A single viral enzyme drives tRNA-dependent hypermodification of DNA at adenine

Domain: biology
Published: 2026-04-03
Editorial outcome: major_revision

Abstract
Nucleic acid modifying enzymes drive diverse defense and counter-defense measures in the evolutionary arms race between viruses and their cellular hosts. Abundant and widespread bacterial viruses (bacteriophage or phage) encode for biosynthetic pathways that install elaborate DNA hypermodifications which protect their genomic DNA from host endonucleases. Here, we establish the molecular basis for the multistep biosynthesis of 6-aminocarboxymethyl-2ʹ-deoxyadenosine (6-NcmdA), a nucleobase hypermodification found in the virion DNA of bacteriophage Mu that leads to restriction evasion in the context of phage-host conflicts. In the first step, we show that Mu-encoded Mom enzyme catalyzes the formation of 6-NcmdA by transferring glycine from charged tRNAGly to the N6 position of adenine within double-stranded DNA. We uncover a second step where the glycyl-dA intermediate undergoes an on-base rearrangement to form 6-NcmdA. Examination of the proposed reaction pathways by quantum chemical calculations confirms the instability of acyl exocyclic groups at N6-adenine and reveals an energetically favorable orientation of 6-NcmdA that restores canonical base pairing. An X-ray structure confirms Mom is a member of the GNAT superfamily and suggests binding sites for both tRNA and DNA. Guided by the Mom structure and patterns of sequence conservation across metagenomic space, we show residues R111 and S124 are essential for catalysis. This work demonstrates that the Mom enzyme defines a new category of acetyltransferases utilizing charged tRNA to modify DNA.

Introduction
Nucleic acid modifying enzymes drive diverse defense and counter-defense measures in the evolutionary arms race between viruses and their cellular hosts1,2. Abundant and widespread bacterial viruses (bacteriophage or phage) encode for biosynthetic pathways that install elaborate DNA hypermodifications which protect their genomic DNA from host endonucleases3–10. The biosynthetic complexity and chemical diversity found within naturally modified DNA from phage, as collectively encoded by the most numerically abundant genetic entity in our biosphere, has only begun to be appreciated. Phage-encoded biosynthetic pathways install structurally complex nucleobase modifications in DNA, termed DNA hypermodifications after those found in tRNA (named by Ross H. Hall in 1971), by harvesting building blocks from host cellular metabolites and appending functional groups pre- and/or post-replicatively, all without affecting the coding capacity of the bases6,7,11. Hypermodifications are found at key positions, or ‘hotspots’, on the nucleobases that do not interfere with Watson-Crick base pairing. These positions are tolerant of bulky, diverse adducts, and study of DNA hypermodifications from phage are expanding our understanding of DNA plasticity. Accordingly, the repertoire of phage enzymes that synthesize hypermodifications exhibit exquisite site-specific precision when conjugating functional groups to nucleobases, and like their bacterial counterparts involved in genome defense, have potential to be engineered for therapeutic and bio-control applications12–14.

One hypermodification of historical significance was found in the genome of phage Mu, a cornerstone of bacterial genetics15, over 40 years ago and subsequently mapped by restriction sensitivity to a loose SASNY consensus sequence in the Mu genome (accounting for 15-25% of total dA, IUPAC code given for consensus sequence)16–22. In 1983, McCloskey and coworkers determined the structure of an unusual aminocarboxymethyl (carbamoylmethyl or glycinamide) group at the N6-position of adenine (abbreviated herein as 6-NcmdA, Fig. 1a); the structure of 6-NcmdA was further supported by MS/MS results in subsequent work21,23–25. The bulky aminocarboxymethyl group does not interfere with Watson-Crick base pairing, though there is a decrease in melting temperature associated with the hypermodification21. These foundational studies on Mu also identified a gene, mom (modification of Mu), necessary for the installation of 6-NcmdA during late stages of Mu infection16–20,23,26–28. A more recent study predicted that Mom belongs to the Gcn5-related N-Acetyltransferase (GNAT) superfamily, and another established that mom is the only Mu gene required for 6-NcmdA to form in vivo in E. coli (referred to colloquially as ‘momylation’)23,28–31. However, it has remained undetermined for over 40 years whether the gene product of mom is an active acyltransferase enzyme, and if so, what co-substrate is used and what functional group is transferred to dA21,23,28. Here, we demonstrate that the phage Mu enzyme Mom is an active aminoacyltransferase that co-opts charged tRNAGly (Gly-tRNAGly) in order to form 6-NcmdA. Moreover, the mechanism uncovered herein reveals that the Mom-catalyzed hypermodification is not formed via simple group transfer, helping to explain how this reaction evaded elucidation for so long.

Results
The adenine hypermodification to 6-NcmdA forms post-replicatively on dsDNA
Since phage enzymes can install hypermodifications before or after replication, we first sought to determine if Mom can modify the DNA polymer directly. We assayed lysates for phage enzyme activity on a biotinylated DNA substrate32,33. We took clarified lysates derived from E. coli cultures heterologously expressing wild-type (WT) Mom, added biotinylated DNA substrates exogenously, incubated, recovered, and analyzed 2'-deoxynucleosides by LC/MS (Fig. 1a, ex vivo assay in Fig. 1b, Supplementary Fig. 1, and Supplementary Table 1). After incubation, a nucleoside identical in mass (308 u) and retention time to the native Mu 2'-deoxynucleoside was observed. This nucleoside product replaced about 20% of dA as compared to DNA incubated in control lysates. Furthermore, PacBio SMRT sequencing and sequence context sensitivity to restriction enzymes show Mu gDNA is specifically modified at dA in the context of an SAS sequence motif accounting for 20% of dA in Mu (Supplementary Fig. 2-5 and Supplementary Table 2)20. Incorporation of a nucleobase modified at the mononucleotide level would be expected to produce a polymer with 6-NcmdA found at random positions. Together, these results establish that momylation involves direct and site-specific modification of double-stranded DNA (dsDNA) that can be achieved in vitro at levels comparable to the native phage.

Adenine hypermodification to 6-NcmdA requires components of transcription/translation
Following the demonstration that Mom acts post-replicatively in lysates, we aimed to reconstitute the momylation reaction in vitro using experimental approaches amenable to systematic dissection of reaction components and potential group donors. Mom was either synthesized in an in vitro transcription-translation (IVTT) system from plasmid template or purified following recombinant expression in E. coli (Supplementary Fig. 6-8). Synthesis of Mom by the IVTT system resulted in active momylation of substrate DNA. By contrast, reconstitution of 6-NcmdA was not achieved with purified Mom until incubation of purified Mom with the IVTT system. Strikingly, installation of 6-NcmdA on DNA substrates by purified Mom is readily observable in reactions containing transcription and translation machinery without templates encoding for Mom. Together, these data show that Mom, along with purified components of the transcription/translation system, are sufficient to reconstitute Mom activity.

Modification of dA by Mom using glycine is a tRNA-dependent process
The IVTT system that supports momylation contains a complex mixture of purified enzymes and cofactors, including tRNA, amino acids, and tRNA synthetases34,35. Some well-established enzymes of the GNAT superfamily use charged tRNA to aminoacylate well-known substrates36–38. We noticed that the aminocarboxymethyl group introduced by Mom and a glycyl group are isomeric, together suggesting that tRNAGly and glycine may be involved in momylation. We tested this tRNAGly hypothesis more directly in an enzyme coupled assay containing purified Mom (10, a construct where the first 10 amino acids have been truncated), tRNAGly/GCC transcribed in vitro (Supplementary Table 3, Supplementary Fig. 9-10, Supplementary Table 4), glycine, ATP, purified E. coli glycyl-tRNA synthetase (GlyRS), and linear dsDNA substrates. As can be seen in Fig. 1c and 1d, modification of DNA by Mom is a tRNA-dependent process: 6-NcmdA can only be detected when tRNAGly/GCC, GlyRS, glycine, and ATP are included in the reactions. The mass of 6-NcmdA formed during Mom assays using glycine labeled with 15N and 13C confirms that Mom transfers an activated glycine to dA (Supplementary Fig. 11).

Gly-tRNAGly is the co-substrate used by Mom to hypermodify dA
Reconstitution of 6-NcmdA enabled us to probe the activated forms of glycine further in order to ascertain the co-substrate used by Mom at the transfer step. Two forms of activated glycine are generated in situ by GlyRS – glycyladenylate as an intermediate and Gly-tRNAGly (charged tRNA) as a product39–43– in the coupled reaction where 6-NcmdA is produced during Mom activity assays. Our strategy involved generating a tRNA analog with a 2'-deoxyadenosine (from 2'-dATP) enzymatically installed by a CCA-adding enzyme at the 3' end of tRNAGly/GCC-CC following in vitro transcription (Fig. 2a, Supplementary Fig. 9, Supplementary Fig. 12-16, and see Methods). All tRNA species were verified by intact mass spectrometry44–46. We additionally verified that GlyRS can charge tRNAGly/GCC-CC-2'-dA47–51, and we note that the charging (percent aminoacylated) for tRNAGly/GCC-CC-2'-dA is comparable to what we observe for the native sequence tRNAGly/GCC-CCA (Supplementary Fig. 17). The activity assays for GlyRS confirm that glycyladenylate and Gly-tRNAGly were generated by GlyRS for both tRNAGly/GCC-CCA and tRNAGly/GCC-CC-2'-dA and that both activated forms of glycine would be available in an activity assay for Mom.
We next tested if tRNAGly/GCC-CC-2ʹ-dA can support momylation. As can be seen in Fig. 2b (n=3 using a pUC19 plasmid substrate), momylation is not observed when tRNAGly/GCC-CC-2ʹ-dA is included in a Mom assay, even after overnight incubation (Supplementary Fig. 18). In controls with tRNAGly/GCC-CCA (n=3 using a pUC19 plasmid substrate), momylation is first detectable after 5 minutes, and the majority of dA’s in an SAS sequence motif (~25% of all dA’s for pUC19) are typically modified around 60 minutes under the conditions tested (Fig. 2b and Supplementary Fig. 19). Though both forms of activated glycine were available to Mom in these activity assays, tRNAGly/GCC-CCA exclusively supports momylation, demonstrating how critical the terminal riboadenosine is for hypermodification of dA. Our activity assays for Mom and GlyRS are in agreement and confirm Gly-tRNAGly-CCA is the activated form of glycine used by Mom to hypermodify DNA.

Mom is selective for Gly-tRNAGly and dsDNA
Given the sensitivity of Mom for the terminal riboadenosine of tRNA, we further explored several features related to the co-substrate and substrate preferences of Mom. We noted that E. coli encodes for three different isoacceptors of tRNAGly – tRNAGly/GCC, tRNAGly/CCC, and tRNAGly/TCC (Supplementary Table 4). While tRNAGly/GCC is reported to be the most frequently-used and most abundant isoacceptor in E. coli52–54, there is precedent for a particular tRNA isoacceptor to be used in a non-canonical role outside of ribosome-dependent protein synthesis55,56. We therefore evaluated if tRNAGly/CCC and tRNAGly/TCC (Supplementary Fig. 9) could support the momylation reaction. As can be seen in Fig. 2c, we see that all isoacceptors of tRNAGly can in fact support momylation. This finding is consistent with the strategy of the viral lifecycle, where available resources are scavenged rapidly in the process of hijacking the host cellular machinery.
We next investigated how selective Mom is for Gly-tRNAGly by utilizing an editing-deficient variant of AlaRS (C666A) to mischarge tRNAAla/TGC (Supplementary Fig. 9, Supplementary Fig. 20)34,57–59. In activity assays for AlaRS, we verified that C666A AlaRS can form Ala-tRNAAla/TGC and Gly-tRNAAla/TGC, while WT AlaRS only forms Ala-tRNAAla/TGC (Supplementary Fig. 21). Under all conditions tested for WT Mom, 6-NcmdA could not be detected in assays where mischarged Gly-tRNAAla/TGC or Ala-tRNAAla/TGC were formed in situ (Fig. 2c). As has been shown for other GNATs60,61, features at the 3ʹ end of tRNA and the amino acid appear to be sufficient to direct co-substrate specificity. Our data here suggest similar drivers of co-substrate recognition for Mom.
We also find that WT Mom exhibits a strong substrate preference between dsDNA and ssDNA (Fig. 2d). In addition to momylating biotinylated DNA and plasmid DNA, WT Mom and a 10 construct hypermodify an 11 bp dsDNA containing a single adenine in the SASNY context with a very high conversion rate (>95%). Mom exhibits activity at A/C MM (93% conversion) and to a lesser extent at an A/A mismatch (MM, 72% conversion per strand). Activity on an A/G MM (10%) is diminished compared to the A/T control. Mom activity on single-stranded DNA from phage M13 or on ssDNA 11-mers could not be detected above background (Supplementary Fig. 22). Taken together, we conclude that Mom is active on dsDNA (even on very short oligomers) but not on ssDNA.
Overall, we establish that Mom exhibits strong preferences to use the terminal riboadenosine of tRNA, Gly-tRNAGly, and target dsDNA. Specificity for co-substrates, particularly tRNAGly, has been attributed to evolutionary pressures on bacterial toxins belonging to the GNAT superfamily62, though a general understanding of the factors that tune co-substrate recognition across GNATs has been elusive possibly due to characteristically high structural homology but very low sequence homology36,37,63,64. Substrate specificity and scope vary even more widely for GNATs. Here we add dsDNA to the multitude of targets modified by this enzyme superfamily.

6-NcmdA forms following an on-base isomerization of the exocyclic glycyl group
Having established that glycine is the group transferred during momylation, we sought to explain the apparent discrepancy between the structures of a modification formed by simple group transfer of the glycyl group to N6 versus the mature hypermodification. As highlighted above, the aminocarboxymethyl group of 6-NcmdA and a glycyl group are structural isomers. We therefore hypothesized that following transfer to the N6 position, the glycyl group necessarily undergoes isomerization. We utilized LC/MS-MS and stable isotope labeling to investigate our hypothesis, focusing on a daughter ion of 6-NcmdA produced during fragmentation by bond breakage, shown as a dashed red line in top reaction of Fig. 3a, at the exocyclic C-C bond (m/z 148, termed [6mA•]+ for clarity, see also Supplementary Fig. 23-25)23. For the coupled Mom activity assay including glycine-1-13C,15N and unlabeled dsDNA supported conversion of dA to 6-NcmdA (m/z = 311) (second reaction in Fig. 3a, Supplementary Fig. 11, Supplementary Fig. 26). Similarly, reactions containing unlabeled glycine and dsDNA labeled with 15N -dA (where all 5 purine nitrogens in dA are labeled with 15N, see Methods for substrate preparation) supported conversion of dA to 6-NcmdA (m/z = 314, third reaction in Fig. 3a, Supplementary Fig. 27 and 28). Fragmentation patterns for 6-NcmdA labeled with glycine-1-13C,15N (m/z = 311) produced a daughter ion with an m/z = 149 that we assigned to [6m-15N-A•]+, one Dalton heavier than the product produced with unlabeled glycine. Given that the labeled nitrogen originated from the primary amine of glycine, the increase in mass here (relative to the unlabeled reaction) can be explained by a rearrangement that must have occurred during the modification reaction. Conversely, reactions utilizing a DNA substrate labeled with 15N -dA (m/z = 314) produced a daughter ion corresponding to [6m-15N -A•]+ (m/z = 152) that is 4 mass units (rather than 5) heavier than [6mA•]+, as is observed in reactions without labeled components (compare, first and third reactions in Fig. 3a).
Because the nitrogen at N6 is a poor nucleophile due to resonance delocalization of the lone pair into the purine ring65, we sought to assess the possibility that the more nucleophilic nitrogen at N166,67 carries out the initial nucleophilic attack, followed by migration of N1-glycyl to N6. Migration of a methyl group from N1 to N6 of adenine has been observed in vitro 68 via a Dimroth rearrangement69, wherein the purine ring opens between N1 and N6 followed by rotation about the C5-C6 bond and ring closure, effectively swapping the N1 and N6 nitrogens together with their associated substituents. To our knowledge, a biologically catalyzed Dimroth rearrangement has not been reported, and it is unclear whether an aminoacyl group instead of an alkyl group could migrate through such a mechanism. We probed the possibility of glycyl addition at N1 by using a DNA substrate where dA is labeled specifically at N6 with 15N (fourth reaction, Fig. 3a, Supplementary Fig. 23A, Supplementary Fig. 29). If N1 were the site of glycyl addition followed by a Dimroth rearrangement, the labeled nitrogen originating at N6 should be retained in the purine ring at N1. However, we observe that the [6mA•]+ daughter ion here is equal in mass (m/z = 148) to mass of the [6mA•]+ daughter ion in the top reaction where unlabeled activity assay components were used. Because we find that the single, labeled nitrogen from N6 exchanged to the distal position and not to the N1 position, we conclude that N6 is the nucleophile that makes the attack on Gly-tRNAGly. Other GNAT superfamily members are known to acylate exocyclic amines. For example, TmcA from E. coli acetylates tRNAMet at the N4 position of the wobble base (C34) to form ac4C, a tRNA modification found in all domains of life36.

Energetics of the on-base rearrangement leading to 6-NcmdA
Our labeling experiments indicate an exchange between the N6 proximal nitrogen and the distal primary amine occurs during momylation (chemically, a transaminoacylation followed by an isomerization). This “flipping” of the transferred group places the glycyl C one atom closer to the nucleobase and apparently displaces the carbonyl oxygen one atom further away from dA. To account for the nitrogen exchange, we propose a mechanism for isomerization that proceeds via a five-membered ring intermediate through ring closure upon attack of C6 by the exocyclic primary amine followed by ring opening through breakage of the initial C6-N6 bond to yield the native hypermodification found in phage Mu (Fig. 3b).
We have not seen an analogous multistep and controlled process – transfer and then rearrangement of the transfer group to one product during hypermodification of a nucleobase – driven by a single enzyme described in the literature. Established acylation chemistry characteristic of GNAT enzymes is notably distinct from the chemistry proposed for isomerization of an exocyclic glycyl group on dA. These factors motivated quantum-chemical exploration of the rearrangement in water in the absence of enzyme using the ORCA package (Supplementary Fig. 30-37)70. We calculated re-arrangement energies for educt and product dihedral angles using the efficient R2SCAN functional (with dispersion correction, in implicit water). We separately considered the neutral (Supplementary Fig. 30-33) and cationic forms (Supplementary Fig. 34-37) allowing for the glycyl-N6 amide bond to occur in the trans- or cis-conformation. A dihedral scan shows that the N1-C6-N6-C angle tends to be close to 0 or 180° (Supplementary Fig. 30 and 34), presumably to conjugate the N6 lone electron pair to the aromatic system of the adenine nucleobase. In the neutral form, the glycyl N6 trans-amide bond is favored (Supplementary Fig. 30 and 31). In the cationic form, the cis-amide, which is poised for the rearrangement reaction, is preferred (Supplementary Fig. 34 and 35). For both neutral and cationic forms, the glycyl is preferably oriented towards the Watson-Crick edge, indicating that without the rearrangement reaction, the glycyl group on N6 would interfere with Watson-Crick base pairing. In the product state, the preference for the Watson-Crick edge over the Hoogsteen edge is mitigated for the neutral form (Supplementary Fig. 30 and 31) or reversed for the cationic form (Supplementary Fig. 34 and 35). Thus, the rearrangement alleviates the impairment of Watson-Crick base pairing from glycyl transfer (Supplementary Fig. 38). Overall, the rearrangement reaction is favorable for both the neutral and cationic forms (Supplementary Fig. 30, 31, 34, 35). In the latter case, deprotonation of the N1 after the rearrangement adds to the favorability. For both the neutral and cationic forms, the free energy of the spirocyclic intermediate is also relatively low, and not much higher than the energy required to push the glycyl group of the educt “out of plane” (Supplementary Fig. 30, 31, 34, 35).
To determine whether the rearrangement reaction could occur spontaneously in water, we analyzed plausible reaction pathways (Supplementary Fig. 32, 33, 36, 37). For the neutral form, we assumed nucleophilic attack of the glycyl Nα on the C6 (Supplementary Fig. 32b) and proton transfer from glycyl Nα to adenine N1 (Supplementary Fig. 32c) to form the spirocyclic reaction intermediate. For the resolution of the intermediate, we tested proton transfer from the adenine N1 to the adenine N6 (Supplementary Fig. 33b), and departure of the N6 leaving group (Supplementary Fig. 33c). For the cationic form, we assumed proton transfer from Nα to N1 (Supplementary Fig. 36b) and Nα to C6 bond formation to form the intermediate (Supplementary Fig. 36c). For the resolution of the intermediate, we postulated proton transfer from N1 to N6 with C6-N6 bond scission (Supplementary Fig. 37b), and then proton transfer from Nα to N1 (Supplementary Fig. 37c). For both the neutral and cationic forms, formation of the spirocyclic intermediate was possible via relatively low-lying saddle points/transition states. In the case of the cationic form, these were sufficiently low (~17 kcal/mol) for a reaction rate above 10/s at room temperature according to Eyring’s equation. By contrast, the barriers for the resolution of the spirocyclic intermediate came out high (Supplementary Fig. 33 and 37). This could indicate either that the optimal reaction path for a spontaneous resolution of the spirocyclic intermediate remains to be identified or that Mom facilitates the rearrangement reaction by general acid-base chemistry or through a concerted protonation/deprotonation step (Supplementary Fig. 39).

The crystal structure of S124A Mom reveals putative active site and distinct features
In order to gain a more detailed picture of momylation, we aimed to solve a crystal structure of Mom. Crystallization screens identified conditions leading to the growth of orthorhombic crystals of Mom(Δ10) S124A (a catalytically dead variant) in space group P2(1)2(1)2(1) that contained two Mom protomers in the asymmetric unit and that diffracted to ~2.0 Å resolution (Fig. 4a). The structure was solved by molecular replacement using an AlphaFold2 generated search model of the Mom protomer and refined with manual edits in Coot (Supplementary Table 5)71–73.
As anticipated23,28,73, Mu Mom protomers have the GNAT fold, but with an atypical insertion spanning residues ~175-209 that visually resembles a helix-turn-helix (HTH) motif (Fig. 4a-c)63,74,75. An insertion can also be seen with superpositions and sequence alignments of Mu Mom with the predicted structures of active homologs mined from metagenome databases (Fig. 4b-c, Supplementary Fig. 40 and Supplementary Fig. 41). Importantly, deletion of the HTH-like insertion leads to complete loss of the activity (Supplementary Fig. 5), highlighting its crucial role in momylation.
Previous work has established that the truncation of 10 N-terminal residues (Δ10) of Mom is well tolerated23, which we also confirm. Notably, however, the truncation of 20 amino acids (Δ20) reduced the activity (Supplementary Fig. 5a, Supplementary Fig. 22a, Supplementary Fig. 42, Methods, and Supplementary Methods). This is surprising because the 11-19 region is unstructured, and our electron map does not contain densities that can be attributed to this stretch of amino acids.
Superposition of 51 diverse GNAT crystal structures solved with CoA or a CoA analogs and Mom suggested residues H58, Y59, R111, S124 D149, R151, and Y159 potentially encompass key active site residues (Fig. 4d, Supplementary Fig. 43, Supplementary Table 6, and Supplementary Table 7). This finding was in line with previous work that showed that alanine mutations of the residues R111, S124, and D149 greatly reduced Mom activity23. As our own in vivo and in vitro data reproduced these observations for R111A and S124A, we decided to carry out a more comprehensive structure guided mutagenesis screen informed by our modeling (Supplementary Fig. 42). In doing so, we identified another essential residue, R151, as the R151A variant lacked any detectable activity in vivo. In addition, the activity of R111K, H58A, Y59A, W48A, Y159A, and W181A variants were drastically reduced.
Superposition of Mom with the subset of those GNAT structures solved with the substrate or product (Fig. 4e) further provide insights to the role of other residues, such as R111, critical for momylation. Superposition of Mom with two GNATS where tRNA is a co-substrate or substrate – FemX (from Weissella viridescens, PDB ID: 4II9) and TacT (from Salmonella typhimurium, PDB ID: 7F36), respectively – reveals two possible binding modes of tRNA each that could position the CCA stem in proximity to residues critical for activity (Fig. 4f, Supplementary Fig. 43, and Supplementary Table 6)76. During momylation, a FemX-like strategy could be used for binding the co-substrate (tRNA), while a TacT-like strategy could be used for binding the substrate (DNA), aided by the positively charged cleft (shown in blue in Fig. 4g-h).
Consistent with size exclusion chromatography results (Supplementary Fig. 8), the structure shows that Mom forms tight dimers with an interface area of ~850 Å2, and a predicted free energy of binding of 4.7 kcal/mol according to the PISA server77. Based on the structure, we have tested the dimer surface by mutating the residues Y28 and W48 to alanine, which we hypothesized were potentially involved in dimerization. While mutation did not have a significant effect for Y28A, W48A severely reduced the activity of Mom in vivo (Supplementary Fig. 5 and Supplementary Fig. 42). To test the role of W48 on Mom dimerization and activity further, we purified the W48A variant and compared its migration on size-exclusion column side-by-side with S124A variant. As predicted, Mom W48A migrated as a monomer (Supplementary Fig. 44a-c) and only had residual activity in vitro (Supplementary Fig. 44d).
The crystal structure of Mom alone combined with structure guided mutagenesis provided key insights in the understanding of residues important for momylation. Because structures of Mom bound to DNA, tRNA, or both have not been solved to date, we utilized AlphaFold3 (AF3)78 modeling of Mom with tRNAGly and dsDNA (Supplementary Fig. 42e-f, Supplementary Results, and Supplementary Methods). To verify that AF3 models were reliable, we also tested several other GNAT members that are known to utilize tRNA as an acyl donor or to modify tRNA (Supplementary Fig. 45, Supplementary Fig. 46, and Supplementary Table 10).
The AF3-generated model of Mom in a ternary complex supports our conclusions on relative orientation of tRNA and DNA based on the extensive structural comparison. Moreover, the accumulation of positive charge in the 11-19 amino acid unstructured region could contribute to tRNA binding, explaining the reduced activity of Δ20 variant (Supplementary Fig. 42g). Satisfyingly, the AF3 model also identifies the positively charged cleft formed between HTH-like insertion and the GNAT part of Mom as DNA binding region, suggesting that HTH-like insertion could interact with DNA through the major groove (Supplementary Fig. 42e-f). The AF3-generated model additionally suggests that substrate dA is flipped-out from the DNA double helix and positioned in close proximity to the 3'-terminal ribose of the CCA stem of tRNA and, importantly, both are surrounded by the essential residues, e.g. R111, D149 and R151 (Supplementary Fig. 42f).

Methods
Phage Mu preparation, genomic DNA isolation, and SMRT Sequencing
Two E. coli strains carrying phage Mu lysogen were obtained from Prof. Stan Hattman and were used to produce Mu phages. Of the two strains, one contains the Mu lysogen (denoted as ER3153; genotype: sup mcrA thi hsdR mcrB zzz::Mu(Cts62)) which produces the Mu phage with the native 6-NcmdA DNA modification phenotype, and the other has a mutant Mu lysogen which produces Mu phages without 6-NcmdA DNA modification but canonical 2´-deoxyadenosine phenotype (denoted as ER3154; genotype: sup mcrA thi hsdR mcrB zzz::Mu(Cts62 mom)).
A heat-shock procedure was performed to induce the production of Mu phage from the lysogen. LB medium was inoculated with the phage lysogen strain from an overnight culture and was incubated at 37 °C with agitation until an OD reading of ~1. The culture was then incubated at 45 °C for 30 minutes and then again at 37 °C with agitation. Phage were harvested when the culture appeared translucent (cells lysed), a process that normally takes about 1.5 – 3 h after heat-shock. The phage lysate solution was then centrifuged at 10000 rcf x g at 4 °C for 10 min, and the supernatant was collected. The phage was then concentrated first by precipitation in a solution of 10% (w/v) poly(ethylene glycol) average MW 8 kDa and 1 M NaCl, followed by pelleting by centrifugation, and finally resuspension in a buffer composed of 50 mM Tris-HCl 7.5, 10 mM MgCl and 75 mM NaCl. The phage solution was stored at 4 °C in the dark. A previously published protocol was followed to extract and purify phage genomic DNA90.
Genomic DNA isolated from phage Mu was used to prepare the single-molecular real-time (SMRT) sequencing libraries. SMRT sequencing reads were analyzed using SMRT Analysis software and pipelines from SMRT Link webtool (version 10.2.0) hosted by Pacific Biosciences. SMRT sequencing raw CCS reads were processed to generate HiFi reads in which the output sequences were used for the de novo assembly of the genome using MEGAHIT assembler software (version 1.2.9) 91. The longest assembled output sequence was used as the reference for subsequent SMRT Analyses steps. Base modification detection and motif analysis were performed with the same SMRT Link version. To detect the base modifications, the Base Modification Analysis application was used to first map the raw reads to the assembled genome and the mapped reads were analyzed further to extract the inter-pulse duration (IPD) kinetics information collected during sequencing course and processed for all pulses aligned to each position in the reference sequence using an in silico kinetic reference and a t-test based kinetic score detection of modified base positions. The per-base IPD ratio mapped outputs were further used to extract the modification motifs. The top three identified modification motif sequences were extracted separately and the sequences for each motif string were used to generate sequence logo by using WebLogo 3 webtool (version 3.7). See Supplementary Fig. 2 and Supplementary Table 2.

DNA Substrates
Biotinylated DNA substrates were prepared by digesting  DNA (NEB) with MseI (NEB) as described32,33,90. Digested  DNA was backfilled with 50 µM Biotin-16-(5-aminoallyl)-dUTP (Jena Bioscience) using Klenow Fragment (3´→5´ exo-) (NEB). Empty plasmids of pUC19 (NEB) and pET28a were used as supplied. Double-stranded DNA oligomers were obtained by heating a 1:1 mix of complementary oligonucleotides at 90 °C for 5 min and annealing by gradual cooling to the room temperature for 30 min. Duplexes were stored at -20 °C until use. Mu 34-mer was resuspended in Duplex Buffer (IDT), complementary strands were mixed in equimoloar ratios, and annealed by heating at 95 °C for 2 minutes and cooling to room temperature over a linear gradient. The 3 kbp substrates were amplified with a standard dNTP mix, 15N -dATP (Cambridge Isotope Laboratories), dGTP, dTTP, and dCTP, or 6-15N-dATP, dGTP, dTTP, and dCTP. 6-15N-dA (Toronto Research Chemicals) was chemically phosphorylated (see Supplemental Methods). See Supplementary Table 1 for sequences.

Restriction digest protection assay
To assay for installation of 6-NcmdA by Mom, Mu gDNA or DNA substrates of Mom were subject to restriction digests with 6-NcmdA-sensitive restriction enzymes HgaI (NEB) or AasI FastDigest (Thermo) according to the manufacturer’s instructions23. The digestion products were resolved on an 0.8% agarose gel and imaged using GelRed (Biotium) staining.

Constructs, cloning, and strains
Escherichia coli NEB® 5-alpha (NEB) or TOP10 strains (Invitrogen) was used for cloning. E. coli T7 Express (NEB) or BL21-CodonPlus (DE3)-RIL strains (Agilent) was used for heterologous protein expression. Plasmids were stored at -20 °C until use. See Supplementary Table 8 and Supplementary Table 9 for cloning primers sequences and a list of expression plasmids.
Mom. Mom was cloned into the pRY plasmid (derived from or pET28c) or pET28a. Expression cells harboring the pRY Mu Mom plasmid were used in downstream in vivo and ex vivo Mom activity assays (see below). Expression cells harboring pET28a plasmids expression WT Mom or Mom variants were used for in vivo Mom activity assays or for purifying enzyme for crystallography.
Glycyl-tRNA synthetase (GlyRS). The E. coli GlyRS expression construct, pET21a-GlyRS-His6, was a gift from Sebastian Maerkl & Takuya Ueda (Addgene plasmid #124110; http://n2t.net/addgene:124110; RRID:Addgene_124110).
Alanyl-tRNA synthetase (AlaRS). C666A AlaRS57–59 was generated on a pEQ expression plasmid using mutagenesis primers given in Supplementary Table 8.
tRNA nucleotidyltransferase cca2 (CCA2). CCA2 from Schizosaccharomyces pombe (S. pombe) was ordered from Twist on a pET28b plasmid with an N-terminal His tag and TEV protease cleavage site.
E. coli tRNA genes. The pUC19 plasmid containing the gene encoding the tRNAGly/GCC isoacceptor from E. coli was kindly gifted by Dr. Haruichi Asahara. The genes encoding for tRNAGly/CCC, tRNAGly/TCC, and tRNAAla/TGC were ordered from GenScript or Twist on a pUC19 plasmid. Sequences of the tRNA isoacceptors from E. coli were accessed at the GtRNAdb Database52,53.

In vivo Mom activity assays
To test the in vivo activity of Mom, variants, and mined homologs (see below), expression plasmids from cells harvested after recombinant expression (as described in the “Protein Expression” section below and in Supplementary Methods) were isolated by miniprepping as described32,92,93. As a negative control, expression strains bearing the pET28a vector with no insert (“Empty”) were used. Plasmids were assayed for the presence of hypermodification as described below.

UHPLC-MS of nucleosides and oligonucleotides and UHPLC-MS/MS analysis of nucleosides
Nucleoside samples were prepared from dsDNA substrates from in vivo, ex vivo, and in vitro Mom activity assays by using the Nucleoside Digest Mix Kit (NEB) according to the manufacturer’s instructions and as previously described32,33,90. Nucleosides were subjected to ultra-high-performance liquid chromatography (UHPLC), UHPLC-mass spectrometry (UHPLC-MS)32,33,90, or LC-MS/MS. Agilent ChemStation software and Adobe Illustrator were used for data processing and visualization. Nucleosides were quantified as previously described94–96. tRNAs were verified by intact mass spectrometry previously described44–46.

Recombinant protein expression of Mom
Mom was heterologously expressed in T7 Express or BL21-CodonPlus (DE3)-RIL strains. A single colony from freshly transformed T7 Express cells was used to inoculate LB/Kan (40 µg/mL) media (+ glucose, 0.1%). The starter culture was grown overnight at 37 °C at 240 rpm. A 1:100 to 1:1000 dilution of the overnight culture was made into fresh expression LB media (- glucose). Cells were grown to an OD of 0.6 at 240 rpm and expression was induced with 100 µM IPTG. Cells were grown for 18 hours at 18 °C after which they were harvested by centrifugation and stored at -20 or -80 °C until use. Mom was also heterologously expressed in the BL21-CodonPlus (DE3)-RIL strain as was GlyRS in LB media under the selective pressure of kanamycin-chloramphenicol and ampicillin-chloramphenicol, respectively. Cells were used to inoculate starter cultures that were grown in the presence of 1% glucose up to a late log phase at 37 °C in a rotary shaker at 140 rpm. A 1:200 dilution of starter culture was used to inoculate expression media. After the OD reached 0.6-0.7, the expression was induced with 250 µM IPTG. Cells were incubated with 140 rpm shaking for 18-20 h at 25 °C. Cells were then collected by centrifugation, washed with PBS (pH 7.4) and stored at -20 °C.

Ex vivo Mom activity assays
Clarified lysates were prepared from cells where WT Mom or variants had been heterologously expressed32,33,90. Ex vivo assay reactions were prepared on ice and incubated with biotinylated DNA substrates at 37 °C for > 15 min as previously described32,33,90. Where indicated, reactions were supplemented with 1 mM 13C2-acetyl-CoA (Sigma Aldrich) (comparable to the intracellular concentration of acetyl-CoA, BNICD 114622)97 that had been stored in 1 M aliquots at -80 °C in water until use. Streptavidin Magnetic Beads (NEB) were used to recover biotinylated DNA substrates from enzymatic reactions. Nucleoside digests were analyzed by UHPLC-MS as detailed above.

Protein purification
All protein purification steps were carried out at the temperature range 0 – 12 °C. All buffers were filtered (0.45 μm) and degassed. Equipment used for protein purification was ÄKTA Purifier FPLC system (GE Healthcare) and Minipuls 3 peristaltic pump (Gilson). Buffer composition for purification of Mom and variants is described in Supplementary Methods. N-ter 6xHis-Tag full-length Mom WT and R111A and 6xHis-SUMO-Δ10 Mom WT and S124A were purified using our original protocol that involved Ni-NTA, Heparin, and size-exclusion chromatography. E. coli GlyRS was also purified according to the protocol from Yoshihiro Shimizu & Takuya Ueda with additional steps34. Detailed protocols can be found in the Supplementary Methods. Aliquots of E. coli WT GlyRS and WT AlaRS were also kindly gifted by Corinna Tuckey, Dr. Ying Zhou, Dr. Bradley J. Landgraf, and Dr. Emily Chen. The expression and purification scheme for WT CC2A from S. pombe was kindly shared by Dr. Andew P. Sikkema and can be found in the Supplementary Methods. A contaminating RNase was removed using an immobilized aptamer98. C666A AlaRS was expressed and purified57–59 (see also Supplementary Methods.).

Mom activity assays with the PURExpress® system
For cell free protein synthesis (also referred as in vitro transcription and translation or IVTT), the PURExpress® system (NEB) was used with 1000 ng of pRY WT Mom plasmid as the template in a reaction incubated at 25 °C for 24 hours according to the manufacturer’s instructions34,35. Nucleosides from recovered plasmids were prepared and analyzed by UHPLC-MS. The PURExpress® system was also utilized for activity assays (without plasmid template), which included PURExpress® kit components, purified WT Mom (2 µM), tRNAGly/GCC (made by in vitro transcription, see below), and 1000 ng of biotinylated DNA substrates where indicated. Reactions were incubated at 25 °C for 24 hours or 37 °C for  2 hours and biotinylated substrates were recovered, digested, and analyzed by UHPLC-MS.

In vitro transcription (IVT) of tRNA
Templates for IVT were amplified by PCR using Q5® High-Fidelity DNA Polymerase or PhusionTM DNA Polymerase (NEB). Unmethylated primers were used for amplification by Q5. Standard IVT reaction conditions using the HiScribe® T7 Quick High Yield RNA Synthesis Kit (NEB) were used for tRNAGly (all isoacceptors) and tRNAAla/TGC (see Supplementary Table 4 for sequences). tRNAGly/GCC was also transcribed using purified T7 polymerase (kindly gifted by Prof. Janusz Bujnicki’s lab) in previously described conditions48,52,53,99. In some cases, repression of nontemplated additions at the 3' end by T7 RNA polymerase was needed. For full-length tRNAGly/GCC-CCA (76 bases), 10% (v/v) DMSO was added to the IVT reaction mixture. For tRNAGly/GCC-CC (75 bases), reverse primers including a 2'-O-methylated base second from the 5' end were used during PCR amplification by Phusion. 10% (v/v) DMSO was also added the IVT reaction mixture containing 2'-O-methylated PCR products. In all cases, GMP was added to a final concentration of 20 mM to the IVT reaction mixture to favor the 5'-monophosphate product. tRNAs were purified using the Monarch® RNA Cleanup Kit (NEB), characterized by urea-PAGE, and where indicated by intact mass spectrometry.

In vitro coupled Mom activity assay with nucleoside analysis
Components for coupled Mom activity assays including GlyRS were mixed on ice in the following order to a final volume of 30 µL and incubated at 37 °C for the indicated durations: activity buffer 1 (AB1, See Supplementary Table 3 for activity buffer compositions); Pyrophosphatase, Inorganic (0.025 units/mL, NEB); GlyRS (2-4 nM); glycine (2 mM); RNase Inhibitor, Murine (20 units, NEB); Mom (2 µM); DNA substrate (typically 1 µg but could range from 250 ng – 10 µg); tRNAGly (~3 µg [4 µM]); and RNase-free water48,61. Where specified, glycine-1-13C,15N (2 mM, Sigma) was used in place of glycine. Reaction components were omitted where specified in the text. After incubation, all reactions treated with 1-3 µL RNase A (NEB) for > 30 min. For time courses, samples were also treated with 1-3 µL of ProtK (NEB). Biotinylated substrates were prepared for UHPLC-MS analysis as described above. The 3 kbp substrates, oligomer substrates, and plasmid substrates were purified using the Monarch® PCR and DNA Cleanup Kit (NEB) with the DNA Cleanup and Concentration and the Oligonucleotide Cleanup protocols, respectively. The 3 kbp substrates, oligomer substrates, and plasmid substrates were prepared for UHPLC-MS analysis as described above. The in vitro activity of Mom on short DNA was tested in Activity Buffer 2 (AB2) in 100 μL reaction volume. The mixture contained 17 μg of 11 bp or 3 bp DNA, 0.6 μM Mom protein (monomer), 1.6 μM tRNA, 0.09 μM GlyRS (hetero-tetramer), 10 mM glycine, and 2 mM ATP. The reaction was incubated at 37 °C for 5 hours followed by nucleoside analysis100. For Mom assays including WT AlaRS or C666A AlaRS, 7 µM of enzyme and 14 µM of tRNA were included34.

In vitro coupled Mom activity assay with restriction challenge
Activity assays with radiolabeled substrates included 2 pmoles of 34-mer dsDNA and other reaction components (in AB2 and assay conditions used for short DNA, see previous section) with an overnight incubation. After incubation, purified DNA was treated by AasI FastDigest (Thermo), and the digestion products were resolved on 20% Urea-PAGE and visualized by autoradiography using Amersham Typhoon Biomolecular Imager. The activity of Mom with and its variants was also tested on 3.6 μg of substrate plasmid (pET28a-empty in activity assay conditions for short DNA). Purified plasmid DNA was subjected to restriction digestion as above.

Single-nucleotide extension of tRNA using CCA2
rA from ATP (NEB) or 2'-dA from 2'-dATP (NEB) was added to the 3' end of tRNAGly/GCC-CC using purified WT CCA2 from S. pombe47,101–103 (see also Supplementary Methods and Supplementary Fig. 14) at 25 °C under single turnover conditions at a ~300 µg scale in activity buffer containing 50 mM Tris (pH 8), 100 mM KCl, 1 mM TCEP, 10 mM magnesium acetate, 1 µM BSA, and 1-2 mM (d)NTP. tRNA was cleaned up using spin columns, treated with apyrase to deplete nucleotide triphosphate carried over during purification, cleaned up using spin columns, and extension was verified by intact mass spectrometry as above.

Charging, labeling, and quantification of tRNA aminoacylation
tRNA (~7 µM) was aminoacylated (charged) for 20-30 minutes at 37 °C in 100 mM HEPES pH 7.6, 2 mM ATP, 20 mM MgCl2, 1 mM DTT, 7 µM aaRS, Pyrophosphatase, Inorganic (NEB, 5 units), and 2 mM amino acid in a total volume of 50 µL as described48–51. Reactions were quenched with 1/10 volume of 3 M sodium acetate (pH 5.2), purified by ethanol precipitation, and resuspended in 40 µL of 5 mM sodium acetate (pH 5.2). The QubitTM Broad Range Assay Kit (InvitrogenTM) was used to quantify the tRNA recovered from the aminoacylation reaction, and samples were stored at -20 °C until use. Roughly 150 pmol (~3.6 µg, ~2.5 µM) of tRNA recovered from the charging reaction was labeled with EZ-LinkTM Sulfo-NHS-LC-Biotin (Thermo ScientificTM) that was freshly prepared in RNase-free water (15 mM) in a total volume of 60 µL on ice. Labeling reactions were cleaned up with a Micro-Bio Spin 6 column pre-equilibrated with 5 mM sodium acetate (pH 5.2). About 8 pmol of recovered tRNA was incubated with 8 µL of streptavidin (1 mg/mL) at room temperature for 20 min. tRNA (200 ng total) was mixed 1:1 with acidic loading dye (100 mM sodium acetate [pH 5.2], 7.5 mM urea, 0.05 bromophenol blue) without heating and resolved by urea-PAGE using 6% NovexTM TBE-Urea Gels, InvitrogenTM at 200 V for 30 min. Gels stained with SYBRTM Gold were visualized using an Amersham Typhoon Biomolecular Imager, and ImageLab (Bio-Rad) was used to quantify band intensities of uncharged and charged (shifted) bands. Percent charging was calculated by (shifted band)/(shifted band + unshifted band). Generally, 20-60% charging was observed.

Mom crystallization and structure determination
Purified Δ10 Mom S124A protein was applied to a Superdex 200 Increase 10/300 GL column equilibrated with Buffer 9 (20 mM HEPES-KOH, pH 7.5, 550 mM NaCl, 50 mM KCl, 5% glycerol, 10 mM 2-ME). The best fractions were combined and concentrated to ~23 mg/mL using an Amicon 10 MWCO centrifugal filter unit. Mom crystals were grown by mixing 2 μL of the protein with 2 μL of the condition H4 of the PACT premier crystal screen (Molecular Dimensions) (0.2 M Potassium Thiocyanate, 0.1 M Bis–Tris propane, pH 8.5, 20% PEG3350) using the vapor diffusion method in hanging drops. For cryo-protection, crystals were soaked in mother liquor containing 25% glycerol and flash-cooled in liquid nitrogen. Mom crystal diffraction data was collected on PETRAIII P11 beamline at the DESY synchrotron. Crystals had a space group P 21 21 2 and diffracted up to 2.03Å at using X-ray wavelength of 1.0332Å. The diffraction data was automatically processed by the XDSAPP program at the synchrotron 104. The structure of Mom was solved by molecular replacement using the Phaser program and the Alphafold 2 predicted model 71,73. The programs Phenix and Coot were used for the refinement and manual building 72,105. The final model had following Ramachandran statistics (%): favored 98.3, allowed 1.7, outliers 0.00. The model contained 1.6% rotamer outliers and had a MolProbity clashscore of 2. The data collection and refinement statistics are presented in Supplementary Table 5. Representative 2mFo-DFc and composite omit electron density maps supporting data quality are depicted in Supplementary Fig. 47. The final model coordinates and the structure factors were deposited in Protein Data Bank under accession code: 8BV8.

3D structural analysis and graphic representation
Structure manipulation and image rendering was performed using UCSF ChimeraX program106. The MatchMaker tool in ChimeraX was used for structure superposition. Secondary structure assessment (Fig. 3b and 3c) was guided by the PDBsum server75. Vector Alignment Search Tool (VAST) was used to search for the structural similarity between Mom and the available GNAT structures on the medium redundancy setting107. GNATs with productively bound CoA and acyl-CoA were used for superposition in Fig. 3d and Fig. 3e and were selected from the top 164 hits (the cut-off was manually determined). A subset (11) of retrieved GNATs with productively bound substrates or products were used for superposition in Fig. 4f. See Supplementary Table 6 and Supplementary Table 7.

Metagenome mining and selection of homologs to screen for activity in vivo
A search for homologs of Mom in metavirome databases was completed as described32,33,92,93. Briefly, using Mu Mom as a query sequence, Geneious Prime software (v 2020.1.1) was used to run a Basic Local Alignment Search Tool (BLAST) search of the Nucleotide collection (nr/nt). 500 sequences only containing Mom domains were used to build an HMM profile with the hmmbuild program from the HMMER software package108. The hmm profile for Mom was used to search two metavirome sequence databases: the Joint Genome Institute’s Integrated Microbial Genomes Viral Resource version 2 (IMG/VR v2)109 and the Global Oceanic Viromes 2.0 (GOV2.0)110. Open reading frames in the recovered contigs (>8,000) were predicted and annotated using Prokka 111 within JGI’s KBase Knowledge Discovery Environment112. A more thorough annotation process using the Pfam database was completed using the hmmscan program113.
All-against-all BLAST comparisons of protein sequences including Mu Mom and >8000 predicted homologs were run using EFI-EST (https://efi.igb.illinois.edu/efi-est/) (Enzyme Function Initiative – Enzyme Similarity Tool)114 with an alignment score threshold of 35115. To facilitate visualization, the final network generated was 95% representative, collapsing sequences of 95% identity into representative nodes (3799 nodes, 589,294 edges, representative of 9,647 sequences). The similarity network was visualized using Cytoscape116. The alignment threshold and representative node percentage reported were selected based on empirical analysis. The SSN for Mom and homologs was used to guide initial screening using the in vivo activity assay. The sequences for Mom Homolog A and Mom Homolog B were selected at random. The structures of Mom Homolog A and Mom Homolog B were predicted using ColabFold117 and superimposed with Chain A of Mom using PyMOL118.

Data availability
The final model of the structure solved in this work was deposited in Protein Data Bank under accession code 8BV8. ORCA output files for all energy minimizations of educts, products, and the spirocyclic intermediate, as well as ORCA output files for the saddle points/transition states, and the corresponding PDB files (translated from the ORCA files using Avogadro) are made available in a Zip drive. A sequencing read archive (SRA) containing BAM files produced by PacBio SMRT sequencing platform and extended to include kinetic data for the modification motif analysis in this work can be obtained at NCBI Bioproject PRJNA1406289. Metagenome information for Mom Homolog A can be found at https://img.jgi.doe.gov/cgi-bin/m/main.cgi?section=TaxonDetail&page=taxonDetail&taxon_oid=3300002484. Metagenome information for Mom Homolog B can be found at https://img.jgi.doe.gov/cgi-bin/m/main.cgi?section=TaxonDetail&page=taxonDetail&taxon_oid=2872672955. All other data supporting the findings of this work are available within the Article, Supplementary Information, or from the corresponding author(s) upon request.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Noteworthy Results: In the manuscript (submitted for consideration as an Research Article in the journal Nature Communications) “A single viral enzyme drives tRNA-dependent hypermodification of DNA at adenine” by Silva et. al, the authors provide important, additional answers to the question of how the bacteriophage Mu forms 6-NcmdA in its DNA. The fact that the gene product of Mu mom is sufficient for the formation of 6-NcmdA was known prior to this study, and Mu Mom had been predicted to belong to the GNAT superfamily. However, the study advances this area of research in the following ways:\nA. The study showed that the molecule that provides the 6-Ncm group for transfer is charged tRNAGly (all three isoacceptors)\nB. Authors showed that after addition of the glycyl group to dsDNA, an isomerization event occurs to form the modification – also providedmechanistic evidence of how that isomerization occurs (N6 acts as the nucleophile)\nC. Computational chemistry showed plausible explanation of how/why the reaction proceeds\nD. The first crystal structure of Mu Mom was solved\nE. Experimental data identifying some of active site residues was provided\nSignificance to the Field: This work is significant because the authors solve unanswered questions regarding a strategy of how a bacteriophage evades its host. Additionally, the results are relevant and of interest to the field of nucleic acid modification. Although the enzyme is part of the fairly well-studied GNAT superfamily of proteins, it has some unique features (use of a charged tRNA as a substrate; insertion of amino acids resembling a helix-turn-helix) which are important to further understand this superfamily of proteins.\nStrength of Conclusions and Claims: Authors claims and conclusions were supported by the data. Authors appeared to be cautious in not overstating what the data showed. For instance, conclusions from data generated from computational experiments were not overstated.\nData Analysis, Interpretation, and Claims: No obvious flaws in analysis and interpretation of data. Data quality was high and was presented well. Authors do a good job of explaining how data fits into the general body of knowledge. Article is clearly written and easy to follow.\nMethodology: The methodology was sound and meets high standards expected in the field. Authors addressed the question using multiple methods.\nInformation in Methods Section: Information in methods section is thorough and provides enough detail to allow for the work to be produced.", "reviewer_2": "The bacteriophage Mu employs a 6-NcmdA modification to protect its DNA from restriction digestion by host bacterial nucleases. Previous studies have identified that the Mom protein is responsible for 6-NcmdA biogenesis; however, the detailed molecular and structural basis for this modification is unclear. In the present work, Silva et al. provided evidence that Mom catalyzes 6-NcmdA via a Gly-tRNAGly-dependent mechanism and solved the structure of apo-Mom with the identification of several residues critical for enzymatic activities. This manuscript can be divided into three aspects, including the establishment of charged tRNAGly as one of the substrates, investigation of the modification and isomerization processes during 6-NcmdA formation and the determination of the Mom structure.\nMajor points:\n1, Various combinations of reaction conditions were included, showing Gly-tRNAGly, but not acetyl-CoA was the potential substrate of 6-NcmdA formation. However, GlyRS is able to generate Gly-adenylate and Gly-tRNAGly in aminoacylation. When compared with the 20% modification efficiency (Fig 1b) with GlyRS, purified Gly-tRNAGly only supported very limited 6-NcmdA modification (around 2%). The authors claimed that low charging level of tRNAGly accounted for the limited modification. On one hand, cognate Gly-tRNAGly preparation could be very efficient and only 20% charging level (Extended Data Fig 4) of tRNAGly by GlyRS was not reasonable, which could be optimized. A higher charging level of tRNAGly should be achieved and included in the hypermodification reaction to see whether elevated amount of Gly-tRNAGly leads to a higher 6-NcmdA biogenesis. On the other hand, the possibility of transferring Gly-moiety from Gly-adenylate to DNA could not be absolutely excluded. It is possible that presence of tRNAGly induces a conformation change of Mom to facilitate the reaction. In this regard, a non-a minoacylated tRNAGly, such as dA76-tRNAGly (with A76 hydroxyl group replaced by H), should be included in the hypermodification reaction to explore whether a non-chargeable tRNAGly is able to stimulate modification. In addition, different GlyRS mutants, such as a Gly-adenylate synthesis-deficiency mutant and a tRNAGly binding deficiency mutant, should be included to understand in detail how the Gly is transferred to the modified A in DNA.\n2, Different tRNAGly isoacceptors were tried in modification reaction with comparable efficiencies. Based on the structure information provided in Fig 3, no residues seem to bind the main structure of tRNA other than CCA end; thus it is puzzling that how amino acid and tRNA specificity was achieved. Other types of amino acids, tRNAs and tRNA synthetases should be explored in the hypermodification to understand the substrate selection. Moreover, editing deficient mutants of alanyl-tRNA synthetase (AlaRS), such as C666A, are able to generate Gly-tRNAAla. Whether Gly-tRNAAla is a substrate of Mom?\n3, It has been reported that certain bacterial acetyltransferases can use acetate as the substrate and the acetyladenylate as the reaction intermediate, in a tRNA-aminoacylation-like mechanism. Have the authors tested the potential use of acetate by Mom?\n4, The apo-Mom structure provided the basic information of the overall folding of Mom and was a valuable progress towards understanding of 6-NcmdA biogenesis. Based on this structure, several key residues have been identified using reconstituted activity determination assays. However, it provided little information in how amino acid, tRNA and DNA were accommodated. Several subdomains were suggested to bind tRNA CCA end and DNA in Fig 3. Point mutations should be performed at potential binding sites of CCA end and DNA helices; affinities between Mom-tRNA and Mom-DNA should be measured to obtain in-depth understanding of protein-nucleic acid interaction in 6-NcmdA formation.\nMinor points:\n1, Abstract, R111A to R111;\n2, Page 7, “mutating the residues Y28A and W48” should be “mutating the residues Y28 and W48”;\n3, Data about dimerization deficiency of the W48A mutant should be shown.\n4, Fig 4 legends, ‘the N6 atom the adenine target’ should be ‘the N6 atom of the adenine target’.", "reviewer_3": "Summary\nIn this study, Silva. et at. set to understand the molecular mechanism of how the DNA hypermodification enzyme mom creates 6-NcmdA. Using in-vivo/vitro activity assays and LC/MS, the authors find that mom uses the charged Gly-tRNAGly as the glycyl group donor and specifically transfers the glycyl to SAS sequence motif of dsDNA. Subsequently, isotope labeling and tracking with LC/MS-MS data suggests that the glycyl group undergoes isomerization to create the final product 6-NcmdA. Quantum chemical calculations are also carried out to support the energetic of such rearrangement. Lastly, the crystal structure of mom with a mutation of S124A (mostly inactive state) is determined, and key residues important for catalysis are identified.\nGeneral remarks\nEnzymes harboring GNAT conserved fold have been reported previously to use the charged tRNA as the aminoacyl donor to modify a variety of substrates, but not DNA. Here Silva. et at. find that mom transfers the glycyl group from charged Gly-tRNAGly to dA and provide evidence that rearrangement is required to create the final product.\nOverall, the authors have done a good job on the biochemical and computational characterization, and the data seem to largely support the conclusions. Crystal structure is also determined, however, I think the authors do not examine deep enough about the molecular details of substrate recognition and reaction mechanism (see below), to explain well enough on the biochemistry observations. Assuming that the authors can make structural hypothesis based on the exist structure models, I believe that the study would be an exciting contribution to the field.\n1, Why Ac-CoA is not an optimal donor for modifying the dA, why mom prefers dsDNA over ssDNA, why mom prefers SAS sequence motif, why all three tRNA isoacceptors are capable as donors, why the first 20 amino acids are important for activity, why mom homo-dimeric state is important for the momylation,. since the authors have the structure model on hand and preform serials of dockings, please at least try to hypothesis/address these open questions from the structure perspective in the result or discussion.\n2, please describe what R111 and S124 function might be during catalysis, seems to me they are far from the potential glycyl transfer active site.\n3, I am curious if the authors have tested that if mom can use Ala-tRNAAla as donor or not.\n4, In Supplementary figure 6, please also show the gel filtration run profile of mom Y28A and W48A, to confirm the mutants are indeed at monomer state.", "reviewer_4": "The authors establish the molecular basis for the biosynthesis of 6-aminocarboxymethyl-2ʹ-deoxyadenosine (6-NcmdA), a nucleobase hypermodification found in the virion DNA of bacteriophage Mu.\nThey demonstrate that the Mu-encoded gene product of Mom is an active aminoacyltransferase that exploits host translational resources to transfer an activated glycyl group from charged tRNAGly to deoxyadenosine in the context of dsDNA.\nUsing stable isotope labelling, they uncover a second step in the biosynthesis of the Mu hypermodification, where a novel on-base rearrangement of the initial aminoacylation product subsequently yields 6-NcmdA. Further examination of the aminoacylated intermediate, transition state, and momylation product by quantum chemistry calculations highlight the characteristic instability of acyl exocyclic groups at N6-adenine and the favourable orientation 6-NcmdA can adopt that facilitates Watson-Crick base pairing.\nMy comments are focused on the methodology employed for the quantum chemical calculations as I am not an expert in the experimental essays and measurements reported.\n1) More details on the computational methods are needed, in particular on the convergence of the basis set employed, which seems to me relatively small for this system.\n2) Details on the structures of reactants, products and educts need to be reported in the supplementary information as pdb, cif or xyz files, in order to make the results reported reproducible.\n3) Quantum effects can be important for estimating the energy and transition states in DNA hydrogen bonds and tautomerisation probabilities. This has been discussed in several recent papers by various groups worldwide. The authors should comment on the possible role of proton delocalisation and, potentially, tunnelling in the systems reported.\n4) It is not clear if dispersion corrections have been included in the calculations. If they have not been included, could the authors please discuss the motivation of their choice?\n5) Details on the energy and force convergence in the geometry optimisations and TS searches need to be reported. RHF geometries are likely to not reflect the B3LYP geometries, therefore I would not take the barrier heights for these XC functionals as anything more than guesswork."}, "author_response": "Dear Reviewer 1: Thank you for your comments. We are grateful that the overall narrative was well received by you. Your summary of the story aligns well with what we hope readers will take away from the work.\nDear Reviewer 2: Thank you for your close reading of our manuscript and for providing helpful criticisms, comments, and suggestions. For the sake of continuity between the points and issues you raise with how we address them, we provide our responses in line with your points.\nMajor points\n1, Various combinations of reaction conditions were included, showing Gly-tRNAGly, but not acetyl-CoA was the potential substrate of 6-NcmdA formation. However, GlyRS is able to generate Gly-adenylate and Gly-tRNAGly in aminoacylation. When compared with the 20% modification efficiency (Fig 1b) with GlyRS, purified Gly-tRNAGly only supported very limited 6-NcmdA modification (around 2%). The authors claimed that low charging level of tRNAGly accounted for the limited modification. On one hand, cognate Gly-tRNAGly preparation could be very efficient and only 20% charging level (Extended Data Fig 4) of tRNAGly by GlyRS was not reasonable, which could be optimized. A higher charging level of tRNAGly should be achieved and included in the hypermodification reaction to see whether elevated amount of Gly-tRNAGly leads to a higher 6-NcmdA biogenesis.\nSince we collected even stronger evidence for our conclusions (see sections below), we have therefore removed Extended Data Fig. 4 and Supplementary Fig. 8 to help focus the manuscript further.\nOn the other hand, the possibility of transferring Gly-moiety from Gly-adenylate to DNA could not be absolutely excluded. It is possible that presence of tRNAGly induces a conformation change of Mom to facilitate the reaction. In this regard, a non-aminoacylated tRNAGly, such as dA76-tRNAGly (with A76 hydroxyl group replaced by H), should be included in the hypermodification reaction to explore whether a non-chargeable tRNAGly is able to stimulate modification.\nReviewer 2 correctly points out that in our coupled reaction, there are two forms of activated glycine generated by GlyRS – glycyladenylate as an intermediate and Gly-tRNAGly as a product. We agree that identifying a tRNA analog that would allow us to distinguish which activated form of glycine is used by Mom would be a great approach here. As suggested, we generated a tRNAGly/GCC analog with a -CCA-2ʹ-dA acceptor stem at the 3ʹ end. We verified that in our hands GlyRS can charge tRNAGly/GCC-CC-2'-dA, as shown previously by Sprinzl and Cramer in 1975 (Supplementary Figure 17)1. As can be seen in Figure 2b, momylation is not observed when tRNA-CC-2ʹ-dA is included in a Mom assay (n=3, no modification detected) even after overnight incubation (Supplementary Figure 18).\nTaken together, we are able to make several important conclusions from these data. First, glycyladenylate and Gly-tRNAGly are generated by GlyRS for both tRNAGly/GCC-CCA and tRNAGly/GCC-CC-2'-dA. For the time courses shown in Figure 2, we can deduce that both forms of activated glycine were available to Mom in the activity assays. Second, tRNAGly/GCC-CC-2'-dA cannot support momylation. Our data are unambiguous here. To emphasize, even in the presence of glycyladenylate and charged tRNAGly/GCC-CC-2'-dA, momylation does not occur, highlighting that the terminal riboadenosine is critical for momylation. Third, we have only observed momylation of DNA at dA when tRNAGly/GCC-CCA is included in the reaction with GlyRS, ATP, and glycine. tRNAGly/GCC-CCA in its native form is absolutely required for momylation. Our time courses with tRNAGly/GCC-CC-2'-dA (Figure 2), GlyRS activity assays (Supplementary Figure 17), and Mom activity assays in the absence of tRNA (again, see Figure 1c, Trace 4 and Figure 1d, Lane 7) are in agreement and strongly support our conclusion that Gly-tRNAGly-CCA is the activated form of glycine used by Mom to hypermodify DNA.\nThis work was nontrivial and required overhauling our existing methods and introducing an enzyme into our work (CCA2) that was not commercially available. Our advancements are detailed in Methods, Supplementary Methods, Figure 2a, Supplementary Figure 9, and Supplementary Figures 12-19. tRNA and tRNA analogs were exceptionally difficult to obtain commercially in acceptable yield and purity despite several attempts with multiple vendors. Guided by the literature and our own empirical testing, we screened and we found new conditions that allowed for isolation of tRNAGly/GCC-CC (75mer) in acceptable yield and purity, screened commercially-available enzymatic approaches to add a single base to the 3' end of tRNAGly/GCC-CC from either ATP or 2'-dATP, and developed methods utilizing WT CCA2 from S. pombe for single-base extension of tRNA. To our knowledge, this is the first demonstration that WT CCA2 from S. pombe can use 2'-dATP to add dA to the 3' end of tRNA. This could be the subject of its own publication.\nOn a final note, we wish to emphasize that there are no known central metabolic reactions which utilize aminoacyl-adenylate intermediates produced by tRNA synthetases (apart from charging tRNA). Aminoacyl-adenylates are transient and strongly sequestered in the enzyme active site. The one area where aminoacyl-adenylates are used in non-proteinogenic biosyntheses is in the synthesis of certain antibiotics, as shown in the work of Jason Crawford (Yale University) and colleagues2. In these examples, the pathways depend on paralogous “defective” tRNA synthetases that have diverged to where they can no longer aminoacylate tRNA and instead have adapted to perform this specialized role within the context of a natural product biosynthetic gene cluster.\nIn addition, different GlyRS mutants, such as a Gly-adenylate synthesis-deficiency mutant and a tRNAGly binding deficiency mutant, should be included to understand in detail how the Gly is transferred to the modified A in DNA.\nA new study focused on characterizing how GlyRS variants affect momylation warrants a separate manuscript.\n2, Different tRNAGly isoacceptors were tried in modification reaction with comparable efficiencies. Based on the structure information provided in Fig 3, no residues seem to bind the main structure of tRNA other than CCA end; thus it is puzzling that how amino acid and tRNA specificity was achieved. Other types of amino acids, tRNAs and tRNA synthetases should be explored in the hypermodification to understand the substrate selection.\nWe have benefitted from insightful reviews and primary literature that have considered the evolution of GNAT superfamily enzymes and the questions of co-substrate and substrate recognition3–9. We direct Reviewer 2 to this literature. Where investigated5,6, the 3ʹ stem of tRNA and the amino acid appear to be sufficient to direct co-substrate specificity for the enzymes studies thus far. For example, the identity of the nucleotide penultimate to the universal -CCA acceptor stem was shown to be important for co-substrate specificity of FemX , a GNAT Wv enzyme that utilizes Ala-tRNAAla for bacterial cell wall biosynthesis. Steric exclusion of bulkier amino acids was also a determinant of specificity.\nComparing the sequence of tRNAGly and tRNAAla at the 3ʹ ends, we find that for all tRNAGly isoacceptors, the nucleotide immediately 5ʹ to the CCA stem is a U, whereas for all tRNAAla isoacceptors, the corresponding position is an A (sequences obtained from GtRNAdb). Analogously for Mom, the terminal glycine and key features at the 3' end of tRNA could reasonably present enough \"information\" to the active site of Mom to control specificity. We think this would be an exciting subject for a future manuscript. More philosophically, we note that many biosynthetic enzymes correctly distinguish glycine from other amino acids. Why should Mom be different? Stated simply, anything larger than glycine might not fit into the enzyme’s active site.\nWith regard to GNAT enzymes, it is striking to us that in this field overall, the factors driving co-substrate and substrate specificity are poorly understood and not frequently studied considering the size of the superfamily (>300,000 enzymes). Little is known why some GNATs recognize acyl-CoA species, why others recognize tRNA charged with a specific amino acid, and why some GNATs are functionally decarboxylases. There is one notable publication on the topic of specificity of bacterial GNAT toxin for tRNAGly, which we briefly discuss and site in section “Mom is selective for tRNAGly, glycine, and dsDNA”3. Again, we posit that the incredible substrate diversity (DNA, tRNA, proteins, lipids, small molecules, etc.) and poor sequence homology10 are contributing to challenges in learning the general ‘rules’ of selectivity for GNAT enzymes.\nAdding to the complexity, for several of the solved structures we have come across in the Protein Data Bank, the native co-substrate and substrate have not been identified. For example, structural alignments of Mom with other GNATs have not provided any insights as to what is driving specificity. The top hit for Mom using the DALI Protein Structure Comparison Server is an engineered glyphosate-N-acetyltransferase (GAT; PDB ID 2BSW); the parent enzyme from B. licheniformis was found in screen to transfer an acetyl group of acetyl-CoA to glyphosate11,12. The rmsd value of the alignment between Mom and GAT is 2.8. We searched Uniprot (Q65LG7) for the latest data on the parent enzyme from B. licheniformis and have not found any more updates. We emphasize here that native co-substrate of GAT is likely acetyl-CoA with an unknown, native target, while our data show that Gly-tRNAGly is the co-substate of Mom with DNA as the target. Even so, GAT and Mom align well. The second DALI hit (rmsd 2.8) is a protein named IAA acetyltransferase from B. cereus. No publication is associated with the crystal structure (1Y9K) and the Uniprot entry (Q81FK8) does not provide additional information about the native co-substrate or substrate. The third hit from the DALI server (rmsd 2.5) is RimI13 from S. typhimurium (2CNT), and this enzyme does have a known co-substrate, acetyl-CoA, and known substrate, ribosomal protein S18. These selected examples highlight that the process of recognition for GNAT may be very dynamic and may require extensive, time-resolved techniques to observe the various complexes formed and various conformations accessed.\nWe can further look to other examples where GNATs evolved to use charged tRNA as substrates that do distinguish one amino acid from another, without reference to an anticodon. The TacT category of GNAT toxins are known to be potent inhibitors of translation by targeting all isoacceptors of Gly-tRNA for acetylation of glycine’s primary amine. Studies of TacT-like toxins show that the amino acid type and the presence of the acceptor stem nucleotides were sufficient determinants by which TacT acetylated Gly-tRNAGly and not other aminoacyl-tRNA species8. Analogously to TacT and FemX, it is entirely plausible that -UCCA and glycine could provide sufficient determinants of specificity in the active site of Mom. In contrast, for tRNA synthetases, the requirement for the enzyme contacting the anticodon loop is likely a quality control measure to ensure accurate aminoacylation specific for the correct tRNA species, one of several key mechanisms supporting translational fidelity.\nWe would additionally like to remind the reviewer that all other aminoacylated forms of tRNA were available to the Mom enzyme both in vivo (Supplementary Fig. 4) and in vitro assays using PURExpress® (Supplementary Fig. 6), and yet only glycine is selected. For now, we hope we have presented evidence from our own work, as well as the literature, to show that amino acid selectivity is a fascinating feature of GNAT enzymes.\nMoreover, editing deficient mutants of alanyl-tRNA synthetase (AlaRS), such as C666A, are able to generate Gly-tRNAAla. Whether Gly-tRNAAla is a substrate of Mom?\nWe thank Reviewer 2 for directing us to literature on AlaRS, an editing-deficient mutant of AlaRS C666A, and for recommending that we test if mischarged Gly-tRNAAla could support momlyation14–16. We did this. From our activity assays where Gly-tRNAAla was generated in situ (Fig. 2c and Supplementary Fig. 20-21), we conclude that mischarged Gly-tRNAAla/TGC is not a substrate of Mom and therefore cannot support momylation.\n3, It has been reported that certain bacterial acetyltransferases can use acetate as the substrate and the acetyladenylate as the reaction intermediate, in a tRNA-aminoacylation-like mechanism. Have the authors tested the potential use of acetate by Mom?\nWe are not sure why it would be necessary to rule out or even test activated forms of acetate as a group donor in the momylation reaction as acetate does not not have the correct stoichiometry (ex. nitrogen) for reconstituting 6-NcmdA in a single transfer step.\nBut considering Reviewer 2’s point, we are making an educated guess that the question above is about ac4C, a tRNA hypermodification where an acetyl group is installed at the N4 position of cytosine in the wobble position of tRNAMet (shown in Figure 5). The GNAT enzyme, TmcA (tRNAMet cytidine acetyltransferase, from E. coli for example), acetylates the exocyclic nitrogen of cytosine using acetyl-CoA as the co-substrate (see citations related to Figure 5 and the associated Discussion given in the main text). However, after investigating the literature further, we learned that an analogous (but unrelated) enzyme, TmcAL (tRNAMet cytidine acetate ligase) from Bacillus subtilis (previously named YlbM), similarly acetylates N4 on cytosine on tRNA. Unlike E. coli TmcA, Bacillus TmcAL uses a different mechanism: ATP-dependent activation of acetate to form acetyladenylate as the intermediate17. Bacillus TmcAL is a homolog of class I aminoacyl-tRNA synthetases, which accounts for the synthesis of an acetyl-adenylate intermediate. Thus, TmcAL activates acetate then transfers the activated acetate. Based on structure determined in this work, Mom firmly belongs to the GNAT superfamily of enzymes, where the chemistry involves transfer of a group, already in an activated form, to a target. For clarity, we have specified in Figure 5 that the example of TmcA we are giving is a GNAT from E. coli.\n4, The apo-Mom structure provided the basic information of the overall folding of Mom and was a valuable progress towards understanding of 6-NcmdA biogenesis. Based on this structure, several key residues have been identified using reconstituted activity determination assays. However, it provided little information in how amino acid, tRNA and DNA were accommodated. Several subdomains were suggested to bind tRNA CCA end and DNA in Fig 3. Point mutations should be performed at potential binding sites of CCA end and DNA helices;\nWe performed extensive structure-guided mutagenesis of residues hypothesized to mediate DNA/tRNA binding, catalysis, and dimerization (Supplementary Fig. 37). Although the mutagenesis preceded the release of AF3, retrospective analysis using an AF3 model of a ternary complex supports our assignments. For example, R62A - but not R61A - reduces activity, consistent with R62 lying more closely to DNA in the model. The same decrease of activity is observed for alanine mutant of R192, which is located in the HTH-like insertion. Of note, deletion of the HTH-like insertion leads to a complete loss of activity. R111, previously identified as essential, may also contribute to DNA binding, although a direct catalytic role cannot be excluded. Based on the modeling, we further hypothesize that S124 is required for tRNA binding and that the positively charged stretch spanning residues 11–19 stabilizes tRNA binding.\naffinities between Mom-tRNA and Mom-DNA should be measured to obtain in-depth understanding of protein-nucleic acid interaction in 6-NcmdA formation.\nOf course, we attempted to crystallize Mom with tRNA and/or DNA. Throughout the course of our studies on Mom, mixing Mom with tRNA or mixing Mom with DNA alone has resulted in visible precipitation. We have tested long-established methods for observing or measuring complexes between macromolecules – native PAGE and size exclusion chromatography (not shown) – under various conditions. So far, Mom:nucleic acid complexes cannot be resolved on a gel even at low concentrations (nM), and a binding curve cannot be obtained. At concentrations needed for detection by SEC, precipitates form that cannot be resolved on a column. We notably do not see precipitation in activity assays. The challenges in studying Mom outside the context of an activity assay does not affect any of the conclusions we are able make about our data.\nThere could be several mechanisms at play here, including aggregation, co-substate- or substrate-induced oligomerization, or other phenomena that are known to occur with protein:nucleic acid complexes, such as phase separation18–20. We anticipate that much more extensive screening and careful testing is needed to establish the molecular origins of this precipitation, which can be the topic of a subsequent publication or publications.\nMinor points:\nAgain, thank you for your close reading.\n1, Abstract, R111A to R111;\nCorrection made.\n2, Page 7, “mutating the residues Y28A and W48” should be “mutating the residues Y28 and W48”;\nCorrection made.\n3, Data about dimerization deficiency of the W48A mutant should be shown.\nData added to Supplementary Fig. 39.\n4, Fig 4 legends, ‘the N6 atom the adenine target’ should be ‘the N6 atom of the adenine target’.\nCorrection made.\nDear Reviewer 3: We are grateful that the overall narrative was well received by you. Thank you for your close reading of our manuscript, collegial comments, and for providing helpful criticisms and suggestions. For the sake of continuity between the points and issues you raise, and how we address them, we provide our responses in line with your points.\nCrystal structure is also determined, however, I think the authors do not examine deep enough about the molecular details of substrate recognition and reaction mechanism (see below), to explain well enough on the biochemistry observations. Assuming that the authors can make structural hypothesis based on the exist structure models, I believe that the study would be an exciting contribution to the field.\nRegarding substrate recognition and reaction mechanism, please see our response below.\n1, Why Ac-CoA is not an optimal donor for modifying the dA,\nAcetyl-CoA cannot be the group donor as it does not contain the necessary complement of atoms matching the hypermodification. Multiple chemical steps and extraordinary chemistry would need to be invoked in order to break bonds in Ac-CoA in such a way as to carve out a nitrogen group. Previously published hypotheses regarding Mom function posited multiple enzymatic steps including radical chemistry and amidation to produce the mature modification. Here we show a single enzyme in the absence of any aminotransferase can accomplish the momylation reaction.\nHowever, we do appreciate the discussion from Reviewer 3 and Reviewer 2 above about what is driving co-substrate specificity for GNAT enzymes. As discussed above in our Response to Reviewer 2, an acetyl group does not have the correct stoichiometry for reconstituting 6-NcmdA in a single transfer step. We also emphasize again here that this is an interesting and open question in the field that we are hopeful will become clearer over time. To date, there are multiple examples of distinct GNAT family members that have convergently evolved the ability to utilize aminoacylated tRNAs as group donors targeting diverse substrates (discussed and referenced in the main text). We present here a new example of a GNAT family member following this pattern and, significantly, the first known and shown in vitro to modify dsDNA. There is a possibility that another GNAT enzyme might exist which uses acetyl-CoA to modify the N6 position of dA within the DNA duplex. This enzyme has not been discovered to our knowledge. In our response to Review 2 above, we discuss TcmA, the enzyme that acetylates N4 of cytidine in tRNA, so it would be mechanistically plausible from an enzyme standpoint for similar chemistry to take place in DNA. However, we note that ac4C is unpaired in tRNA and in the context of DNA, an acetyl group could cause steric clash with the cognate base-pair. Such a steric clash is mitigated in 6-NcmdA through greater degrees of freedom afforded by an additional carbon between N6 of dA and the planar carbonyl of the carbamoyl moiety. Another factor that needs to be considered here is the microbial arms race between bacteria and their viruses. The modification ac4C has been reported to undergo spontaneous deacetylation in vitro at room temperature at neutral pH21. Considering the extreme selection pressure phage are under by their hosts, a potentially more labile modification may not be advantageous for protecting against host defenses (e.g. restriction enzymes).\nwhy mom prefers dsDNA over ssDNA,\nThe preference for dsDNA is not unusual or unexpected. Mu is a dsDNA phage, and Mom is post-replicatively active (see main text). Many phage-encoded DNA modifying enzymes operate on dsDNA post-replicatively (e.g. T4-BGT). Furthermore, ssDNA would be only transiently available at the replication fork. As with understanding co-substrate preference, understanding the structural basis for the substrate preference can be explored in future work.\nwhy mom prefers SAS sequence motif,\nThis is also an open-ended question that has both mechanistic and biological aspects. We do not yet know what amino acids at the protein-DNA interface dictate sequence specificity. There is also a biological “why” here. Does the SAS sequence allow for maximum protection against multiple endonuclease-based antiphage systems targeting different sequences? Or does SAS sequence preference also impact the biology of the host, perhaps by overlapping with dcm? Does the SAS sequence allow for sufficient modification (~15-20%) of the Mu genome without negatively impacting the biophysical properties of the gDNA? These questions, while fascinating, are beyond the scope of the work presented here and do not impact our findings.\nwhy all three tRNA isoacceptors are capable as donors,\nThis result suggests that Mom contacts tRNA at the acceptor stem of Gly-tRNAGly/GCC, which is identical in sequence among all isoacceptors in E. coli. This result is also consistent with our observed dimensions of Mom. Unlike GlyRS, which must enforce a “proofreading” function of tRNA aminoacylation, Mom is not long enough to reach the anticodon loop. Put another way, while there may be a negative fitness cost to a mischarged tRNA finding its way into protein translation (hence GlyRS editing) there would be no cost to Mu for using glycine from any tRNA for modifying DNA. From standpoint of an opportunistic virus, it is advantageous to harvest glycine from all sources available.\nwhy the first 20 amino acids are important for activity,\nComparison of the structures of Mom to the structure of FemX (Fig. 4d-f) and our modeling experiments (Supplementary Fig. 40) suggest these residues could be important for tRNA binding.\nwhy mom homo-dimeric state is important for the momylation,.\nThis is an interesting question to which there is no obvious answer. The literature reports that GNATs enzymes can be found as monomers, five different types of dimers, trimers, tetramers, hexamers, and dodecamers13,22–29. Dimers have been noted to be the most common form of GNATs observed30. We are careful not to attach more mechanistic significance to this observation and look forward to addressing the mechanistic role of monomer versus dimer in future work.\nsince the authors have the structure model on hand and preform serials of dockings, please at least try to hypothesis/address these open questions from the structure perspective in the result or discussion.\nWe have complemented our structural comparisons with AlphaFold3 modeling during the revision. Both approaches agree on the relative orientation of tRNA and DNA. Moreover, we have mapped the active site, and using quantum chemical approaches, deepened our understanding of the reaction dynamics. However, given the multistep nature of the momylation reaction, we remain cautious about more detailed speculations, such as assigning exact roles to individual amino acid residues within the active site.\n2, please describe what R111 and S124 function might be during catalysis, seems to me they are far from the potential glycyl transfer active site.\nUsing overlays with other GNATs and AlphaFold3, we have modeled putative sites where tRNA and DNA may interreact with Mom. We acknowledge that this system is complex, and we were careful not to claim that we had identified the regions of Mom that interact with tRNA and DNA (see page 7, lines 22-34 and Fig 3f). Without a more definitive understanding of how Mom interacts with tRNA and with DNA, a proposed mechanism would be highly speculative. If the binding sites differ from the model or if there are significant conformational changes during the reaction, any proposal may need to be revisited. Examining the specifics of the transfer mechanism warrants its own manuscript.\n3, I am curious if the authors have tested that if mom can use Ala-tRNAAla as donor or not.\nIn a mixture of tRNAs charged with all twenty proteinogenic amino acids (the activity assays using the PURExpress product, Supplementary Fig. 6), only glycine is utilized. In experiments described in our response to Reviewer 2 with alanyl-tRNA synthetase and an editing-deficient variant, C666A, we produced Ala-tRNAAla/TGC and mischarged Gly-tRNAAla/TGC and showed that neither is able to support DNA modification by Mom.\n4, In Supplementary figure 6, please also show the gel filtration run profile of mom Y28A and W48A, to confirm the mutants are indeed at monomer state.\nAs we did not observe a loss of activity for Y28A in vivo, we focused on W48A, which showed a pronounced reduction in activity. Purified W48A eluted as a monomer by size-exclusion chromatography (SEC) and exhibited only trace activity in vitro. These data have been added to the Supplementary Information (Supplementary Fig. 39). We thank the reviewer for pointing this out.\nThe quantum chemistry section on the uncatalyzed rearrangement of N6-glycyl adenine to methylated adenine in water has undergone drastic changes. First, we now consider the rearrangement reaction for both the neutral and cationic forms. We systematically scan the educt and product conformations to validate our intuition regarding the location of the low-energy states. We also performed more sophisticated calculations on the educts, products, and tricyclic intermediate in ORCA at the wb97X-D3-def2-SVP level (see Supplementary Fig. 31), as well as more calculations to locate transition states/saddle points.\n1) More details on the computational methods are needed, in particular on the convergence of the basis set employed, which seems to me relatively small for this system.\nAs additional checks, we have carried out calculations in GAUSSIAN and in GAMESS, with relatively simple base sets of different complexities (down to 3-21G), with either pure RHF or B3LYP hybrid calculations. The qualitative conclusions that\n• overall the reaction is favorable,\n• among the educts, the trans-WC configuration has lowest energy, and\n• the tricylic rearrangement intermediate has a relatively low energy that is however higher than the energy of educts and products\nare all independent of the exact method used for calculation (see the figure below).\nUltimately, we performed more sophisticated calculations using the wb97X-D3-def-SVP hybrid method with a hybrid exchange and dispersion correction and CPMC correction. Furthermore, we switched from calculating energies to calculating chemically more relevant Gibbs free energies. To avoid cluttering the manuscript with redundant results, we only report the highest-level wb97X-D3-def-SVP CPCM calculations. Additionally, we confirmed that the rearrangement reaction is favorable for the neutral form, but also the cationic form of N6-glycyl adenine.\n2) Details on the structures of reactants, products and educts need to be reported in the supplementary information as pdb, cif or xyz files, in order to make the results reported reproducible.\nWe now offer a TAR archive containing all the coordinates of the low-energy reactant and product states, the tricyclic intermediate, and saddle points. We provide .xyz files and the original orca output files, so that interested readers can also verify (a) geometric convergence, (b) vibrational modes, including the presence of only one imaginary (negative) frequency, and (c) agreement of the imaginary normal modes with the proposed reaction pathway.\n3) Quantum effects can be important for estimating the energy and transition states in DNA hydrogen bonds and tautomerisation probabilities. This has been discussed in several recent papers by various groups worldwide. The authors should comment on the possible role of proton delocalisation and, potentially, tunnelling in the systems reported.\nThe referee’s concern was relevant to the previous version of the manuscript. That version had an incorrect transition state that was not fully converged or numerically stable. It was also not analyzed for normal modes and could reasonably be expected to be affected by a tunneling possibility for hydrogen atoms. It is now evident that the profile of the rearrangement reaction is more intricate. However, it is now clear that proton transfer reactions are not rate limiting either in the neutral or the cationic state. Therefore, we do not expect tunneling to be a significant factor in the calculations.\n4) It is not clear if dispersion corrections have been included in the calculations. If they have not been included, could the authors please discuss the motivation of their choice?\nDispersion corrections were not included in the original version of the manuscript but have now been added. However, they are small and do not affect the chemical conclusions from the quantum chemical data.\n5) Details on the energy and force convergence in the geometry optimisations and TS searches need to be reported. RHF geometries are likely to not reflect the B3LYP geometries, therefore I would not take the barrier heights for these XC functionals as anything more than guesswork.\nThe referee was right regarding the transition state, which we noted to be unstable, and which indeed had a convergence problem. All structures reported in the current manuscript meet the ORCA criteria for convergence (as noted in the manuscript). Specifically, these are: All minima and saddle points/transition states met the ORCA criteria for convergence of geometry optimizations, i.e root mean square gradient (RMSG) below 1 * 10-4 Eh/au, maximum gradient (MaxG) below 3 * 10-4 Eh/au, energy change (TolE) below 5 * 10-6 Eh, RMS displacement (RMSD) below 2 * 10-3 au, and maximum displacement (MaxD) below 4 * 10-3 au, where Eh is 1 Hartree (~627.5 kcal/mol), and au is one atomic unit (one Bohr, 0.52 Å). Of note, we noted that ORCA does not output Gibbs free energies when convergence criteria are not met, even if their calculation is requested. Hence, the mere fact that free energies could be calculated suggests that the criteria are met. Since the ORCA output is now provided as a Suppl., the interested reader can directly verify convergence."}, {"round": 2, "reviewer_comments": {"reviewer_2": "This reviewer appreciates that authors have added large amount of data to support a tRNA dependent dA DNA modification. Most of my concerns have been addressed. However, this reviewer still emphasizes that adding a GlyRS mutant, defective in Gly-tRNAGly formation, to the in vitro DNA modification reaction, would be valuable to strengthen this work. This addition would demonstrate that it is the aminoacylation activity of GlyRS that mediates the phage specific DNA modification.\nMinor points:\n1. The present abstract was significantly shortened compared with that of the first version. However, it lacks any description about background and unresolved scientific questions.\n2. Abstract, R111A still not corrected to R111 despite it was claimed in the rebuttal letter.\n3. Page 8, “the residues Y28A and W48A…”not corrected, despite it was claimed in the rebuttal letter.\n4. Figure S13 is of poor resolution, making impossible to get clear spectrometry data.", "reviewer_3": "I appreciate the authors for addressing the suggestions I made. I have no further concerns regarding the publication of this article. Thank you, and congratulations to the authors.", "reviewer_4": "The authors have answered my questions."}, "author_response": "We addressed Reviewer 2’s point with our activity assays by engineering tRNA through precise incorporation of 2'-deoxyadenosine at the 3' end using CCA2, an enzyme we had not worked with prior to this revision (see Figure 2a and 2b, Supplementary Fig. 17, and associated text). We demonstrated experimentally that WT GlyRS charges tRNA ending in a 2'-deoxyadenosine, confirming the production of the glycyl-adenylate intermediate necessary for tRNA charging. However, as shown in Figure 2b, we observed that this tRNA ending in a 2'-deoxyadenosine could not support momylation, allowing us to conclude that aminoacylation activity of GlyRS and a native terminal riboadenosine are necessary for momylation. The experiment indicates that glycyl-adenylate, which is formed under our reaction conditions, is not sufficient for momylation. The simplest explanation is that charged tRNA is the group donor in the momylation reaction. Furthermore, our observations are consistent with what is known about multiple, independently evolved GNAT enzymes that use charged tRNA as a group donor1,2. Additional arguments against the formal possibility of glycyl-adenylate being the group donor are presented in the manuscript’s main text. We contend at this time that our chemoenzymatic approach for “mutating” the tRNA acceptor stem, which was originally suggested by Reviewer 2 and employed for this revision, is more elegant and informative for Mom function than working with an unspecified and/or poorly characterized mutant of GlyRS3 .\nMinor points:\nAgain, thank you for your close reading.\n1. The present abstract was significantly shortened compared with that of the first version. However, it lacks any description about background and unresolved scientific questions.\nWe shortened the abstract to comply with word count requirements.\n2. Abstract, R111A still not corrected to R111 despite it was claimed in the rebuttal letter.\nWe apologize for our oversight. This typo has been corrected.\n3. Page 8, “the residues Y28A and W48A…”not corrected, despite it was claimed in the rebuttal letter.\nWe apologize for our oversight. This typo has been corrected.\n4. Figure S13 is of poor resolution, making impossible to get clear spectrometry data.\nThe resolution of Supplementary Fig. 13 is of high quality on our end and does not lose resolution upon zooming into the image. We re-exported the data and include it below. Should our manuscript be accepted, we will of course provide the raw data files as well."}, {"round": 3, "reviewer_comments": {"reviewer_2": "The authors have addressed all my concerns. The paper is now ready for publication."}, "author_response": ""}]

Decision letter