in addition to their AP endonuclease activity, were shown to possess the “nucleotide incision repair” (NIR) activity that cleaves DNA 5’ to many lesions of different chemical nature including quite bulky ones such as 1, *N*⁶-etheno-dA, 3, *N*⁴-etheno-dC, and 3, *N*⁴-benzetheno-dC (41,58-62). Since the pH, buffer and ionic composition optimal for BER and NIR are enzyme-specific, we tested the ability of these AP endonucleases to cleave APPXL-containing substrates under both conditions (Supplementary Table S2).

APE1 efficiently hydrolyzed the natural AP site but did not yield any detectable product of the APPXL-I substrate cleavage (Figure 5A). However, the APPXL-Y substrate was modestly hydrolyzed by APE1, especially under NIR conditions (Figure 6A). This result was rather surprising, since the bulkier APPXL-Y adduct was expected to have larger inhibitory effect, and suggested that the chemical nature of the peptide adduct may be important for its recognition by AP endonucleases. Another member of the EEP superfamily, Xth, also showed low activity on substrates of both types (Figure 5C, 6C). The degradation product observed at the highest enzyme concentrations migrates slower than the expected APPXL cleavage product and is likely due to the robust 3'→5'-exonuclease activity of Xth evident on the AP substrate. In contrast to APE1 and Xth, both Nfo and Apn1p efficiently processed the substrates containing APPXL-I or APPXL-Y adducts, cleaving the DNA at the cross-link site and showing some additional exonucleolytic degradation (Figure 5B, 5D, 6B, 6D). The cleavage was evident under either BER or NIR conditions. Thus, both TIM barrel AP endonucleases cleaved APPXL-containing DNA significantly better than did EEP superfamily AP endonucleases.
To compare the efficiency of AP endonucleases processing APPXLs with their established substrates in a quantitative way, we have measured the steady-state kinetic parameters (K<sub>M</sub>, k<sub>cat</sub>, and the catalytic efficiency k<sub>cat</sub>/K<sub>M</sub>) values using duplex oligonucleotides containing APPXL-I, APPXL-Y, (3-hydroxytetrahydrofuran-2-yl)methyl phosphate (THF, an AP site analog resistant to β-elimination and often used as a model AP site in BER studies) or 5-hydroxy-2'-deoxyuridine (OHU, a typical NIR substrate) (61,62). As shown in Table 1 and Supplementary Figure S6, the apparent k<sub>cat</sub>/K<sub>M</sub> values confirm APPXL-Y is a better substrate compared to APPXL-I for all studied AP endonucleases. For the TIM barrel enzymes, APPXL-Y cleavage activity was comparable with (~0.5-fold for Apn1p) or even significantly higher than (~13-fold for Nfo) the OHU cleavage, strongly suggesting that these AP endonucleases may be biologically relevant for processing of AP site–peptide adducts. Moreover, even under BER conditions the efficiency of APPXL-Y cleavage by Nfo was comparable with THF cleavage (THF favored only ~2-fold). More efficient processing of APPXL-Y than of APPXL-I may also be of biological importance, as peptides covalently bound to the AP site through an internal residue are statistically more likely to arise in the cell after protein conjugation followed by proteolysis.

We also tested the ability of several bifunctional DNA glycosylases capable of AP site cleavage, namely *E. coli* Fpg and Nei, human OGG1, NEIL1 and NEIL2, to cleave the APPXL adducts. However, none of them was able to process cross-links of either type to any significant degree (Supplementary Figure S7).

## Strand with a hanging 5'-terminal peptide cross-link can be displaced by DNA polymerases

AP endonucleases nick DNA 5' of the lesion producing a hanging 5'-terminal 2'-deoxyribophosphate (or another lesion remnant if the substrate was something else than an aldehydic AP site) and a free 3'-OH, which then serves as a primer for a DNA polymerase. To see whether the nick with a 5'-terminal peptide adduct can be processed in the same way, we have first treated the APPXL-I or APPXL-Y substrate with Nfo and then added KF and all four dNTPs (Figure 7A, 7B). Although in both cases a pause was observed after the incorporation of one nucleotide, eventually the full-length primer extension product was synthesized. Thus, even if the peptide part of the cross-link interferes with polymerase binding or the early steps of primer extension, once the flap is >1 nt there is apparently no obstacles for further strand displacement.

Furthermore, we have reconstituted the full BER cycle with APPXL-I (annealed opposite to G; 40compG template strand), Nfo, full-length *E. coli* DNA polymerase I, and DNA ligase. Unlike KF, Pol I degrades the strand ahead by a combined exo/endonucleolytic action as it extends the primer (63). To prevent full degradation of the downstream strand, we have omitted dATP and dGTP from the reaction mixture, which should cause the polymerase to stop after incorporating 14 nt, yielding a 30-mer product (Figure 7C). Nfo cleaved the duplex at the adduct, producing the 16-mer with the 3'-OH end, which served as an entry point for Pol I (Figure 7C). In the absence of dNTPs, the 3'→5' exonuclease activity of Pol I degraded the primer, whereas addition of dCTP and dTTP allowed primer extension to the position of the first pyrimidine in the template. Finally, if DNA ligase was present, the product had a higher electrophoretic mobility than the DNA–peptide adduct, migrating
approximately at the same level as an undamaged 40-mer (Figure 7C), which clearly indicated that the repair took place.

These observations support the possibility of APPXL removal via the long-patch BER subpathway (Figure 7D). Obviously, short-patch repair is also possible if a non-stabilized 5'-terminal conjugate can be processed by a dRP lyase.

## DISCUSSION

For many years, AP sites were considered a fairly well studied type of DNA damage, but recent discoveries regarding new features of their enzymatic processing and biologically relevant reactions of AP sites with non-specific DNA-bound proteins (18,25-27) put these lesions in an unexpected perspective. There is mounting evidence that native and oxidized AP sites in the cell can covalently capture proteins to form highly toxic DNA-protein cross-links. Several breakthrough works in recent years have outlined the general repair scheme for DNA damage of this type. DPXLs mainly serve as substrates for a specialized repair pathway, which is initiated by proteolytic degradation of the protein part of the cross-link by the proteasome or specialized replication-associated proteases (SPRTN1, FAM111A, DDI1, or others yet to be discovered) to a small peptide (30-33). What happens after that, however, remains poorly understood. The studied models of naturally occurring DNA-peptide cross-links include peptide conjugates with exocyclic amino groups of G and A modified through (i) a γ-hydroxypropano bridge (a product of acrolein-induced DNA damage) (35,64-66) or (ii) through a C2 linker derived from alkylation of Cys residues by 1,2-dibromoethane (67), and (iii) conjugates at the aldehyde group of 5-formylcytosine (68-70). In addition, unnatural adducts that are readily obtained synthetically, such as products of azide-alkyl addition to the functionalized C5 position of thymine (71) and cross-links to 7-deaza-7-(2-oxoethyl)guanine (72), were used as model systems.

The major difference between peptide cross-links through nucleobases and through AP sites is the inability of the latter to form complementary bonds. Until now, there have been no reports in the literature regarding the levels of APPXLs, their effect on DNA polymerases or the ways to repair such residual damage. However, recent data on the reactivity of aldehyde and oxidized AP sites suggest that the number of cross-links with the AP site can be comparable to the number of cross-links with bases, or even exceed them. Moreover, some conjugates with oxidized AP sites are much more stable than reversible imine conjugates with aldehydic AP sites. In this work, we developed a model to generate cross-links of AP sites with peptides of different structure based on the reduction of the Schiff base intermediate formed by DNA glycosylases Fpg and OGG1 followed by trypsinolysis. Fpg carries an N-terminal nucleophilic amino group and, after proteolysis, leaves behind a decapeptide attached via its end (APPXL-I), while OGG1 uses the internal Lys249 for catalysis, and upon trypsinolysis yields a longer peptide remnant, in which the cross-link is formed at an internal position (APPXL-Y). This peptide adduct may be of greater biological relevance because DPXLs in the cell are statistically much more likely to be attached within the polypeptide chain, and protease cleavage immediately next to the cross-link point may be hindered by DNA. Although the diversity of proteins capable of cross-linking to DNA dictates the universality of the mechanisms for APPXL repair, the size
and properties of the peptide remnant might be expected to affect the APPXL interactions with DNA polymerases and the repair machinery.

A key aspect of APPXL biochemistry determining their biological consequences is the ability to block DNA polymerases or guide incorporation of particular dNMPs. Although no single DNA-peptide adduct was studied with a fully representative set of DNA polymerases, in vitro experiments with peptides conjugated through nucleobases show that bypass of such lesions depends both on their size and the preservation of complementarity. DNA polymerases bind DNA in the minor groove and leave the major groove mostly accessible, so bulky moieties occupying the major groove may be better tolerated. For example, 5-formylcytosine adducts or peptides attached to the C5 position of dU through click chemistry, which protrude to the major groove and do not interfere with base pairing, are quite easily bypassed by both replicative and translesion human DNA polymerases, although longer peptides may present a problem (e. g., 10-mer peptides were bypassed, while 23-mers were not) (70,71,73). In another report, even a large 31-mer peptide conjugated to 5-formylcytosine was bypassed by translesion DNA polymerases κ and η (68). Similarly, conjugates through position C7 of 7-deazaG are bypassed by translesion polymerases in a size-dependent manner (74). Peptides adducted through the exocyclic amino group of A, which may either eliminate or keep base pairing depending on the orientation of the peptide, completely block E. coli replicative DNA polymerase III but demonstrate low-to-full bypass by E. coli DNA polymerases I, II, IV and V, human translesion DNA polymerases η, ι, κ, and v, and replicative yeast DNA polymerase δ (65-67). On the other hand, peptide adducts with the exocyclic group of G, which either disrupt base pairing or protrude into the minor groove, are strongly blocking for most polymerases and were efficiently bypassed only by Family Y translesion DNA polymerases (E. coli Pol IV and human Pol κ) (64,65). In the cell, mutagenic bypass of peptide adducts requires translesion DNA polymerases (69,70,75).

Bypass of natural AP sites and their modified varieties is often described by the "A-rule", which states that dAMP insertion is preferred opposite a non-instructive lesion. Interestingly, the structural reasons underlying this preference are different between DNA polymerases. For example, the structure of the Stoffel fragment of Taq DNA polymerase (which corresponds to KF in the E. coli enzyme) shows templating by a conservative Tyr residue inserted in place of a template nucleotide and acting as a thymine mimic (76,77). RBpol, on the other hand, relies on the favorable stacking between the incoming dATP and the extended planar system formed by the terminal primer nucleotide and its pairing partner in the absence of the template nucleobase (78,79). However, in the structures of both polymerases with AP-DNA, C1' of the AP site resides is buried at the protein-DNA interface, and the template strand would require extensive conformational rearrangement for a bypass if a large peptide is attached to this position. The steric clash could be relieved by flipping the template nucleotide around its flanking phosphates, as often observed in structures of Families X and Y DNA polymerases stabilizing a misaligned primer-template junction (80-82). In the "open" state of the enzyme-DNA complex, Family A DNA polymerases, including KF, also rotate the downstream template nucleotides outside of the helix, and it has been suggested that this movement helps prevent the template bases from misalignment in the absence of an incoming dNTP (83). As we do not see preferential incorporation of a dNMP corresponding to +1 template nucleotide, characteristic of the
primer–template misalignment bypass mechanism, we hypothesize that favorable stacking can guide dAMP and dGMP insertion opposite extrahelical APPXLs in the rare bypass events. On the other hand, structures of Dpo4 bypassing an abasic site, demonstrate extensive conformational flexibility allowing for primer/template misalignment and generation of mismatches, +1 and –1 frameshifts (84). In the structure with misalignment between the primer end and the +1 position in the template (which was observed in our experiments), the AP site is looped out of the helix into a gap between the fingers and the little finger domains, providing plenty of space for accommodation of a cross-linked peptide. No structure of PolX in the frameshift mode is available but POLβ, a prototypical member of Family X, can also loop out a template nucleotide to match the primer end to the +1 position in the template (82).

As we used representative DNA polymerases of non-human origin in our study, it remains to be seen which DNA polymerases may participate in APPXL bypass in human cells. Based on the *in vitro* insertion specificity data, it has long been held that natural AP sites are bypassed by DNA polymerase η (POLη) (85,86). On the other hand, AP site bypass in POLη-deficient human cells is almost as efficient as in wild-type cells (87). An alternative model was proposed, based on yeast mutation spectra and *in vitro* biochemistry of human and yeast DNA polymerase ζ (POLζ), that the POLζ/REV1 complex acting both as an inserter and an extender polymerase provides mutagenic bypass of AP sites (reviewed in (88)). However, mutation spectra induced by AP sites in yeast and human cells seem to be different (89), and human REV1 also interacts with DNA polymerases η, κ, and ι (88), so the nature of the inserter polymerase in human cells exposed to AP sites or their derivatives, including APPXLs, needs further investigation.

The high proficiency of TIM barrel AP endonucleases in nicking DNA at APPXLs was rather unexpected, since even the known substrates for the NIR activity of these enzymes are much less bulky. However, inspection of the structures of DNA-bound Nfo (90,91) reveals that C1' is solvent-accessible in both enzyme–substrate and enzyme–product complexes (Supplementary Figure S8) and the attached peptide could possibly be accommodated in the major groove of the DNA molecule, which is sharply bent upon Nfo binding. The structure of yeast Apn1p has not been solved but a similar C1' exposure is observed in a DNA-bound African swine fever virus AP endonuclease (92), another TIM barrel superfamily member. At the same time, the C1' atom of the target nucleotide is fully buried in the complex of APE1 with AP site-containing DNA, the nicked AP product, or 3'-terminal mismatched dNMPs that are substrates for the exonuclease activity of APE1 (93-95). The structure of DNA-bound *E. coli* Xth is not available but in the structurally related AP endonuclease from *Neisseria meningitidis* the C1' atom is also inaccessible (96). Thus, structural reasons may explain the lack of cleavage of the APPXL substrates by Xth and the mediocre cleavage by APE1 despite its NIR activity being capable of processing bulky etheno adducts (59,62). The remaining activity of APE1 on APPXL-Y may be due to a more pronounced duplex destabilization by the larger peptide, which would make easier the access to the phosphate of the damaged nucleotide without fully moving the peptide part into the AP site-recognition pocket.

Overall, our results suggest that, at least in bacteria and budding yeast, APPXL may be repaired by the BER pathway. After the initial nick by Nfo/Apn1p, the repair could proceed along the long-patch
branch displacing or degrading a flap decorated with the 5'-terminal adduct. Alternatively, it is possible
that 2'-deoxyribo-5'-phosphate lyases, such as RecJ or Fpg in E. coli (97,98) and Trf4 in yeast (99), or
exonucleases resolving topoisomerase II-type adducts (100,101), can catalyze excision of 5'-terminal
imine-peptide AP adducts, or that such conjugates undergo spontaneous β-elimination (102,103),
allowing the repair to continue through the short-patch subpathway. As for human cells, given that
under NIR conditions APE1 processed APPXL-Y and OHU at about the same order of magnitude,
BER could also contribute, at least partly, to APPXL repair. Possible alternatives include NER
(104,105), homologous recombination (106), or direct adduct uncoupling by HMCES-SRAP proteins
(107). Interestingly, Nfo/Apn1p homologs are present in fish and amphibians, raising a possibility that
BER of APPXL may be evolutionarily conserved and occur even in vertebrates.

DATA AVAILABILITY

All data are provided in the paper and the supplementary materials.

SUPPLEMENTARY DATA

Supplementary Data are available at NAR online.

ACKNOWLEDGEMENT

Mass spectrometry was performed at the SB RAS Institute of Chemical Biology and Fundamental
Medicine Joint Center for Genomic, Proteomic and Metabolomics Studies. We thank Dr. Alexander A.
Chernonosov for helpful discussion.

FUNDING

This work was supported by the Russian Science Foundation (grant 21-74-00061 to A.V.Y., all
biochemical experiments) and Russian Ministry of Higher Education and Science (grant
121031300056-8 to D.O.Z.). D.V.K. is supported by a graduate student fellowship from the Russian
Foundation for Basic Research (No. 20-34-90092).

CONFLICT OF INTEREST

None declared.
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TABLE AND FIGURES LEGENDS

**Table 1.** Steady-state kinetics of cleavage of APPXL substrates by AP endonucleases.

**Figure 1.** Scheme of the DNA–peptide cross-link preparation. 8-OxoG-containing DNA duplexes were treated with bifunctional DNA glycosylases Fpg (top) or OGG1 (bottom) to produce a Schiff base conjugate with the nascent aldehydic AP site through a terminal amino group in case of Fpg or an internal amino group in case of OGG1. The enzyme–DNA intermediates were stabilized with $NaBH_4$ to yield covalent DNA-protein cross-links (DPXLs). Then, DPXL were subjected to full trypsinolysis to obtain AP-peptide cross-links.

**Figure 2.** Stalling of KF and RBpol by AP–peptide cross-links. A, APPXL-I, primer–template; B, APPXL-Y, primer–template; C, APPXL-I, primer–downstream strand–template; D, APPXL-Y, primer–downstream strand–template. The $^{32}$P-labeled primer is shown in red, the downstream strand is in lowercase. Grey arrows mark the sites of synthesis termination at the lesion. Reaction time and the nature of the lesion are indicated under the gel images. In all panels, lanes 1–3 are size markers corresponding to the primer (11 nt), full-size product (40 nt), and primer extended to the cross-link site (23 nt). “–”, no enzyme added; K1, undamaged primer–template, 30 min; K2, undamaged primer–template–downstream strand, 30 min. The inset shows KF pause sites at a lower band intensity.

**Figure 3.** Stalling of Dpo4 and PolX by AP–peptide cross-links. (A), primer–template; (B), primer–downstream strand–template. The nature of the polymerase and the cross-link is indicated over and below the gel images, respectively. The $^{32}$P-labeled primer is shown in red, the downstream strand is in lowercase. Grey arrows mark the sites of synthesis termination at the lesion. Reaction time and the nature of the lesion are indicated under the gel images. In all panels, lanes 1–3 are size markers corresponding to the primer (11 nt), full-size product (40 nt), and primer extended to the cross-link site (23 nt). “–”, no enzyme added; K1, undamaged primer–template, 30 min; K2, undamaged primer–template–downstream strand, 30 min. No bypass was observed for PolX.

**Figure 4.** Incorporation of dNMPs opposite APPXLs by KF (A), RBpol (B), Dpo4 (C) and PolX (D). The nature of the substrate and dNTP is indicated below the gel images. The $^{32}$P-labeled primer is shown in red. “–”, no enzyme or dNTPs added; N, all dNTPs added; K1, undamaged primer–template, all dNTPs, 30 min.

**Figure 5.** Cleavage of APPXL-I by AP endonucleases: APE1 (A), Apn1p (B), Xth (C), and Nfo (D). For panels A–C: lane 1, APPXL-I, no enzyme; lanes 2–5, APPXL-I cleavage under the BER conditions; lanes 6–9, APPXL-I cleavage under the NIR conditions; lanes 10–13, AP site cleavage under the BER conditions; lanes 14–17, AP site cleavage under the NIR conditions; lane 18, AP site treated with NaOH; lane 19, AP site, no enzyme. Panel D: lanes 1–4, APPXL-I cleavage under the BER conditions; lanes 5–8, APPXL-I cleavage under the NIR conditions; lanes 9–12, AP site cleavage under the BER conditions; lane 13, AP site treated with NaOH; lane 14, AP site, no enzyme. BER and NIR conditions for the individual AP endonucleases are specified in Supplementary Table S2.
**Figure 6.** Cleavage of APPXL-Y by AP endonucleases: APE1 (A), Apn1p (B), Xth (C), and Nfo (D). For panels A–C: lane 1, APPXL-Y, no enzyme; lanes 2–5, APPXL-Y cleavage under the BER conditions; lanes 6–9, APPXL-Y cleavage under the NIR conditions; lanes 10–13, AP site cleavage under the BER conditions; lanes 14–17, AP site cleavage under the NIR conditions; lane 18, AP site untreated (A) or treated with NaOH (B, C). Panel D: lane 1, APPXL-Y, no enzyme; lanes 2–5, APPXL-Y cleavage under the BER conditions; lanes 6–9, APPXL-Y cleavage under the NIR conditions; lanes 10–13, AP site cleavage under the BER conditions. BER and NIR conditions for the individual AP endonucleases are specified in Supplementary Table S2.

**Figure 7.** Base excision repair continuation after the cleavage of AP site–peptide cross-links. A, B, Extension by KF of the primer with the displacement of the downstream strand bearing a 5'-terminal cross-link after Nfo cleavage of APPXL-I (A) and APPXL-Y (B). Lane 1, cross-linked strand; lane 2, APPXL treated with Nfo; lanes 3–5, APPXL treated with Nfo followed by KF extension for the indicated time; lane 6, size marker corresponding to the unmodified strand. C, Reconstitution of the full BER cycle of APPXL-I with Nfo, Pol I, and DNA ligase. Lane 1 and 2, unmodified and cross-linked strands, respectively; lane 3, substrate cleaved by Nfo; lane 4, substrate cleaved by Nfo in the presence of Pol I without dNTPs; lane 5, substrate cleaved by Nfo and extended by Pol I; lane 6, substrate cleaved by Nfo, extended by Pol I and ligated. D General scheme of possible BER-dependent repair of AP site–peptide cross-links.
<table><caption>**Table 1.** Steady-state kinetics of cleavage of APPXL substrates by AP endonucleases.</caption><thead><tr><th>Enzyme</th><th>Substrate</th><th>Conditions</th><th>K<sub>M</sub>, nM</th><th>k<sub>cat</sub>, s<sup>-1</sup><br>(×10<sup>-5</sup>)</th><th>k<sub>cat</sub>/K<sub>M</sub>, nM<sup>-1</sup>·s<sup>-1</sup><br>(×10<sup>-8</sup>)</th></tr></thead><tbody><tr><td>Apn1p</td><td>APPXL-I</td><td>BER</td><td>190 ± 60</td><td>2.0 ± 0.3</td><td>10 ± 3</td></tr><tr><td>Apn1p</td><td>APPXL-I</td><td>NIR</td><td>21 ± 8</td><td>1.2 ± 0.1</td><td>58 ± 24</td></tr><tr><td>Apn1p</td><td>APPXL-Y</td><td>BER</td><td>110 ± 30</td><td>47 ± 5</td><td>430 ± 130</td></tr><tr><td>Apn1p</td><td>APPXL-Y</td><td>NIR</td><td>200 ± 80</td><td>65 ± 13</td><td>330 ± 150</td></tr><tr><td>Apn1p</td><td>THF</td><td>BER</td><td>8.8 ± 3.0</td><td>160 ± 20</td><td>18600 ± 6700</td></tr><tr><td>Apn1p</td><td>OHU</td><td>NIR</td><td>61 ± 21</td><td>38 ± 4</td><td>620 ± 220</td></tr><tr><td>Nfo</td><td>APPXL-I</td><td>BER</td><td>n/s<sup>1</sup></td><td>n/s</td><td>5.2 ± 1.0</td></tr><tr><td>Nfo</td><td>APPXL-I</td><td>NIR</td><td>130 ± 50</td><td>6.5 ± 1.0</td><td>50 ± 20</td></tr><tr><td>Nfo</td><td>APPXL-Y</td><td>BER</td><td>n/s</td><td>n/s</td><td>200 ± 30</td></tr><tr><td>Nfo</td><td>APPXL-Y</td><td>NIR</td><td>61 ± 17</td><td>260 ± 20</td><td>4200 ± 1200</td></tr><tr><td>Nfo</td><td>THF</td><td>BER</td><td>150 ± 60.0</td><td>66 ± 12</td><td>430 ± 180</td></tr><tr><td>Nfo</td><td>OHU</td><td>NIR</td><td>140 ± 40.0</td><td>46 ± 5</td><td>320 ± 90</td></tr><tr><td>APE1</td><td>APPXL-I</td><td>BER</td><td>n/c<sup>2</sup></td><td>n/c</td><td>n/c</td></tr><tr><td>APE1</td><td>APPXL-I</td><td>NIR</td><td>n/c</td><td>n/c</td><td>n/c</td></tr><tr><td>APE1</td><td>APPXL-Y</td><td>BER</td><td>n/c</td><td>n/c</td><td>n/c</td></tr><tr><td>APE1</td><td>APPXL-Y</td><td>NIR</td><td>44 ± 19</td><td>1.6 ± 0.2</td><td>37 ± 16</td></tr><tr><td>APE1</td><td>THF</td><td>BER</td><td>21 ± 7</td><td>580 ± 70</td><td>28200 ± 10000</td></tr><tr><td>APE1</td><td>OHU</td><td>NIR</td><td>170 ± 80</td><td>19 ± 4</td><td>110 ± 50</td></tr></tbody></table>

<footnote n="1">¹ n/s, not saturated, saturation with substrate could not be achieved; *k*<sub>cat</sub>/K<sub>M</sub> determined from the initial phase of reaction velocity vs substrate concentration plots.</footnote>

<footnote n="2">² n/c, not cleaved, cleavage absent or too low to reliably estimate kinetic constants.</footnote>
<figure><img src="image_2.png" /><figcaption>**Figure 1.** Scheme of the DNA–peptide cross-link preparation. 8-OxoG-containing DNA duplexes were treated with bifunctional DNA glycosylases Fpg (top) or OGG1 (bottom) to produce a Schiff base conjugate with the nascent aldehydic AP site through a terminal amino group in case of Fpg or an internal amino group in case of OGG1. The enzyme–DNA intermediates were stabilized with $NaBH_4$ to yield covalent DNA-protein cross-links (DPXLs). Then, DPXL were subjected to full trypsinolysis to obtain AP-peptide cross-links.</figcaption></figure>
<figure><img src="image_3.png" /><figcaption>**Figure 2.** Stalling of KF and RBpol by AP-peptide cross-links. A, APPXL-I, primer-template; B, APPXL-Y, primer-template; C, APPXL-I, primer-downstream strand-template; D, APPXL-Y, primer-downstream strand-template. The ³²P-labeled primer is shown in red, the downstream strand is in lowercase. Grey arrows mark the sites of synthesis termination at the lesion. Reaction time and the nature of the lesion are indicated under the gel images. In all panels, lanes 1–3 are size markers</figcaption></figure>
corresponding to the primer (11 nt), full-size product (40 nt), and primer extended to the cross-link site (23 nt). “-”, no enzyme added; K1, undamaged primer–template, 30 min; K2, undamaged primer–template–downstream strand, 30 min. The inset shows KF pause sites at a lower band intensity.
<figure><img src="image_4.png" /><figcaption>**Figure 3.** Stalling of Dpo4 and PolX by AP-peptide cross-links. (A), primer-template; (B), primer-downstream strand-template. The nature of the polymerase and the cross-link is indicated over and below the gel images, respectively. The ³²P-labeled primer is shown in red, the downstream strand is in lowercase. Grey arrows mark the sites of synthesis termination at the lesion. Reaction time and the nature of the lesion are indicated under the gel images. In all panels, lanes 1–3 are size markers corresponding to the primer (11 nt), full-size product (40 nt), and primer extended to the cross-link site (23 nt). “-”, no enzyme added; K1, undamaged primer-template, 30 min; K2, undamaged primer-template-downstream strand, 30 min. No bypass was observed for PolX.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Figure 4.** Incorporation of dNMPs opposite APPXLs by KF (A), RBpol (B), Dpo4 (C) and PolX (D). The nature of the substrate and dNTP is indicated below the gel images. The ³²P-labeled primer is shown in red. "-", no enzyme or dNTPs added; N, all dNTPs added; K1, undamaged primer–template, all dNTPs, 30 min.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Figure 5.** Cleavage of APPXL-I by AP endonucleases: APE1 (A), Apn1p (B), Xth (C), and Nfo (D). For panels A–C: lane 1, APPXL-I, no enzyme; lanes 2–5, APPXL-I cleavage under the BER conditions; lanes 6–9, APPXL-I cleavage under the NIR conditions; lanes 10–13, AP site cleavage under the BER conditions; lanes 14–17, AP site cleavage under the NIR conditions; lane 18, AP site treated with NaOH; lane 19, AP site, no enzyme. Panel D: lanes 1–4, APPXL-I cleavage under the BER conditions; lanes 5–8, APPXL-I cleavage under the NIR conditions; lanes 9–12, AP site cleavage under the BER conditions; lane 13, AP site treated with NaOH; lane 14, AP site, no enzyme. BER and NIR conditions for the individual AP endonucleases are specified in Supplementary Table S2.</figcaption></figure>
<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /><figcaption>**Figure 6.** Cleavage of APPXL-Y by AP endonucleases: APE1 (A), Apn1p (B), Xth (C), and Nfo (D). For panels A–C: lane 1, APPXL-Y, no enzyme; lanes 2–5, APPXL-Y cleavage under the BER conditions; lanes 6–9, APPXL-Y cleavage under the NIR conditions; lanes 10–13, AP site cleavage under the BER conditions; lanes 14–17, AP site cleavage under the NIR conditions; lane 18, AP site untreated (A) or treated with NaOH (B, C). Panel D: lane 1, APPXL-Y, no enzyme; lanes 2–5, APPXL-Y cleavage under the BER conditions; lanes 6–9, APPXL-Y cleavage under the NIR conditions; lanes 10–13, AP site cleavage under the BER conditions. BER and NIR conditions for the individual AP endonucleases are specified in Supplementary Table S2.</figcaption></figure>
<figure><img src="image_11.png" /><figcaption>**Figure 7.** Base excision repair continuation after the cleavage of AP site–peptide cross-links. A, B, Extension by KF of the primer with the displacement of the downstream strand bearing a 5'-terminal cross-link after Nfo cleavage of APPXL-I (A) and APPXL-Y (B). Lane 1, cross-linked strand; lane 2, APPXL treated with Nfo; lanes 3–5, APPXL treated with Nfo followed by KF extension for the indicated time; lane 6, size marker corresponding to the unmodified strand. C, Reconstitution of the full BER cycle of APPXL-I with Nfo, Pol I, and DNA ligase. Lane 1 and 2, unmodified and cross-linked strands, respectively; lane 3, substrate cleaved by Nfo; lane 4, substrate cleaved by Nfo in the presence of Pol I without dNTPs; lane 5; substrate cleaved by Nfo and extended by Pol I; lane 6, substrate cleaved by Nfo, extended by Pol I and ligated. D General scheme of possible BER-dependent repair of AP site–peptide cross-links.</figcaption></figure>
# Abasic site–peptide cross-links are blocking lesions repaired by AP endonucleases

Anna V. Yudkina<fnref n="1" />,<fnref n="2" />*, Nikita A. Bulgakov<fnref n="1" />, Daria V. Kim<fnref n="1" />,<fnref n="2" />, Svetlana V. Baranova<fnref n="1" />, Alexander A. Ishchenko<fnref n="3" />, Murat K. Saparbaev<fnref n="3" />, Vladimir V. Koval<fnref n="1" /> and Dmitry O. Zharkov<fnref n="1" />,<fnref n="2" />,*

## KEY POINTS

* Abasic (AP) site–peptide cross-links arise in the proteolytic repair of AP site–protein cross-links

* AP site–peptide cross-links are blocking and follow the A-rule or induce primer misalignment

* AP site–peptide cross-links can be repaired by the TIM barrel superfamily AP endonucleases

## ABSTRACT

Apurinic/apyrimidinic (AP) sites are abundant DNA lesions arising from spontaneous hydrolysis of the N-glycosidic bond and as base excision repair (BER) intermediates. AP sites and their derivatives readily trap DNA-bound proteins, resulting in DNA–protein cross-links. Those are subject to proteolysis but the fate of the resulting AP–peptide cross-links (APPXLs) is unclear. Here we report two *in vitro* models of APPXLs *synthesized* by cross-linking of DNA glycosylases Fpg and OGG1 to DNA followed by trypsinolysis. The reaction with Fpg produces a 10-mer peptide cross-linked through its N-terminus, while OGG1 yields a 23-mer peptide attached through an internal lysine. Both adducts strongly blocked Klenow fragment, phage RB69 polymerase, *Saccharolobus solfataricus* Dpo4, and African swine fever virus PolX. In the residual lesion bypass, mostly dAMP and dGMP were incorporated by Klenow and RB69 polymerases, while Dpo4 and PolX used primer/template misalignment. Of AP endonucleases involved in BER, *E. coli* endonuclease IV and its yeast homolog Apn1p efficiently hydrolyzed both adducts. In contrast, *E. coli* exonuclease III and human APE1 showed little activity on APPXL substrates. Our data suggest that APPXLs produced by proteolysis of AP site-trapped proteins may be removed by the BER pathway, at least in bacterial and yeast cells.

<footnote n="1">¹SB RAS Institute of Chemical Biology and Fundamental Medicine, Novosibirsk, 630090, Russia</footnote>

<footnote n="2">²Department of Natural Sciences, Novosibirsk State University, Novosibirsk, 630090, Russia</footnote>

<footnote n="3">³Groupe "Mechanisms of DNA Repair and Carcinogenesis", Equipe Labellisée LIGUE 2016, CNRS UMR9019, Université Paris-Saclay, Gustave Roussy Cancer Campus, F-94805 Villejuif, France</footnote>

<footnote>* To whom correspondence should be addressed. Tel: +7 383 363 5187; Fax: +7 383 363 5128; Email: dzharkov@niboch.nsc.ru. Correspondence may also be addressed to Anna Yudkina. Tel: +7 383 363 5188; Fax: +7 383 363 5128; Email: ayudkina@niboch.nsc.ru.</footnote>
## INTRODUCTION

Apurinic/apyrimidinic (AP) sites occur by hydrolysis of the N-glycosidic bond in deoxyribonucleotides and are among the most abundant spontaneous and induced DNA lesions in the cell (1,2). They are also formed as intermediates in the base excision repair pathway, often being more genotoxic and mutagenic than the original lesion and thus requiring tight control over their processing (3,4). Chemically, AP sites are a family of lesions, which includes the much-studied aldehydic AP site as well as its derivatives oxidized at C1' (deoxyribolactone, DRL) (5-7), C2' (C2-AP) (8), C4' (C4-AP) or C5' position (5'-(2-phosphoryl-1,4-dioxobutane), DOB) (9,10).

Normally, AP sites are quickly removed from the genome by the base excision DNA repair (BER) system. However, there are important differences in the processing of different types of AP sites. The repair of aldehydic AP sites follows the classical short-patch BER branch, which, in human cells, involves the AP endonuclease (APE1) followed by polymerase and deoxyribophosphate lyase (dRPase) activities of DNA polymerase β (POLβ) (11). At the same time, C2-AP is not a substrate for the dRPase activity of POLβ and is removed through the long-patch branch of BER: the DNA flap containing the lesion at the 5'-end is displaced during the repair DNA synthesis and hydrolyzed by FEN1 nuclease (8,12). DRL repair is even more problematic: this lesion forms DNA-protein cross-links (DPXLs) with Lys residues in the active sites of DNA glycosylase Nth and POLβ (13,14), and the β-elimination product of DRL can similarly trap DNA glycosylases Fpg and NEIL1 (14). The presence of a 1,4-dicarbonyl moiety in C4-AP and DOB also promotes covalent capture of POLβ and DNA polymerase λ (15,16). The irreversible inhibition of the main DNA repair enzymes leads to significant cytotoxicity of modified AP sites (7).

Both aldehydic and oxidized AP sites readily form DPXLs. The covalent capture of many DNA-binding proteins by AP sites has been reported, including histones (6,17,18), integrase (19), DNA ligase (20), Ku antigen (21), as well as RNA-binding proteins (22,23), chaperones (22) and enzymes of carbohydrate metabolism (22,24). Recently, a unique mechanism for the repair of AP sites in single-stranded DNA was discovered (25-27), based on the formation of cross-links between the AP site and the HMCES protein (in human cells) or YedK protein (in E. coli), followed by switching to the proteasome-dependent pathway of DPXL repair. Thus, DPXL with AP sites are highly relevant adducts in vivo.

DPXLs are not limited to cross-links with AP sites and appear through a multitude of mechanisms including reactions with aldehydes and heavy metals, UV and ionizing radiation, and failed action of enzymes such as topoisomerases or C5-methyltransferases (28,29). Thus, a special pathway dedicated to DPXL repair has evolved. It is tightly coupled to replication and involves proteolytic degradation of the protein part of the cross-link (30-33). However, at the moment it remains poorly understood how the last peptide remnant conjugated with DNA is finally removed. It was suggested that this role can be played by nucleotide excision repair (NER), which normally protects the genome from bulky adducts (34-38). Also, Fpg/Nei-like DNA glycosylases are able to excise oxidized purine residues cross-linked to peptides (38). Recombination repair does not appear to be involved in the removal of peptide conjugates from DNA, although it may play a role in tolerance to larger DPXLs (31,36). It should be noted that all these data were obtained on cross-links chemically different from
those formed by AP sites, and peptide adducts of this type remain uncharacterized with respect to their repair and their effect on replication. While the studied peptide adducts with nucleobases usually retain some base-pairing ability, the bulky nature and non-instructing properties of AP site–peptide cross-links make these lesions more problematic for the cell. Here we report a model of stable, chemically determined AP site–peptide cross-links (APPXLs), conjugated through either a terminal or an internal position in the peptide, and show that they block DNA polymerases and can be repaired by AP endonucleases.

## MATERIAL AND METHODS

### Oligonucleotides and enzymes

E. coli formamidopyrimidine–DNA glycosylase (Fpg) (39), endonuclease VIII (Nei) (40) and endonuclease IV (Nfo) (41), human AP endonuclease (APE1) (42), 8-oxoguanine–DNA glycosylase (OGG1) (43), endonuclease VIII-like protein 1 (NEIL1) (44) and endonuclease VIII-like protein 2 (NEIL2) (45), yeast AP endonuclease (Apn1p) (41), 3'→5'-exonuclease-deficient (exo⁻) Klenow fragment of E. coli DNA polymerase I (KF) (46) and exo⁻ bacteriophage RB69 DNA polymerase (RBpol) (47) were overexpressed and purified essentially as described. Cloning and purification of DNA polymerase X from African swine fever virus (ASFV PolX) is described below. E. coli exonuclease III (Xth) was purchased from Roche (Basel, Switzerland), E. coli uracil–DNA glycosylase (Ung) and exonuclease-proficient KF, from SibEnzyme (Novosibirsk, Russia), full-length E. coli DNA polymerase I (Pol I) and Saccharolobus (Sulfolobus) solfataricus DNA polymerase IV (Dpo4), from New England Biolabs (Ipswich, MA) and trypsin, from Samson-Med (St. Petersburg, Russia). Purity of the DNA polymerases and AP endonucleases, except for those obtained commercially, is illustrated in Supplementary Figure S1. Oligonucleotides (Supplementary Table S1) were synthesized in-house from commercially available phosphoramidites (Glen Research, Sterling, VA).

### ASFV PolX cloning and purification

The coding frame of PolX optimized for expression in E. coli was synthesized by Gene Universal (Newark, DE, USA) and confirmed by Sanger sequencing. The insert was subcloned into the pET-24b vector (Merck, Darmstadt, Germany) at Ndel/XhoI sites. The plasmid was subsequently introduced into the E. coli Rossetta 2(DE3) strain (Merck). One liter of LB medium supplemented with 100 µg/ml of kanamycin was inoculated with 5 ml of an overnight culture. The cells were grown with vigorous shaking at 37°C to A₆₀₀ = 0.6, isopropyl-β-D-thiogalactopyranoside was added to 1 mM, and the growth was continued for 4 h at 37°C. The cells were harvested by centrifugation at 12,000×g at 4°C for 20 min and stored at –72°C. Before the purification, the pellet was thawed on ice in 40 ml of Buffer A consisting of 20 mM sodium phosphate (pH 7.5), 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM dithiothreitol (DTT) and supplemented with 500 mM NaCl and 1 mM phenylmethylsulfonyl fluoride. The cells were sonicated, and the lysate was clarified by centrifugation at 12,000×g at 4°C for 30 min. The supernatant was filtered through a 0.45-µm polyvinylidene difluoride membrane, diluted with four volumes of Buffer A and loaded onto a 5-ml SP Sepharose
HiTrap column (GE Healthcare, Chicago, IL) equilibrated in Buffer A plus 100 mM NaCl. The column was washed with Buffer A supplemented with 100 mM NaCl, and PolX was eluted with a NaCl gradient at ~650 mM. The fractions containing PolX were pooled, diluted with six volumes of Buffer A and loaded onto a 5-ml heparine Sepharose HiTrap column (GE Healthcare) equilibrated in Buffer A plus 100 mM NaCl. The column was washed with the same buffer, and PolX was eluted with a NaCl gradient at ~730 mM. The fractions containing PolX were pooled, diluted with six volumes of Buffer A and additionally purified on a 1-ml MonoS column (GE Healthcare) previously equilibrated in Buffer A plus 100 mM NaCl. PolX was eluted with a NaCl gradient at ~600 mM. The fractions containing >90% homogeneous protein were pooled, dialyzed against the buffer containing 20 mM sodium phosphate (pH 7.5), 400 mM NaCl, 1 mM EDTA, 1 mM DTT, and 50% glycerol, and stored at -20°C.

## Synthesis of model peptide conjugates with AP sites

DNA cross-links to Fpg or OGG1 proteins were obtained as described (48-50). Briefly, ~2 nmol of the oligonucleotide duplex (40oG//28down or 28oG//21comp; Supplementary Table S1) was treated with 3-6 nmol of the enzyme in 50 mM sodium phosphate (pH 6.8), 1 mM EDTA, 1 mM DTT and 100 mM NaBH₄ in a total volume of 40 µl. The reaction was allowed to proceed at 37°C for 2 h. The resulting DPXL was denatured at 95°C for 10 min, cooled, and pH of the solution was adjusted to ~8.0 by adding Tris base. The mixture was supplemented with 2 µg of trypsin and incubated for 4 h at 37°C. The oligonucleotide-peptide conjugate was separated by electrophoresis in 20% polyacrylamide/7 M urea, excised under UV illumination, desalted by reverse-phase chromatography on Isolute C18 sorbent (Biotage, Uppsala, Sweden) and characterized by MALDI MS. If necessary, the oligonucleotides for the following experiments were 5'-labeled using γ[³²P]ATP and annealed to a 1.5-fold molar excess of the complementary strand.

## DNA polymerase assays

For the standing-start assay, the substrates were assembled from the 40-mer template strand containing an APPXL and the ³²P-labeled primer (13pri; Supplementary Table S1) or the 28-mer template strand containing an APPXL and the ³²P-labeled primer (16pri; Supplementary Table S1). The reaction mixture (10 µl) contained 20 nM pre-annealed substrate, DNA polymerase (100 nM KF or RBpol, 400 nM PolX, 0.1 U/µl Dpo4), dNTPs (either 200 µM individual dATP, dCTP, dGTP, or dTTP, or a mixture of all four dNTPs each at 200 µM) in the reaction buffer (50 mM Tris-HCl (pH 7.5), 5 mM MgCl₂ and 1 mM DTT for KF, RBpol and PolX; 20 mM Tris-HCl (pH 8.8), 10 mM (NH₄)₂SO₄, 10 mM KCl, 2 mM MgSO₄, 0.1% Triton X-100 for Dpo4). The reaction was allowed to proceed at 37°C for 30 min, quenched with an equal volume of the loading solution (80% formamide, 20 mM Na-EDTA, 0.1% xylene cyanol, 0.1% bromophenol blue), and heated for 1 min at 95°C. The reaction products were resolved by 20% denaturing PAGE and visualized on a Typhoon FLA 9500 imager (GE Healthcare). The bands were quantified using Quantity One v4.6.8 (Bio-Rad Laboratories, Hercules, CA). For the running-start assay, the substrates were assembled from the 40-mer template strand containing an APPXL and the ³²P-labeled primer (11pri; Supplementary Table S1). The substrates
were taken at 50 nM, and the enzymes, at 50 nM (KF or RBpol), 200 nM (PolX) or 0.1 U/µl (Dpo4);
aliquots were taken at 2, 5, and 30 min; otherwise the reactions were run and processed as above.

## AP endonuclease assay

The substrates were assembled from the ³²P-labeled 40-mer strand containing an APPXL and the complementary 40-mer strand (40compC; Supplementary Table S1). The reaction mixture (10 µl) contained 50 nM substrate prepared as above and 10–1000 nM Xth, APE1, Apn1p or Nfo in the respective buffer optimal for BER or NIR (Supplementary Table S2). As a control, instead of the APPXL, we used the same oligonucleotide construct with an aldehydic AP site, freshly prepared by treatment of the U-containing substrate with Ung at 37°C for 20 min. The endonuclease reaction was allowed to proceed at 37°C for 30 min, treated and analyzed as above.

## AP endonuclease kinetics

The reaction mixture (10 µl) contained increasing concentrations of the ³²P-labeled 40-mer APPXL substrates or control substrates 23OHU:23comp or 20THF:20comp (1–500 nM) in the BER or NIR buffer. Concentrations of AP endonucleases were optimized for each substrate and buffer to produce ≤20% cleavage and were in the 5 pM – 0.5 nM range. The reaction was allowed to proceed for 10 min at 37°C, treated and analyzed as above. The data were fitted by nonlinear regression to the Michaelis–Menten equation using Sigmaplot v11.0 (Systat Software, Chicago, IL). The reported values are averages of 3–5 independent experiments.

## AP endonuclease / DNA polymerase assay

The reaction mixture (10 µl) contained 200 nM ³²P-labeled duplex 40-mer APPXL substrate and 50–150 nM Nfo in the BER buffer (Supplementary Table S2). After 10 min, the reaction mixture was supplemented with dNTPs (200 µM each), 5 mM MgCl₂, and 50–250 nM KF. The reaction was allowed to proceed at 37°C for 2, 5, or 30 min, treated and analyzed as above.

## DNA glycosylase assay

The reaction mixture (10 µl) contained 10 nM labeled duplex substrate (see above), 1 µM DNA glycosylase (E. coli Fpg or Nei, human OGG1, NEIL1, or NEIL2), 50 mM Tris–HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA and 1 mM DTT. The reaction was allowed to proceed at 37°C for 30 min, treated and analyzed as above.

# RESULTS

## Synthesis and characterization of DNA–peptide cross-links with AP site

Since AP sites can form cross-links to a variety of proteins, which can be further processed to APPXLs, it is usually assumed that the repair of such adducts does not depend on the exact nature of the cross-linked peptide. Hence, to obtain model APPXLs, we made use of two enzymes, *E. coli* Fpg and human OGG1 DNA glycosylases, that form transient covalent intermediates with AP sites during
the catalysis. To excise the damaged base (8-oxoguanine; 8-oxoG) from DNA, Fpg and OGG1 carry out a nucleophilic attack by an amino group at the C1' atom of the target nucleotide forming a labile Schiff base, which may be nearly quantitatively reduced by NaBH₄ or NaBH₃CN to a stable conjugate (Figure 1). This reaction was successfully used to crystallize covalent Fpg–DNA and OGG1–DNA complexes (39,51) and to study the interaction of full-length DPXLs with the replication and repair machinery (36,48-50). Thus, we first prepared DPXLs by treatment of 8-oxoG-containing duplexes with Fpg or OGG1 in the presence of NaBH₄ and subjected them to trypsinolysis (Supplementary Figures S2A, S2B). Fpg and OGG1 belong to different structural superfamilies and use different nucleophilic residues for catalysis: N-terminal Pro1 in Fpg and Lys249 in OGG1. Hence, after trypsinolysis, the resulting peptide conjugates are expected to have different structures (Figure 1). The Fpg–DNA conjugate gives an adduct with the decapeptide (P)ELPEVETSR (the residue that forms the cross-link is in parentheses), in which the peptide forms a bond with DNA through its N-terminus. On the other hand, trypsinolysis of the OGG1 conjugate should give an adduct with the 23-mer peptide ALCILPGVGT(K)VADCICLMALDK, cross-linked through an internal position of the peptide. Based on the visual resemblance (Figure 1), we will further call these AP site–peptide cross-links APPXL-I (terminally adducted, linear peptide) and APPXL-Y (an adduct through an internal residue with two peptide branches).

APPXL-I and APPXL-Y were purified by denaturing polyacrylamide gel electrophoresis followed by reverse-phase chromatography and characterized by MALDI-TOF mass spectrometry (Supplementary Figures S2C, S2D). The yield of the homogeneous stable APPXLs was ~5–15% of the initial 8-oxoG-containing oligonucleotide. In addition to the cross-link with the full-length modified strand, we have observed some products that likely corresponded to products of strand cleavage by Fpg/OGG1, which were easily separated from the major adduct by electrophoresis (Supplementary Figures S2A, S2B). Despite the long oligonucleotide parts, of the cross-links, their masses were better resolved by MALDI mass spectrometry in a positive mode and were within a few Da of the expected values. **No peaks of the starting material (expected [M+H⁺], 12138.9 Da) was evident, indicating good separation of the APPXLs by electrophoresis.** In the APPXL-I mass spectrum, we observed two peaks corresponding to the expected molecular weight of the adduct of the 40-mer oligonucleotide with the (P)ELPEVETSR peptide (observed [M+H⁺], 13131.0 Da, expected, 13127.9 Da) and (P)ELPEVETSRR peptide (observed [M+H⁺], 13285.8 Da, expected, 13284.0 Da), most likely resulting from incomplete trypsin cleavage at two adjacent Arg residues (Supplementary Figure S2C). As heterogeneity of cross-linked peptides may be naturally expected from proteolysis of DPXLs in the cell, we have proceeded to characterize the biochemical properties of our adducts.

## AP site–peptide cross-links block DNA polymerases

To investigate the ability of DNA polymerases to bypass APPXLs, we annealed the cross-linked template oligonucleotide with a labeled primer with its 3'-terminus placed 12 bases upstream of the cross-link site ("running start" conditions, see the substrate schemes in Figures 2A, 2B, 3A). This distance exceeds the footprints of the full-length proteins from which the cross-linked peptides were derived, and thus the peptide was not expected to prevent DNA polymerase binding. We also tested
the ability of DNA polymerases to carry out synthesis through the APPXL in the context of DNA duplex when the polymerase has to displace the downstream strand (Figures 2C, 2D, 3B). As APPXLs are non-instructive, both APPXL-I and APPXL-Y are expected to be strongly blocking.

Therefore, we used a natural freshly prepared AP site as a control to determine which events would result from DNA polymerases blocking due to the lack of a base or due to the nature of cross-linked peptide. Non-damaged primer–template or primer–downstream strand–template constructs were used as polymerase activity controls.

Four DNA polymerases, representing a range of structural and functional features, were investigated. As we were interested in the general ability of DNA polymerases to bypass APPXLs, we opted to use easily accessible representative members of various structural families originating from bacteria, archaea, and viruses, rather than human enzymes. The Klenow fragment of E. coli DNA polymerase I (KF) belongs to Family A of DNA polymerases, acting in DNA repair and replication. The homologous human enzymes are DNA polymerases γ, θ, and v. Bacteriophage RB69 DNA polymerase (RBpol) is a high-fidelity replicative polymerase from Family B and is often taken as a convenient model of structurally related human replicative DNA polymerases α, δ, and ε, and a translesion DNA polymerase ζ. In both cases, we have used 3'→5'-exonuclease-deficient versions of the polymerases to prevent primer degradation; exonuclease-proficient KF was also brought in for comparison. DNA polymerase X (PolX) from African swine fever virus (ASFV) belongs to structural Family X, mostly engaged in DNA repair and encompassing human DNA polymerases β, λ, and μ. Finally, DNA polymerase IV from Saccharolobus (formerly Sulfolobus) solfataricus (Dpo4) is a representative of Family Y translesion polymerases, of which human cells possess DNA polymerases η, ι, and κ.

Under the running start conditions, the blocking properties of APPXL-I were comparable with the properties of the natural AP site: KF demonstrated slightly better bypass of APPXL-I compared to AP site in comparison with RBpol, which was slightly more efficient on AP site-containing substrates. After 30 min, KF was able to fully elongate 6.8% of the primer if APPXL-I was in the template, and only 4.6% if an AP site was in the template whereas RB69 demonstrated 6.6% bypass of APPXL-I and 11% of the AP site (Figure 2A). Moreover, two strong pause points were evident one nucleotide before the cross-linking site and directly opposite the cross-link (23- and 24-nt long products; marked by grey arrows in Figures 2 and 3), indicating low ability of DNA polymerases both to incorporate a dNMP opposite APPXL-I and to extend the primer after such incorporation. The absence of any additional pause points suggests that the bulky peptide does not hamper moving DNA polymerase access to the cross-link site. The aldehyde AP site produced a single pause point one nucleotide before the lesion. Exonuclease-proficient KF demonstrated the same behavior as KF exo⁻ except for noticeable primer degradation at longer reaction times (Supplementary Figure S3). Thus, APPXL-I, while strongly blocking, still allows better incorporation of a dNMP than the smaller non-instructive AP site. PolX, which is known to be nearly fully blocked by AP sites (52), demonstrated no bypass of the APPXL-I as well as the natural AP site, showing a single pause point corresponding to the 23-mer synthesis product (Figure 3A). Blocking of PolX may be due to its distributive mode of synthesis, which facilitates DNA release and complicates polymerase binding near the blocking lesion.
Translesion DNA polymerase Dpo4, as expected, demonstrated the strongest ability to bypass the AP site (Dpo4 was able to fully elongate ~28% of the primer) and APPXL-I (~30% of the bypass), producing the same two pause points in the latter case (Figure 3A). The presence of a downstream strand complicated the bypass of the blocking lesion, yet the synthesis pause points and the relative efficiency of lesion bypass seen with the primer–template system were preserved (Figure 2C, Figure 3B).

Strong polymerase inhibition and two pronounced pause points for KF, RBpol and Dpo4 were also evident for substrates containing the branched APPXL-Y adduct (Figures 2B, 2D, 3, Supplementary Figure S3). The bulkier APPXL-Y turned out to be even more blocking than APPXL-I. The pause point corresponding to the 24-nt product for KF and RBpol was weaker than the corresponding pause for APPXL-I, and the full-length product decreased significantly for KF and Dpo4.

We have also addressed polymerase-blocking ability of AP site–peptide cross-links present in an internal position of the non-template downstream strand. However, only for PolX was a minor pause point observed, while KF and RBpol displaced the cross-linked strand without effort, and Dpo4, while having lower strand displacement activity, did not pause at the cross-link (Supplementary Figure S4).

Interestingly, when different substrates were compared, we systematically observed primer utilization efficiency decreasing in the order AP site > APPXL-I > APPXL-Y. Possibly, the peptide part may partially interfere with DNA binding by the polymerase even when the primer end is 12 nt away from the cross-link site, for example, by obstructing non-specific DNA binding and facilitated diffusion used by polymerases to locate the primer end (53). The only exception was Dpo4 with APPXL-Y in the downstream strand, in which case the bulky peptide adduct might destabilize the duplex and improve the poor strand displacement ability of Dpo4.

## AP site–peptide cross-links mostly direct A and G incorporation and can induce primer–template misalignment

Since we observed some dNMP insertion opposite APPXLs, we sought to explore the spectra of the incorporated nucleotides. To do this, we moved the primer to place its 3'-end immediately next to the site of the lesion and presented the polymerases with the individual dNMPs as well as with their mixture ("standing start" conditions, Figure 4). Here, KF utilized the APPXL-I adducted template much better than the APPXL-Y adduct, whereas for RBpol the efficiency was somewhat better for the APPXL-Y. Notably, in all cases the primer elongation was significantly lower than under the running start conditions, most likely because the bulky peptide interferes with DNA polymerases' binding to the primer/template junction. Nevertheless, the polymerase preferences for dNMP incorporation opposite APPXLs were clear. Both KF and RBpol predominantly incorporated dAMP, and, to a lesser extent, dGMP (Figures 4A, 4B). We observed the preference of dNMP incorporation in the order A > G > C > T, which is consistent with the "A rule", the preference pattern for better dAMP incorporation opposite the AP site (54-56). dGMP incorporation could possibly be explained by template/primer slippage since the next base after the lesion in the template is C. To address this possibility, we repeated the experiment with the substrate of another sequence featuring G as the next base (Supplementary Figure S5), for which template/primer slippage would guide the
incorporation of dCMP. However, the same general dNMP incorporation pattern A > G > C > T for KF and RBpol was observed (Supplementary Figures S5A, S5B), confirming that these preferences characterize the miscoding properties of the peptide adducts rather than the tendency for template misalignment. The order of preference of dNTP incorporation by the Family Y Dpo4 was G >> A > C, T (Figure 4C); however, when the substrate was switched, the preference for dNTP usage completely to C >> A > G, T (Supplementary Figure S5C), indicating that Dpo4 use +1 nucleotide template misalignment mechanism rather than the “A rule” during the synthesis on the AP-peptide APPXL-containing substrates. Dpo4 tended to misincorporate several nucleotides, in agreement with its reported low fidelity characteristic of Family Y DNA polymerases (57). PolX also bypassed the peptide adduct by +1 slippage mechanism, albeit with a low efficiency: the enzyme preferred G > A, T, C on the substrate presenting C as the +1 template nucleotide while changing the specificity to C > A >> T, G on the substrate presenting G at this position (Figure 4D, Supplementary Figure 5D).

## AP site–peptide cross-links can be processed by AP endonucleases

Aldehydic AP sites and many of their natural and synthetic modifications are removed from DNA through the BER pathway initiated by AP endonucleases. These enzymes belong to two major unrelated structural groups: the exonuclease–endonuclease–phosphatase (EEP) superfamily and the triosephosphate isomerase (TIM) barrel superfamily. Therefore, we have tested representative members of each group – *E. coli* exonuclease III (Xth) and the main human AP endo/exonuclease 1 (APE1) from the EEP superfamily and *E. coli* endonuclease IV (Nfo) and yeast endonuclease Apn1p from the TIM barrel superfamily – for their ability to cleave oligonucleotide substrates containing APPXL-I or APPXL-Y. Notably, all these enzymes but Xth, in addition to their AP endonuclease activity, were shown to possess the “nucleotide incision repair” (NIR) activity that cleaves DNA 5’ to many lesions of different chemical nature including quite bulky ones such as 1,*N*⁶-etheno-dA, 3,*N*⁴-etheno-dC, and 3,*N*⁴-benzetheno-dC (41,58-62). Since the pH, buffer and ionic composition optimal for BER and NIR are enzyme-specific, we tested the ability of these AP endonucleases to cleave APPXL-containing substrates under both conditions (Supplementary Table S2).

APE1 efficiently hydrolyzed the natural AP site but did not yield any detectable product of the APPXL-I substrate cleavage (Figure 5A). However, the APPXL-Y substrate was modestly hydrolyzed by APE1, especially under NIR conditions (Figure 6A). This result was rather surprising, since the bulkier APPXL-Y adduct was expected to have larger inhibitory effect, and suggested that the chemical nature of the peptide adduct may be important for its recognition by AP endonucleases. Another member of the EEP superfamily, Xth, also showed low activity on substrates of both types (Figure 5C, 6C). The degradation product observed at the highest enzyme concentrations migrates slower than the expected APPXL cleavage product and is likely due to the robust 3'→5'-exonuclease activity of Xth evident on the AP substrate. In contrast to APE1 and Xth, both Nfo and Apn1p efficiently processed the substrates containing APPXL-I or APPXL-Y adducts, cleaving the DNA at the cross-link site and showing some additional exonucleolytic degradation (Figure 5B, 5D, 6B, 6D). The cleavage was evident under either BER or NIR conditions. Thus, both TIM barrel AP endonucleases cleaved APPXL-containing DNA significantly better than did EEP superfamily AP endonucleases.
To compare the efficiency of AP endonucleases processing APPXLs with their established substrates in a quantitative way, we have measured the steady-state kinetic parameters ($K_M$, $k_{cat}$, and the catalytic efficiency $k_{cat}/K_M$) values using duplex oligonucleotides containing APPXL-I, APPXL-Y, (3-hydroxytetrahydrofuran-2-yl)methyl phosphate (THF, an AP site analog resistant to β-elimination and often used as a model AP site in BER studies) or 5-hydroxy-2'-deoxyuridine (OHU, a typical NIR substrate) (61,62). As shown in Table 1 and Supplementary Figure S6, the apparent $k_{cat}/K_M$ values confirm APPXL-Y is a better substrate compared to APPXL-I for all studied AP endonucleases. For the TIM barrel enzymes, APPXL-Y cleavage activity was comparable with (~0.5-fold for Apn1p) or even significantly higher than (~13-fold for Nfo) the OHU cleavage, strongly suggesting that these AP endonucleases may be biologically relevant for processing of AP site–peptide adducts. Moreover, even under BER conditions the efficiency of APPXL-Y cleavage by Nfo was comparable with THF cleavage (THF favored only ~2-fold). More efficient processing of APPXL-Y than of APPXL-I may also be of biological importance, as peptides covalently bound to the AP site through an internal residue are statistically more likely to arise in the cell after protein conjugation followed by proteolysis.

We also tested the ability of several bifunctional DNA glycosylases capable of AP site cleavage, namely *E. coli* Fpg and Nei, human OGG1, NEIL1 and NEIL2, to cleave the APPXL adducts. However, none of them was able to process cross-links of either type to any significant degree (Supplementary Figure S7).

## Strand with a hanging 5'-terminal peptide cross-link can be displaced by DNA polymerases

AP endonucleases nick DNA 5' of the lesion producing a hanging 5'-terminal 2'-deoxyribophosphate (or another lesion remnant if the substrate was something else than an aldehydic AP site) and a free 3'-OH, which then serves as a primer for a DNA polymerase. To see whether the nick with a 5'-terminal peptide adduct can be processed in the same way, we have first treated the APPXL-I or APPXL-Y substrate with Nfo and then added KF and all four dNTPs (Figure 7A, 7B). Although in both cases a pause was observed after the incorporation of one nucleotide, eventually the full-length primer extension product was synthesized. Thus, even if the peptide part of the cross-link interferes with polymerase binding or the early steps of primer extension, once the flap is >1 nt there is apparently no obstacles for further strand displacement.

Furthermore, we have reconstituted the full BER cycle with APPXL-I (annealed opposite to G; 40compG template strand), Nfo, full-length *E. coli* DNA polymerase I, and DNA ligase. Unlike KF, Pol I degrades the strand ahead by a combined exo/endonucleolytic action as it extends the primer (63). To prevent full degradation of the downstream strand, we have omitted dATP and dGTP from the reaction mixture, which should cause the polymerase to stop after incorporating 14 nt, yielding a 30-mer product (Figure 7C). Nfo cleaved the duplex at the adduct, producing the 16-mer with the 3'-OH end, which served as an entry point for Pol I (Figure 7C). In the absence of dNTPs, the 3'→5' exonuclease activity of Pol I degraded the primer, whereas addition of dCTP and dTTP allowed primer extension to the position of the first pyrimidine in the template. Finally, if DNA ligase was present, the product had a higher electrophoretic mobility than the DNA-peptide adduct, migrating
approximately at the same level as an undamaged 40-mer (Figure 7C), which clearly indicated that the repair took place.

These observations support the possibility of APPXL removal via the long-patch BER subpathway (Figure 7D). Obviously, short-patch repair is also possible if a non-stabilized 5'-terminal conjugate can be processed by a dRP lyase.

DISCUSSION

For many years, AP sites were considered a fairly well studied type of DNA damage, but recent discoveries regarding new features of their enzymatic processing and biologically relevant reactions of AP sites with non-specific DNA-bound proteins (18,25-27) put these lesions in an unexpected perspective. There is mounting evidence that native and oxidized AP sites in the cell can covalently capture proteins to form highly toxic DNA-protein cross-links. Several breakthrough works in recent years have outlined the general repair scheme for DNA damage of this type. DPXLs mainly serve as substrates for a specialized repair pathway, which is initiated by proteolytic degradation of the protein part of the cross-link by the proteasome or specialized replication-associated proteases (SPRTN1, FAM111A, DDI1, or others yet to be discovered) to a small peptide (30-33). What happens after that, however, remains poorly understood. The studied models of naturally occurring DNA-peptide cross-links include peptide conjugates with exocyclic amino groups of G and A modified through (i) a y-hydroxypropano bridge (a product of acrolein-induced DNA damage) (35,64-66) or (ii) through a C2 linker derived from alkylation of Cys residues by 1,2-dibromoethane (67), and (iii) conjugates at the aldehyde group of 5-formylcytosine (68-70). In addition, unnatural adducts that are readily obtained synthetically, such as products of azide-alkyl addition to the functionalized C5 position of thymine (71) and cross-links to 7-deaza-7-(2-oxoethyl)guanine (72), were used as model systems.

The major difference between peptide cross-links through nucleobases and through AP sites is the inability of the latter to form complementary bonds. Until now, there have been no reports in the literature regarding the levels of APPXLs, their effect on DNA polymerases or the ways to repair such residual damage. However, recent data on the reactivity of aldehyde and oxidized AP sites suggest that the number of cross-links with the AP site can be comparable to the number of cross-links with bases, or even exceed them. Moreover, some conjugates with oxidized AP sites are much more stable than reversible imine conjugates with aldehydic AP sites. In this work, we developed a model to generate cross-links of AP sites with peptides of different structure based on the reduction of the Schiff base intermediate formed by DNA glycosylases Fpg and OGG1 followed by trypsinolysis. Fpg carries an N-terminal nucleophilic amino group and, after proteolysis, leaves behind a decapeptide attached via its end (APPXL-I), while OGG1 uses the internal Lys249 for catalysis, and upon trypsinolysis yields a longer peptide remnant, in which the cross-link is formed at an internal position (APPXL-Y). This peptide adduct may be of greater biological relevance because DPXLs in the cell are statistically much more likely to be attached within the polypeptide chain, and protease cleavage immediately next to the cross-link point may be hindered by DNA. Although the diversity of proteins capable of cross-linking to DNA dictates the universality of the mechanisms for APPXL repair, the size
and properties of the peptide remnant might be expected to affect the APPXL interactions with DNA polymerases and the repair machinery.

A key aspect of APPXL biochemistry determining their biological consequences is the ability to block DNA polymerases or guide incorporation of particular dNMPs. Although no single DNA-peptide adduct was studied with a fully representative set of DNA polymerases, in vitro experiments with peptides conjugated through nucleobases show that bypass of such lesions depends both on their size and the preservation of complementarity. DNA polymerases bind DNA in the minor groove and leave the major groove mostly accessible, so bulky moieties occupying the major groove may be better tolerated. For example, 5-formylcytosine adducts or peptides attached to the C5 position of dU through click chemistry, which protrude to the major groove and do not interfere with base pairing, are quite easily bypassed by both replicative and translesion human DNA polymerases, although longer peptides may present a problem (e. g., 10-mer peptides were bypassed, while 23-mers were not) (70,71,73). In another report, even a large 31-mer peptide conjugated to 5-formylcytosine was bypassed by translesion DNA polymerases κ and η (68). Similarly, conjugates through position C7 of 7-deazaG are bypassed by translesion polymerases in a size-dependent manner (74). Peptides adducted through the exocyclic amino group of A, which may either eliminate or keep base pairing depending on the orientation of the peptide, completely block E. coli replicative DNA polymerase III but demonstrate low-to-full bypass by E. coli DNA polymerases I, II, IV and V, human translesion DNA polymerases η, ι, κ, and v, and replicative yeast DNA polymerase δ (65-67). On the other hand, peptide adducts with the exocyclic group of G, which either disrupt base pairing or protrude into the minor groove, are strongly blocking for most polymerases and were efficiently bypassed only by Family Y translesion DNA polymerases (E. coli Pol IV and human Pol κ) (64,65). In the cell, mutagenic bypass of peptide adducts requires translesion DNA polymerases (69,70,75).

Bypass of natural AP sites and their modified varieties is often described by the "A-rule", which states that dAMP insertion is preferred opposite a non-instructive lesion. Interestingly, the structural reasons underlying this preference are different between DNA polymerases. For example, the structure of the Stoffel fragment of Taq DNA polymerase (which corresponds to KF in the E. coli enzyme) shows templating by a conservative Tyr residue inserted in place of a template nucleotide and acting as a thymine mimic (76,77). RBpol, on the other hand, relies on the favorable stacking between the incoming dATP and the extended planar system formed by the terminal primer nucleotide and its pairing partner in the absence of the template nucleobase (78,79). However, in the structures of both polymerases with AP-DNA, C1' of the AP site resides is buried at the protein-DNA interface, and the template strand would require extensive conformational rearrangement for a bypass if a large peptide is attached to this position. The steric clash could be relieved by flipping the template nucleotide around its flanking phosphates, as often observed in structures of Families X and Y DNA polymerases stabilizing a misaligned primer-template junction (80-82). In the "open" state of the enzyme-DNA complex, Family A DNA polymerases, including KF, also rotate the downstream template nucleotides outside of the helix, and it has been suggested that this movement helps prevent the template bases from misalignment in the absence of an incoming dNTP (83). As we do not see preferential incorporation of a dNMP corresponding to +1 template nucleotide, characteristic of the
primer–template misalignment bypass mechanism, we hypothesize that favorable stacking can guide dAMP and dGMP insertion opposite extrahelical APPXLs in the rare bypass events. On the other hand, structures of Dpo4 bypassing an abasic site, demonstrate extensive conformational flexibility allowing for primer/template misalignment and generation of mismatches, +1 and –1 frameshifts (84). In the structure with misalignment between the primer end and the +1 position in the template (which was observed in our experiments), the AP site is looped out of the helix into a gap between the fingers and the little finger domains, providing plenty of space for accommodation of a cross-linked peptide. No structure of PolX in the frameshift mode is available but POLβ, a prototypical member of Family X, can also loop out a template nucleotide to match the primer end to the +1 position in the template (82).

As we used representative DNA polymerases of non-human origin in our study, it remains to be seen which DNA polymerases may participate in APPXL bypass in human cells. Based on the *in vitro* insertion specificity data, it has long been held that natural AP sites are bypassed by DNA polymerase η (POLη) (85,86). On the other hand, AP site bypass in POLη-deficient human cells is almost as efficient as in wild-type cells (87). An alternative model was proposed, based on yeast mutation spectra and *in vitro* biochemistry of human and yeast DNA polymerase ζ (POLζ), that the POLζ/REV1 complex acting both as an inserter and an extender polymerase provides mutagenic bypass of AP sites (reviewed in (88)). However, mutation spectra induced by AP sites in yeast and human cells seem to be different (89), and human REV1 also interacts with DNA polymerases η, κ, and ι (88), so the nature of the inserter polymerase in human cells exposed to AP sites or their derivatives, including APPXLs, needs further investigation.

The high proficiency of TIM barrel AP endonucleases in nicking DNA at APPXLs was rather unexpected, since even the known substrates for the NIR activity of these enzymes are much less bulky. However, inspection of the structures of DNA-bound Nfo (90,91) reveals that C1' is solvent-accessible in both enzyme–substrate and enzyme–product complexes (Supplementary Figure S8) and the attached peptide could possibly be accommodated in the major groove of the DNA molecule, which is sharply bent upon Nfo binding. The structure of yeast Apn1p has not been solved but a similar C1' exposure is observed in a DNA-bound African swine fever virus AP endonuclease (92), another TIM barrel superfamily member. At the same time, the C1' atom of the target nucleotide is fully buried in the complex of APE1 with AP site-containing DNA, the nicked AP product, or 3'-terminal mismatched dNMPs that are substrates for the exonuclease activity of APE1 (93-95). The structure of DNA-bound *E. coli* Xth is not available but in the structurally related AP endonuclease from *Neisseria meningitidis* the C1' atom is also inaccessible (96). Thus, structural reasons may explain the lack of cleavage of the APPXL substrates by Xth and the mediocre cleavage by APE1 despite its NIR activity being capable of processing bulky etheno adducts (59,62). The remaining activity of APE1 on APPXL-Y may be due to a more pronounced duplex destabilization by the larger peptide, which would make easier the access to the phosphate of the damaged nucleotide without fully moving the peptide part into the AP site-recognition pocket.

Overall, our results suggest that, at least in bacteria and budding yeast, APPXL may be repaired by the BER pathway. After the initial nick by Nfo/Apn1p, the repair could proceed along the long-patch
branch displacing or degrading a flap decorated with the 5'-terminal adduct. Alternatively, it is possible
that 2'-deoxyribo-5'-phosphate lyases, such as RecJ or Fpg in E. coli (97,98) and Trf4 in yeast (99), or
exonucleases resolving topoisomerase II-type adducts (100,101), can catalyze excision of 5'-terminal
imine-peptide AP adducts, or that such conjugates undergo spontaneous β-elimination (102,103),
allowing the repair to continue through the short-patch subpathway. As for human cells, given that
under NIR conditions APE1 processed APPXL-Y and OHU at about the same order of magnitude,
BER could also contribute, at least partly, to APPXL repair. Possible alternatives include NER
(104,105), homologous recombination (106), or direct adduct uncoupling by HMCES-SRAP proteins
(107). Interestingly, Nfo/Apn1p homologs are present in fish and amphibians, raising a possibility that
BER of APPXL may be evolutionarily conserved and occur even in vertebrates.

DATA AVAILABILITY

All data are provided in the paper and the supplementary materials.

SUPPLEMENTARY DATA

Supplementary Data are available at NAR online.

ACKNOWLEDGEMENT

Mass spectrometry was performed at the SB RAS Institute of Chemical Biology and Fundamental
Medicine Joint Center for Genomic, Proteomic and Metabolomics Studies. We thank Dr. Alexander A.
Chernonosov for helpful discussion.

FUNDING

This work was supported by the Russian Science Foundation (grant 21-74-00061 to A.V.Y., all
biochemical experiments) and Russian Ministry of Higher Education and Science (grant
121031300056-8 to D.O.Z.). D.V.K. is supported by a graduate student fellowship from the Russian
Foundation for Basic Research (No. 20-34-90092).

CONFLICT OF INTEREST

None declared.
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TABLE AND FIGURES LEGENDS

**Table 1.** Steady-state kinetics of cleavage of APPXL substrates by AP endonucleases.

**Figure 1.** Scheme of the DNA–peptide cross-link preparation. 8-OxoG-containing DNA duplexes were treated with bifunctional DNA glycosylases Fpg (top) or OGG1 (bottom) to produce a Schiff base conjugate with the nascent aldehydic AP site through a terminal amino group in case of Fpg or an internal amino group in case of OGG1. The enzyme–DNA intermediates were stabilized with NaBH<sub>4</sub> to yield covalent DNA-protein cross-links (DPXLs). Then, DPXL were subjected to full trypsinolysis to obtain AP-peptide cross-links.

**Figure 2.** Stalling of KF and RBpol by AP–peptide cross-links. A, APPXL-I, primer–template; B, APPXL-Y, primer–template; C, APPXL-I, primer–downstream strand–template; D, APPXL-Y, primer–downstream strand–template. The <sup>32</sup>P-labeled primer is shown in red, the downstream strand is in lowercase. Grey arrows mark the sites of synthesis termination at the lesion. Reaction time and the nature of the lesion are indicated under the gel images. In all panels, lanes 1–3 are size markers corresponding to the primer (11 nt), full-size product (40 nt), and primer extended to the cross-link site (23 nt). “–”, no enzyme added; K1, undamaged primer–template, 30 min; K2, undamaged primer–template–downstream strand, 30 min. The inset shows KF pause sites at a lower band intensity.

**Figure 3.** Stalling of Dpo4 and PolX by AP–peptide cross-links. (A), primer–template; (B), primer–downstream strand–template. The nature of the polymerase and the cross-link is indicated over and below the gel images, respectively. The <sup>32</sup>P-labeled primer is shown in red, the downstream strand is in lowercase. Grey arrows mark the sites of synthesis termination at the lesion. Reaction time and the nature of the lesion are indicated under the gel images. In all panels, lanes 1–3 are size markers corresponding to the primer (11 nt), full-size product (40 nt), and primer extended to the cross-link site (23 nt). “–”, no enzyme added; K1, undamaged primer–template, 30 min; K2, undamaged primer–template–downstream strand, 30 min. No bypass was observed for PolX.

**Figure 4.** Incorporation of dNMPs opposite APPXLs by KF (A), RBpol (B), Dpo4 (C) and PolX (D). The nature of the substrate and dNTP is indicated below the gel images. The <sup>32</sup>P-labeled primer is shown in red. “–”, no enzyme or dNTPs added; N, all dNTPs added; K1, undamaged primer–template, all dNTPs, 30 min.

**Figure 5.** Cleavage of APPXL-I by AP endonucleases: APE1 (A), Apn1p (B), Xth (C), and Nfo (D). For panels A–C: lane 1, APPXL-I, no enzyme; lanes 2–5, APPXL-I cleavage under the BER conditions; lanes 6–9, APPXL-I cleavage under the NIR conditions; lanes 10–13, AP site cleavage under the BER conditions; lanes 14–17, AP site cleavage under the NIR conditions; lane 18, AP site treated with NaOH; lane 19, AP site, no enzyme. Panel D: lanes 1–4, APPXL-I cleavage under the BER conditions; lanes 5–8, APPXL-I cleavage under the NIR conditions; lanes 9–12, AP site cleavage under the BER conditions; lane 13, AP site treated with NaOH; lane 14, AP site, no enzyme. BER and NIR conditions for the individual AP endonucleases are specified in Supplementary Table S2.
**Figure 6.** Cleavage of APPXL-Y by AP endonucleases: APE1 (A), Apn1p (B), Xth (C), and Nfo (D). For panels A–C: lane 1, APPXL-Y, no enzyme; lanes 2–5, APPXL-Y cleavage under the BER conditions; lanes 6–9, APPXL-Y cleavage under the NIR conditions; lanes 10–13, AP site cleavage under the BER conditions; lanes 14–17, AP site cleavage under the NIR conditions; lane 18, AP site untreated (A) or treated with NaOH (B, C). Panel D: lane 1, APPXL-Y, no enzyme; lanes 2–5, APPXL-Y cleavage under the BER conditions; lanes 6–9, APPXL-Y cleavage under the NIR conditions; lanes 10–13, AP site cleavage under the BER conditions. BER and NIR conditions for the individual AP endonucleases are specified in Supplementary Table S2.

**Figure 7.** Base excision repair continuation after the cleavage of AP site–peptide cross-links. A, B, Extension by KF of the primer with the displacement of the downstream strand bearing a 5'-terminal cross-link after Nfo cleavage of APPXL-I (A) and APPXL-Y (B). Lane 1, cross-linked strand; lane 2, APPXL treated with Nfo; lanes 3–5, APPXL treated with Nfo followed by KF extension for the indicated time; lane 6, size marker corresponding to the unmodified strand. C, Reconstitution of the full BER cycle of APPXL-I with Nfo, Pol I, and DNA ligase. Lane 1 and 2, unmodified and cross-linked strands, respectively; lane 3, substrate cleaved by Nfo; lane 4, substrate cleaved by Nfo in the presence of Pol I without dNTPs; lane 5, substrate cleaved by Nfo and extended by Pol I; lane 6, substrate cleaved by Nfo, extended by Pol I and ligated. D General scheme of possible BER-dependent repair of AP site–peptide cross-links.
<table><caption>**Table 1.** Steady-state kinetics of cleavage of APPXL substrates by AP endonucleases.</caption><thead><tr><th>Enzyme</th><th>Substrate</th><th>Conditions</th><th>K<sub>M</sub>, nM</th><th>k<sub>cat</sub>, s<sup>-1</sup><br>(×10<sup>-5</sup>)</th><th>k<sub>cat</sub>/K<sub>M</sub>, nM<sup>-1</sup>·s<sup>-1</sup><br>(×10<sup>-8</sup>)</th></tr></thead><tbody><tr><td>Apn1p</td><td>APPXL-I</td><td>BER</td><td>190 ± 60</td><td>2.0 ± 0.3</td><td>10 ± 3</td></tr><tr><td>Apn1p</td><td>APPXL-I</td><td>NIR</td><td>21 ± 8</td><td>1.2 ± 0.1</td><td>58 ± 24</td></tr><tr><td>Apn1p</td><td>APPXL-Y</td><td>BER</td><td>110 ± 30</td><td>47 ± 5</td><td>430 ± 130</td></tr><tr><td>Apn1p</td><td>APPXL-Y</td><td>NIR</td><td>200 ± 80</td><td>65 ± 13</td><td>330 ± 150</td></tr><tr><td>Apn1p</td><td>THF</td><td>BER</td><td>8.8 ± 3.0</td><td>160 ± 20</td><td>18600 ± 6700</td></tr><tr><td>Apn1p</td><td>OHU</td><td>NIR</td><td>61 ± 21</td><td>38 ± 4</td><td>620 ± 220</td></tr><tr><td>Nfo</td><td>APPXL-I</td><td>BER</td><td>n/s<sup>1</sup></td><td>n/s</td><td>5.2 ± 1.0</td></tr><tr><td>Nfo</td><td>APPXL-I</td><td>NIR</td><td>130 ± 50</td><td>6.5 ± 1.0</td><td>50 ± 20</td></tr><tr><td>Nfo</td><td>APPXL-Y</td><td>BER</td><td>n/s</td><td>n/s</td><td>200 ± 30</td></tr><tr><td>Nfo</td><td>APPXL-Y</td><td>NIR</td><td>61 ± 17</td><td>260 ± 20</td><td>4200 ± 1200</td></tr><tr><td>Nfo</td><td>THF</td><td>BER</td><td>150 ± 60.0</td><td>66 ± 12</td><td>430 ± 180</td></tr><tr><td>Nfo</td><td>OHU</td><td>NIR</td><td>140 ± 40.0</td><td>46 ± 5</td><td>320 ± 90</td></tr><tr><td>APE1</td><td>APPXL-I</td><td>BER</td><td>n/c<sup>2</sup></td><td>n/c</td><td>n/c</td></tr><tr><td>APE1</td><td>APPXL-I</td><td>NIR</td><td>n/c</td><td>n/c</td><td>n/c</td></tr><tr><td>APE1</td><td>APPXL-Y</td><td>BER</td><td>n/c</td><td>n/c</td><td>n/c</td></tr><tr><td>APE1</td><td>APPXL-Y</td><td>NIR</td><td>44 ± 19</td><td>1.6 ± 0.2</td><td>37 ± 16</td></tr><tr><td>APE1</td><td>THF</td><td>BER</td><td>21 ± 7</td><td>580 ± 70</td><td>28200 ± 10000</td></tr><tr><td>APE1</td><td>OHU</td><td>NIR</td><td>170 ± 80</td><td>19 ± 4</td><td>110 ± 50</td></tr></tbody></table>

<footnote n="1">¹ n/s, not saturated, saturation with substrate could not be achieved; $k_{cat}/K_M$ determined from the initial phase of reaction velocity vs substrate concentration plots.</footnote>

<footnote n="2">² n/c, not cleaved, cleavage absent or too low to reliably estimate kinetic constants.</footnote>
<figure><img src="image_12.png" /><figcaption>**Figure 1.** Scheme of the DNA–peptide cross-link preparation. 8-OxoG-containing DNA duplexes were treated with bifunctional DNA glycosylases Fpg (top) or OGG1 (bottom) to produce a Schiff base conjugate with the nascent aldehydic AP site through a terminal amino group in case of Fpg or an internal amino group in case of OGG1. The enzyme–DNA intermediates were stabilized with NaBH₄ to yield covalent DNA-protein cross-links (DPXLs). Then, DPXL were subjected to full trypsinolysis to obtain AP-peptide cross-links.</figcaption></figure>
<figure><img src="image_13.png" /><figcaption>**Figure 2.** Stalling of KF and RBpol by AP-peptide cross-links. A, APPXL-I, primer-template; B, APPXL-Y, primer-template; C, APPXL-I, primer-downstream strand-template; D, APPXL-Y, primer-downstream strand-template. The ³²P-labeled primer is shown in red, the downstream strand is in lowercase. **Grey arrows mark the sites of synthesis termination at the lesion. Reaction time and the nature of the lesion are indicated under the gel images.** In all panels, lanes 1–3 are size markers</figcaption></figure>
corresponding to the primer (11 nt), full-size product (40 nt), and primer extended to the cross-link site (23 nt). “-”, no enzyme added; K1, undamaged primer–template, 30 min; K2, undamaged primer–template–downstream strand, 30 min. The inset shows KF pause sites at a lower band intensity.
<figure><img src="image_14.png" /><figcaption>**Figure 3.** Stalling of Dpo4 and PolX by AP-peptide cross-links. (A), primer-template; (B), primer-downstream strand-template. The nature of the polymerase and the cross-link is indicated over and below the gel images, respectively. The ³²P-labeled primer is shown in red, the downstream strand is in lowercase. Grey arrows mark the sites of synthesis termination at the lesion. Reaction time and the nature of the lesion are indicated under the gel images. In all panels, lanes 1–3 are size markers corresponding to the primer (11 nt), full-size product (40 nt), and primer extended to the cross-link site (23 nt). “-”, no enzyme added; K1, undamaged primer-template, 30 min; K2, undamaged primer-template-downstream strand, 30 min. No bypass was observed for PolX.</figcaption></figure>
<figure><img src="image_15.png" /><figcaption>**Figure 4.** Incorporation of dNMPs opposite APPXLs by **KF (A)**, **RBpol (B)**, **Dpo4 (C)** and **PolX (D)**. The nature of the substrate and dNTP is indicated below the gel images. The ³²P-labeled primer is shown in red. "-", no enzyme or dNTPs added; N, all dNTPs added; K1, undamaged primer–template, all dNTPs, 30 min.</figcaption></figure>
<figure><img src="image_16.png" /><figcaption>**Figure 5.** Cleavage of APPXL-I by AP endonucleases: APE1 (A), Apn1p (B), Xth (C), and Nfo (D). For panels A–C: lane 1, APPXL-I, no enzyme; lanes 2–5, APPXL-I cleavage under the BER conditions; lanes 6–9, APPXL-I cleavage under the NIR conditions; lanes 10–13, AP site cleavage under the BER conditions; lanes 14–17, AP site cleavage under the NIR conditions; lane 18, AP site treated with NaOH; lane 19, AP site, no enzyme. Panel D: lanes 1–4, APPXL-I cleavage under the BER conditions; lanes 5–8, APPXL-I cleavage under the NIR conditions; lanes 9–12, AP site cleavage under the BER conditions; lane 13, AP site treated with NaOH; lane 14, AP site, no enzyme. BER and NIR conditions for the individual AP endonucleases are specified in Supplementary Table S2.</figcaption></figure>
<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /><figcaption>**Figure 6.** Cleavage of APPXL-Y by AP endonucleases: APE1 (A), Apn1p (B), Xth (C), and Nfo (D). For panels A–C: lane 1, APPXL-Y, no enzyme; lanes 2–5, APPXL-Y cleavage under the BER conditions; lanes 6–9, APPXL-Y cleavage under the NIR conditions; lanes 10–13, AP site cleavage under the BER conditions; lanes 14–17, AP site cleavage under the NIR conditions; lane 18, AP site untreated (A) or treated with NaOH (B, C). Panel D: lane 1, APPXL-Y, no enzyme; lanes 2–5, APPXL-Y cleavage under the BER conditions; lanes 6–9, APPXL-Y cleavage under the NIR conditions; lanes 10–13, AP site cleavage under the BER conditions. BER and NIR conditions for the individual AP endonucleases are specified in Supplementary Table S2.</figcaption></figure>
<figure><img src="image_21.png" /></figure>

**Figure 7.** Base excision repair continuation after the cleavage of AP site–peptide cross-links. A, B, Extension by KF of the primer with the displacement of the downstream strand bearing a 5'-terminal cross-link after Nfo cleavage of APPXL-I (A) and APPXL-Y (B). Lane 1, cross-linked strand; lane 2, APPXL treated with Nfo; lanes 3–5, APPXL treated with Nfo followed by KF extension for the indicated time; lane 6, size marker corresponding to the unmodified strand. C, Reconstitution of the full BER cycle of APPXL-I with Nfo, Pol I, and DNA ligase. Lane 1 and 2, unmodified and cross-linked strands, respectively; lane 3, substrate cleaved by Nfo; lane 4, substrate cleaved by Nfo in the presence of Pol I without dNTPs; lane 5; substrate cleaved by Nfo and extended by Pol I; lane 6, substrate cleaved by Nfo, extended by Pol I and ligated. D General scheme of possible BER-dependent repair of AP site–peptide cross-links.
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<figure><img src="image_22.png" /><figcaption>Figure 1. Scheme of the DNA-peptide cross-link preparation. 8-OxoG-containing DNA duplexes were treated with bifunctional DNA glycosylases Fpg (top) or OGG1 (bottom) to produce a Schiff base conjugate with the nascent aldehydic AP site through a terminal amino group in case of Fpg or an internal amino group in case of OGG1. The enzyme-DNA intermediates were stabilized with NaBH₄ to yield covalent DNA-protein cross-links (DPXLs). Then, DPXL were subjected to full trypsinolysis to obtain AP-peptide cross-links.</figcaption></figure>

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<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /></figure>

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<figure><img src="image_26.png" /></figure>

Figure 2. Stalling of KF and RBpol by AP-peptide cross-links. A, APPXL-I, primer-template; B, APPXL-Y, primer-template; C, APPXL-I, primer-downstream strand-template; D, APPXL-Y, primer-downstream strand-template. The 32P-labeled primer is shown in red, the downstream strand is in lowercase. Grey arrows mark the sites of synthesis termination at the lesion. Reaction time and the nature of the lesion are indicated under the gel images. In all panels, lanes 1–3 are size markers corresponding to the primer (11 nt), full-size product (40 nt), and primer extended to the cross-link site (23 nt). “-”, no enzyme added; K1, undamaged primer-template, 30 min; K2, undamaged primer-template-downstream strand, 30 min. The inset shows KF pause sites at a lower band intensity.

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<figure><img src="image_27.png" /><figcaption>Figure 3. Stalling of Dpo4 and PolX by AP-peptide cross-links. (A), primer-template; (B), primer-downstream strand-template. The nature of the polymerase and the cross-link is indicated over and below the gel images, respectively. The 32P-labeled primer is shown in red, the downstream strand is in lowercase. Grey arrows mark the sites of synthesis termination at the lesion. Reaction time and the nature of the lesion are indicated under the gel images. In all panels, lanes 1-3 are size markers corresponding to the primer (11 nt), full-size product (40 nt), and primer extended to the cross-link site (23 nt). “-”, no enzyme added; K1, undamaged primer-template, 30 min; K2, undamaged primer-template-downstream strand, 30 min. No bypass was observed for PolX.</figcaption></figure>

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<figure><img src="image_28.png" /><figcaption>Figure 4. Incorporation of dNMPs opposite APPXLs by KF (A), RBpol (B), Dpo4 (C) and PolX (D). The nature of the substrate and dNTP is indicated below the gel images. The 32P-labeled primer is shown in red. "-", no enzyme or dNTPs added; N, all dNTPs added; K1, undamaged primer-template, all dNTPs, 30 min.</figcaption></figure>

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<figure><img src="image_32.png" /><figcaption>Figure 5. Cleavage of APPXL-I by AP endonucleases: APE1 (A), Apn1p (B), Xth (C), and Nfo (D). For panels A–C: lane 1, APPXL-I, no enzyme; lanes 2–5, APPXL-I cleavage under the BER conditions; lanes 6–9, APPXL-I cleavage under the NIR conditions; lanes 10–13, AP site cleavage under the BER conditions; lanes 14–17, AP site cleavage under the NIR conditions; lane 18, AP site treated with NaOH; lane 19, AP site, no enzyme. Panel D: lanes 1–4, APPXL-I cleavage under the BER conditions; lanes 5–8, APPXL-I cleavage under the NIR conditions; lanes 9–12, AP site cleavage under the BER conditions; lane 13, AP site treated with NaOH; lane 14, AP site, no enzyme. BER and NIR conditions for the individual AP endonucleases are specified in Supplementary Table S2.</figcaption></figure>

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<figure><img src="image_33.png" /></figure>

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<figure><img src="image_36.png" /><figcaption>Figure 6. Cleavage of APPXL-Y by AP endonucleases: APE1 (A), Apn1p (B), Xth (C), and Nfo (D). For panels A–C: lane 1, APPXL-Y, no enzyme; lanes 2–5, APPXL-Y cleavage under the BER conditions; lanes 6–9, APPXL-Y cleavage under the NIR conditions; lanes 10–13, AP site cleavage under the BER conditions; lanes 14–17, AP site cleavage under the NIR conditions; lane 18, AP site untreated (A) or treated with NaOH (B, C). Panel D: lane 1, APPXL-Y, no enzyme; lanes 2–5, APPXL-Y cleavage under the BER conditions; lanes 6–9, APPXL-Y cleavage under the NIR conditions; lanes 10–13, AP site cleavage under the BER conditions. BER and NIR conditions for the individual AP endonucleases are specified in Supplementary Table S2.</figcaption></figure>

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<figure><img src="image_37.png" /><figcaption>Figure 7. Base excision repair continuation after the cleavage of AP site–peptide cross-links. A, B, Extension by KF of the primer with the displacement of the downstream strand bearing a 5’-terminal cross-link after Nfo cleavage of APPXL-I (A) and APPXL-Y (B). Lane 1, cross-linked strand; lane 2, APPXL treated with Nfo; lanes 3–5, APPXL treated with Nfo followed by KF extension for the indicated time; lane 6, size marker corresponding to the unmodified strand. C, Reconstitution of the full BER cycle of APPXL-I with Nfo, Pol I, and DNA ligase. Lane 1 and 2, unmodified and cross-linked strands, respectively; lane 3, substrate cleaved by Nfo; lane 4, substrate cleaved by Nfo in the presence of Pol I without dNTPs; lane 5, substrate cleaved by Nfo and extended by Pol I; lane 6, substrate cleaved by Nfo, extended by Pol I and ligated. D, General scheme of possible BER-dependent repair of AP site–peptide cross-links.</figcaption></figure>

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Supplementary materials for

Abasic site–peptide cross-links are blocking lesions repaired by AP
endonucleases

Anna V. Yudkina<fnref n="1" />,<fnref n="2" />,*, Nikita A. Bulgakov<fnref n="1" />, Svetlana V. Baranova<fnref n="1" />, Alexander A. Ishchenko<fnref n="3" />,
Murat K. Saparbaev<fnref n="3" />, Vladimir V. Koval<fnref n="1" /> and Dmitry O. Zharkov<fnref n="1" />,<fnref n="2" />,*

This file contains

* Supplementary Tables S1–S2

* Supplementary Figures S1–S8

* A list of Supplementary References

<footnote n="1">¹SB RAS Institute of Chemical Biology and Fundamental Medicine, Novosibirsk, 630090, Russia</footnote>

<footnote n="2">²Department of Natural Sciences, Novosibirsk State University, Novosibirsk, 630090, Russia</footnote>

<footnote n="3">³Groupe “Mechanisms of DNA Repair and Carcinogenesis”, Equipe Labellisée LIGUE 2016, CNRS UMR9019, Université Paris-Saclay, Gustave Roussy Cancer Campus, F-94805 Villejuif, France</footnote>

<footnote>* To whom correspondence should be addressed. Tel: +7 383 363 5187; Fax: +7 383 363 5128; Email: dzharkov@niboch.nsc.ru. Correspondence may also be addressed to Anna Yudkina. Tel: +7 383 363 5188; Fax: +7 383 363 5128; Email: ayudkina@niboch.nsc.ru.</footnote>
<table><caption>**Supplementary Table S1.** Oligonucleotides used in this work.</caption><thead><tr><th>ID</th><th>Sequence, 5'→3'</th></tr></thead><tbody><tr><td colspan="2"><em>Assembly of substrates for cross-linking and enzyme assays</em></td></tr><tr><td>40oG</td><td>GCTCTGGAATTCCTTCXCTTCTTTCCCTCTCGACGGTCTCG; X = oxoG</td></tr><tr><td>40U</td><td>GCTCTGGAATTCCTTCXCTTCTTTCCCTCTCGACGGTCTCG; X = U</td></tr><tr><td>40G</td><td>GCTCTGGAATTCCTTCGCTTCTTTCCCTCTCGACGGTCTCG</td></tr><tr><td>40compC</td><td>CGAGACCGTCGAGAGGAAAGAAGCGAAGGAATTCCAGAGC</td></tr><tr><td>40compG</td><td>CGAGACCGTCGAGAGGAAAGAAGGGAAGGAATTCCAGAGC</td></tr><tr><td>28down</td><td>GAGGAAAGAAGCGAAGGAATTCCAGAGC</td></tr><tr><td>28oG</td><td>GAGGAAAGAAGXGAAGGAATTCCAGAGC; X = oxoG</td></tr><tr><td>21comp</td><td>GGAATTCCTTCCCTTCTTTCC</td></tr><tr><td colspan="2"><em>AP endonuclease kinetics</em></td></tr><tr><td>23OHU</td><td>TCTCCTTTCCCTCXCTTCCCTCTC; X = OHU</td></tr><tr><td>23comp</td><td>AGAGGAAAGGAGCGAAGGGAGAG</td></tr><tr><td>20THF</td><td>GAGTGAGAXGTCAAGTTGGG; X = THF</td></tr><tr><td>20comp</td><td>CCCAACTTGACGTCTCACTC</td></tr><tr><td colspan="2"><em>Polymerase primers</em></td></tr><tr><td>16pri</td><td>GCTCTGGAATTCCTTC</td></tr><tr><td>13pri</td><td>GAGAGGAAAGAAG</td></tr><tr><td>11pri</td><td>CGAGACCGTCG</td></tr><tr><td colspan="2"><em>Electrophoretic mobility markers</em></td></tr><tr><td>40m</td><td>CGAGACCGTCGAGAGGAAAGAAGCGAAGGAATTCCAGAGC</td></tr><tr><td>23m</td><td>CGAGACCGTCGAGAGGAAAGAAG</td></tr><tr><td>11m</td><td>CGAGACCGTCG</td></tr></tbody></table>
<table><caption>**Supplementary Table S2.** Composition of BER and NIR buffers.</caption><thead><tr><th>Enzyme</th><th>BER buffer (1x)</th><th>NIR buffer (1x)</th></tr></thead><tbody><tr><td>APE1</td><td>20 mM HEPES-KOH (pH 8.0)<br>100 mM KCl<br>5 mM MgCl<sub>2</sub><br>100 µg/ml BSA<br>1 mM DTT<br>Reference: (1)</td><td>20 mM HEPES-KOH (pH 6.8)<br>25 mM KCl<br>0.5 mM MgCl<sub>2</sub><br>100 µg/ml BSA<br>1 mM DTT<br>References: (1-3)</td></tr><tr><td>Apn1p</td><td>20 mM HEPES-KOH (pH 7.6)<br>100 mM KCl<br>5 mM MgCl<sub>2</sub><br>100 µg/ml BSA<br>1 mM DTT<br>Reference: (4)</td><td>20 mM HEPES-KOH (pH 7.6)<br>50 mM KCl<br>5 mM MgCl<sub>2</sub><br>100 µg/ml BSA<br>1 mM DTT<br>References: (4,5)</td></tr><tr><td>Nfo</td><td>20 mM HEPES-KOH (pH 7.6)<br>100 mM KCl<br>100 µg/ml BSA<br>1 mM DTT<br>Reference: (6)</td><td>20 mM HEPES-KOH (pH 7.6)<br>50 mM KCl<br>100 µg/ml BSA<br>1 mM DTT<br>Reference: (6)</td></tr><tr><td>Xth</td><td>20 mM HEPES-KOH (pH 7.6)<br>100 mM KCl<br>5 mM MgCl<sub>2</sub><br>100 µg/ml BSA<br>1 mM DTT<br>Reference: (4)</td><td>20 mM HEPES-KOH (pH 7.6)<br>25 mM KCl<br>5 mM CaCl<sub>2</sub><br>100 µg/ml BSA<br>1 mM DTT<br>Reference: (7)</td></tr></tbody></table>
<figure><img src="image_38.png" /><figcaption>**Supplementary Figure S1.** Images of Coomassie-stained gels of the preparations of KF, RBpol, PolX, APE1, Apn1p and Nfo used in this work. In all panels, lane 1 is a set of molecular weight markers (sizes indicated next to the leftmost panel), lane 2 is the purified protein.</figcaption></figure>
<figure><img src="image_39.png" /></figure>

<figure><img src="image_40.png" /></figure>

<figure><img src="image_41.png" /></figure>

<figure><img src="image_42.png" /><figcaption>**Supplementary Figure S2.** Preparation of AP–peptide cross-links. (A), (B), purification of 40-nt adducts APPXL-I (A) and APPXL-Y (B) on a denaturing 20% polyacrylamide gel. Lanes 1 and 3 in (A) and 1 in (B) contain molecular weight markers (40 nt and 28 nt). (C), (D), MALDI mass spectra of APPXL-I (C) and APPXL-Y (D). Mass spectra were obtained on a Bruker Autoflex Speed mass spectrometer equipped with a VSL-337 ND $N_2$ laser (337 nm) at 1–5 ns pulse duration, positive linear mode within the m/z range 3000–22000, laser frequency 60 Hz, ion source 1 voltage 19 kV, ion source 2 voltage 16.7 kV, lens voltage 8.75 kV, and 5000 laser shots at 200 Hz.</figcaption></figure>
<figure><img src="image_43.png" /></figure>

A

<figure><img src="image_44.png" /></figure>

<figure><img src="image_45.png" /></figure>

C

<figure><img src="image_46.png" /></figure>

<figure><img src="image_47.png" /></figure>

B

<figure><img src="image_48.png" /></figure>

<figure><img src="image_49.png" /></figure>

D

<figure><img src="image_50.png" /></figure>

**Supplementary Figure S3.** Stalling of 3'→5' exonuclease-proficient KF by AP-peptide cross-links. A, APPXL-I, primer-template; B, APPXL-Y, primer-template; C, APPXL-I, primer-downstream strand-template; D, APPXL-Y, primer-downstream strand-template. The ³²P-labeled primer is shown in red, the downstream strand is in lowercase. Reaction time and the nature of the lesion are indicated under the gel images. In all panels, lanes 1–3 are size markers corresponding to the primer (11 nt), full-size product (40 nt), and primer extended to the cross-link site (23 nt). “-”, no enzyme added; K1, undamaged primer-template, 30 min; K2, undamaged primer-template-downstream strand, 30 min.
A

KF

<figure><img src="image_51.png" /></figure>

C

RBpol

<figure><img src="image_52.png" /></figure>

E

Dpo4

<figure><img src="image_53.png" /></figure>

B

KF

<figure><img src="image_54.png" /></figure>

D

RBpol

<figure><img src="image_55.png" /></figure>

F

Dpo4

<figure><img src="image_56.png" /></figure>

G

PolX

<figure><img src="image_57.png" /></figure>

<figure><img src="image_58.png" /></figure>

**Supplementary Figure S4.** Displacement of a downstream strand bearing an AP-peptide cross-link by DNA polymerases. A, APPXL-I, KF; B, APPXL-Y, KF; C, APPXL-I, RBpol; D, APPXL-Y, RBpol; E, APPXL-I, Dpo4; F, APPXL-Y, Dpo4; G, APPXL-I and APPXL-Y, PolX. The ³²P-labeled primer is shown in red, the downstream strand is in lowercase. Reaction time and the nature of the lesion are indicated under the gel images. In all panels, lanes 1–3 are size markers corresponding to the primer (11 nt), full-size product (40 nt), and primer extended to the cross-link site (23 nt). “-”, no enzyme
added; K1, undamaged primer-template, 30 min; K2, undamaged primer-template-downstream
strand, 30 min.
<figure><img src="image_59.png" /><figcaption>**Supplementary Figure S5.** Incorporation of dNMPs opposite APPXL-I and APPXL-Y by KF (A), RBpol (B), Dpo4 (C) and PolX (D) in a different sequence context. The nature of the DNA and dNTP substrate is indicated below the gel image. The ³²P-labeled primer is shown in red. "-", no enzyme or dNTPs added; N, all dNTPs added; K1, undamaged primer–template, all dNTPs, 30 min.</figcaption></figure>
<figure><img src="image_60.png" /><figcaption>**Supplementary Figure S6.** Graphical summary of the kinetic data from Table 1 showing a comparison of the enzymes' catalytic efficiency ($k_{cat}/K_M$) on different substrates under different conditions. A, Apn1p; B, Nfo; C, APEX1. Black bars correspond to the canonical substrates (THF for the BER conditions, OHU for the NIR conditions), red bars, APPXL-I, green bars, APPXL-Y.</figcaption></figure>
A

<figure><img src="image_61.png" /></figure>

B

<figure><img src="image_62.png" /></figure>

**Supplementary Figure S7.** Resistance of APPXL-I (A) and APPXL-Y (B) to bifunctional DNA glycosylases. The nature of the enzyme and the substrate is indicated below the gel image. In both panels, lanes 1–6 are APPXL substrates, lanes 7–12, are the substrate containing a natural AP site.
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<figure><img src="image_63.png" /><figcaption>**Supplementary Figure S8.** Structures of DNA-bound *E. coli* Nfo (A; 1QUM, Ref. 8), African swine fever virus AP endonuclease (B; 6KI3, Ref. 9), human APEX1 (C; 1DEW, Ref. 10) and *N. meningitides* AP endonuclease (D; 4B5F, Ref. 11). The structures are aligned to present DNA (yellow) in the same orientation. The surfaces are built with a 1.4 Å probe radius. The C1' atom of the AP site is shown as a red ball. The Figure is made in PyMol (Schrödinger, New York, NY).</figcaption></figure>
Supplementary References

1. Gros, L., Ishchenko, A.A., Ide, H., Elder, R.H. and Saparbaev, M.K. (2004) The major human AP endonuclease (Ape1) is involved in the nucleotide incision repair pathway. *Nucleic Acids Res., 32*, 73-81.

2. Daviet, S., Couve-Privat, S., Gros, L., Shinozuka, K., Ide, H., Saparbaev, M. and Ishchenko, A.A. (2007) Major oxidative products of cytosine are substrates for the nucleotide incision repair pathway. *DNA Repair*, **6**, 8-18.

3. Redrejo-Rodríguez, M., Vigouroux, A., Mursalimov, A., Grin, I., Alili, D., Koshenov, Z., Akishev, Z., Maksimenko, A., Bissenbaev, A.K., Matkarimov, B.T. et al. (2016) Structural comparison of AP endonucleases from the exonuclease III family reveals new amino acid residues in human AP endonuclease 1 that are involved in incision of damaged DNA. *Biochimie*, **128-129**, 20-33.

4. Ishchenko, A.A., Yang, X., Ramotar, D. and Saparbaev, M. (2005) The 3'→5' exonuclease of Apn1 provides an alternative pathway to repair 7,8-dihydro-8-oxodeoxyguanosine in *Saccharomyces cerevisiae*. *Mol. Cell. Biol.*, **25**, 6380-6390.

5. Ishchenko, A.A., Sanz, G., Privezentzev, C.V., Maksimenko, A.V. and Saparbaev, M. (2003) Characterisation of new substrate specificities of *Escherichia coli* and *Saccharomyces cerevisiae* AP endonucleases. *Nucleic Acids Res.*, **31**, 6344-6353.

6. Ishchenko, A.A., Ide, H., Ramotar, D., Nevinsky, G. and Saparbaev, M. (2004) α-Anomeric deoxynucleotides, anoxic products of ionizing radiation, are substrates for the endonuclease IV-type AP endonucleases. *Biochemistry*, **43**, 15210-15216.

7. Ischenko, A.A. and Saparbaev, M.K. (2002) Alternative nucleotide incision repair pathway for oxidative DNA damage. *Nature*, **415**, 183-187.

8. Hosfield, D.J., Guan, Y., Haas, B.J., Cunningham, R.P. and Tainer, J.A. (1999) Structure of the DNA repair enzyme endonuclease IV and its DNA complex: Double-nucleotide flipping at abasic sites and three-metal-ion catalysis. *Cell*, **98**, 397-408.

9. Chen, Y., Chen, X., Huang, Q., Shao, Z., Gao, Y., Li, Y., Yang, C., Liu, H., Li, J., Wang, Q. et al. (2020) A unique DNA-binding mode of African swine fever virus AP endonuclease. *Cell Discov.*, **6**, 13.

10. Mol, C.D., Izumi, T., Mitra, S. and Tainer, J.A. (2000) DNA-bound structures and mutants reveal abasic DNA binding by APE1 and DNA repair coordination. *Nature*, **403**, 451-456.

11. Lu, D., Silhan, J., MacDonald, J.T., Carpenter, E.P., Jensen, K., Tang, C.M., Baldwin, G.S. and Freemont, P.S. (2012) Structural basis for the recognition and cleavage of abasic DNA in *Neisseria meningitidis*. *Proc. Natl Acad. Sci. U.S.A.*, **109**, 16852-16857.