# Rev1, Rev3, or Rev7 siRNA Abolishes Ultraviolet Light-Induced Translesion Replication in HeLa Cells: A Comprehensive Study Using Alkaline Sucrose Density Gradient Sedimentation

Authors: Jun Takezawa; Yukio Ishimi; Naomi Aiba; Kouichi Yamada
Journal: Journal of Nucleic Acids
 (2010)

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## Abstract

When a replicative DNA polymerase stalls upon encountering a lesion on the template strand, it is relieved by other low-processivity polymerase(s), which insert nucleotide(s) opposite the lesion, extend by a few nucleotides, and dissociate from the 3′-OH. The replicative polymerase then resumes DNA synthesis. This process, termed translesion replication (TLS) or replicative bypass, may involve at least five different polymerases in mammals, although the participating polymerases and their roles have not been entirely characterized. Using siRNAs originally designed and an alkaline sucrose density gradient sedimentation technique, we verified the involvement of several polymerases in ultraviolet (UV) light-induced TLS in HeLa cells. First, siRNAs to Rev3 or Rev7 largely abolished UV-TLS, suggesting that these 2 gene products, which comprise Polζ, play a main role in mutagenic TLS. Second, Rev1-targeted siRNA also abrogated UV-TLS, indicating that Rev1 is also indispensable to mutagenic TLS. Third, Polη-targeted siRNA also prevented TLS to a greater extent than our expectations. Forth, although siRNA to Polι had no detectable effect, that to Polκ delayed UV-TLS. To our knowledge, this is the first study reporting apparent evidence for the participation of Polκ in UV-TLS.


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## Body

## 1. Introduction

Multiple systems have evolved to manage the genomic photoproducts generated by harmful UV light. One such system is nucleotide excision repair (NER), which eliminates photoproducts from DNA strands by dual incision on both sides of a damaged base. The NER system cannot, however, remove all UV-damaged bases. When a replicative DNA polymerase stalls upon encountering a residual photoproduct on the template strand, it is relieved by other low-processivity polymerase(s), which incorporate nucleotide(s) opposite the lesion, extend by a few nucleotides and dissociate from the 3′-OH. The replicative polymerase then resumes DNA synthesis. This process, termed translesion replication (TLS) or replicative bypass (reviewed in [1]), is also one of the subtle systems that have evolved for the management of genomic photoproducts.UV-C (100–290 nm wavelength) induces 2 main photoproducts [2]: the more frequent cyclobutane pyrimidine dimer (CPD) and the several-fold lower pyrimidine-pyrimidone (6-4) photoproduct ((6-4)PP). cis-syn CPD, a predominant form of the multiple configurations, contains 2 adjacent pyrimidines that are covalently linked in parallel. Although the frequency of CPD varies with nucleotide composition, a ratio of T-T to C-T to T-C to C-C of 68 : 13 : 16 : 3 is obtained from UV-irradiated plasmid DNA. Cytosines within CPD are unstable, and are deaminated to uracil or 5-methylcytosine, and further deaminated to thymine [3]. The helical distortion caused by CPD is so inconspicuous that almost half of the lesions remain unrepaired by NER, even 6 hours after UV irradiation in the case of CHO cells [2].The (6-4)PPs from T-C, C-C, and, less frequently, T-T sequences are detected in UV-irradiated DNA whereas that of C-T are not. In (6-4)PP, linkage between C-6 of one pyrimidine and C-4 of the adjacent pyrimidine cause the 2 bases to be in nearly perpendicular position. Consequently, formation of this lesion causes a major distortion in the double helix. NER preferentially removes (6-4)PP more rapidly than it removes CPD from the genome in human and rodent cells [4].At least 5 mammalian DNA polymerases are suggested to be implicated in UV-induced TLS: Polsη, ι, ζ, κ, and Rev1, all of which belong to the Y family except for Polζ (B family) (reviewed in [1, 5, 6]). However, the participating polymerases and their roles have not been entirely characterized.Patients with the autosomal recessive disorder, xeroderma pigmentosum variant (XP-V), have a predisposition to skin cancer, and XP-V cells demonstrate hypermutability after UV irradiation (reviewed in [7]). The defective gene in XP-V encodes Polη, which was first purified from a HeLa cell extract as an activity that complements TLS defect in XP-V cell extract [8]. Human Polη catalysed DNA synthesis past TT-CPD very efficiently and in a relatively accurate manner, as demonstrated by the lesion-bypass assay [7, 9]. When template DNA contained a (6-4)TT-PP, Polη incorporated one (random) nucleotide opposite the first thymine and another nucleotide opposite the second thymine of the lesion, but rarely continued across the lesion [7, 9].Human Polη was also identified via a search for the homolog of yeast Saccharomyces cerevisiae  Rad30 gene, which encodes an error-free bypass protein [10]. Various XP-V causative mutations have been found in the Polη gene, hRAD30A, of XP-V patients [10, 11].Polι (RAD30B) is the other mammalian homolog of yeast Polη, isolated by a similar approach [12]. In contrast to Polη, Polι is less efficient and less accurate [13].Polκ was obtained by cloning of a human homolog of the E. coli  dinB gene, encoding DNA Pol IV [14]. Polκ was reported to be unable to bypass either CPD or (6-4)PP [15, 16].Originally, Rev1, 3, and 7 were cloned fromS. cerevisiae isolates, in which the frequency of UV-induced reversion from cyc1 mutations was reduced [17]. Human and mouse homologs (Rev1, 3, 7 gene) were later isolated [18–20]. Polζ is a comprex of the Rev3 and Rev7 gene products, which act as the catalytic and regulatory subunit, respectively. Yeast Polζ is shown to be responsible for 98% of UV-induced base substitutions and 90% of frameshift mutations, in addition to spontaneous mutations [21]. Nonetheless, yeast Polζ itself was revealed to be too faithful to incorporate opposite CPD. Instead, it can efficiently extend from a matched or mismatched 3′-end (reviewed in [5, 6]). Human or mouse Polζ is assumed to have similar enzymatic properties to that of yeast, because several lines of antisense RNA expression or siRNA knockdown experiments in human or mouse cells have proven that Polζ is involved in mutagenic TLS [22–24].Yeast and human Rev1 encode highly specialized DNA polymerases that preferentially insert a C residue opposite an abasic site in the template. This deoxycytidyl (dC) transferase activity is, however, unlikely to be required for UV-TLS, judging by observation in a yeastrev1-1 mutant strain [25], which retains much of its dC transferase activity, but has a missense mutation (G193R) in the N-terminal BRCT domain. Rev1 protein also contains ubiquitin-binding motifs (UBMs) that interact with monoubiquitinated PCNA (a DNA polymerase sliding clamp) [26]. In the downstream C-terminal region, Rev1 interacts not only with Rev7 but also with other bypass-polymerases [27], suggesting that Rev1 acts as a mediator and physical bridges between PCNA and Polζ.Following DNA damage, such as that caused by UV and MMS, monoubiquitins are conjugated to PCNA arrested at the lesion-site by RAD6/RAD18 and recruit bypass-polymerases [28, 29]. In addition to Rev1, Pols η, ι, and κ possess UBMs and physically interact with PCNA [1, 5, 6]. Stalled replicative Polδ is replaced, in turn, with one of these bypass polymerases bound on the PCNA by yet unknown “polymerase switching” mechanisms (reviewed in [1, 30]).Translesion replication is typically detected with an alkaline sucrose density gradient centrifugation (ASDG) technique. Pulse-labelled replication products are smaller in UV-irradiated XP-V cells than in unirradiated cells; however, on prolonged incubation, the replication products in the irradiated cells eventually attain a high molecular weight similar to that in unirradiated cells. This conversion is interpreted that DNA synthesis is temporarily retarded by UV photoproducts, and then continues beyond the lesion, leaving a gap that is subsequently sealed [31]. The initial size of the newly synthesized DNA is approximately equal to the average distance between lesions in the template strands [32]. This means the gaps in the newly synthesized DNA are opposite the photoproducts [33]. Therefore, sealing of the gaps, by translesion or other postreplication repair mechanisms, can be observed by monitoring the molecular weight of labelled DNA.Using a modified ASDG technique [34], we precisely detected the elongation of pulse-labelled replication products in the irradiated XP-V cells, showing that UV-TLS is delayed in the cells, but not completely abolished [35]. The marginal TLS is markedly prevented by caffeine at millimolar concentrations, as Lehmann et al. pointed out [31], and by proteasome inhibitors as well (unpublished results). In contrast, these agents do not retard UV-TLS in normal diploid cells. To know more about the inefficient polymerase(s) in vivo, we added specific DNA polymerase inhibitors. Butylphenyldeoxyguanosine (BuPGdR) inhibited TLS in XP-V cells [35], suggesting that Polζ may be involved in this Polη-independent bypass.We recently reported that caffeine or proteasome inhibitors inhibit UV-TLS also in human cancer cells [36], and that, similar to XP-V cells, UV-TLS was much slower than in normal cells. These results suggested that UV bypass in cancer cells is predominantly of the Polη-independent type. Therefore, we expected that Polζ plays a major part in UV-TLS in cancer cells. Although Polη exists in normal quantity in these cells, it was supposed to be inactivated by some reasons. We explored these hypotheses here.


## 2. Materials and Methods

### 2.1. Design of siRNAs

All siRNAs duplexes were synthesized by JBioS (Japan) (Table1). We designed the sequences for siRNA according to the JBioS guide (http://www.JBioS.co.jp/RNAiselect.htm). Selection of target sequence for each siRNA proceeds as follows: first, an AAG or AAC sequence is found at least 75 nucleotides (nt) downstream from the start codon; the AAG (or AAC) and the following 18 nt sequence (N18) are picked up. Ideally, the 2 nt following the N18 are TT, TN, NT, or AA. GC content of N18 is recommended to be about 40% to 50%. If GC content is less than 30%, or greater than 70%, avoid the sequence. The N18 had better be AT-rich in the 3′ half and especially at the 3′ end (2 nt).Table 1
siRNAs used.
siRNASense strand(a),(b)Antisense strand(a),(b)Prevention of UV-TLS (result)siRev3-AgcuuuacaugagauacaaaTTuuuguaucucauguaaagcTTsignificantsiRev3-BgacaguuuucagucaagauTTaucuugacugaaaacugucTTsignificantsiRev3-CgagguaugauccugauauuTTaauaucaggaucauaccucTTsignificantsiRev3-DguauugacuuaugucggauTTauccgacauaagucaauacTTsignificantsiRev3cont-A(6nt mismatches)gguuugaguaauguacgauTTaucguacauuacucaaaccTTno effectsiRev3cont-B (4nt mismatches)ggauugaguuauguacgauTTaucguacauaacucaauccTTno effectsiRev3cont-C (2nt mismatches)guauugaguuauguccgauTTaucggacauaacucaauacTTno effectsiRev7-AgauccaggucaucaaggauTTauccuugaugaccuggaucTTpartialsiRev7-BgaugcagcuuuacguggaaTTuuccacguaaagcugcaucTTsignificantsiRev7-CcacugucugucucaaauacTTguauuugagacagacagugTTsignificantsiRev7cont-AgaugcagguuuacgucgaaTTuucgacguaaaccugcaucTTno effectsiRev1-AsiRev1-BsiRev1-CsiRev1-DsiRev1cont-EcacauauuauugccacaaaTTgaagauugaaacggaaaauTTccuucagacugcaauuuuaTTgugugaauugacugaguuuTTccuucacaccgcaacguuaTTuuuguggcaauaauaugugTTauuuuccguuucaaucuucTTuaaaauugcagucugaaggTTaaacucagucaauucacacTTuaacguugcggugugaaggTTsignificantsignificantsignificantsignificantlittle to no effectsiPolη-4siPolη-AsiPolη-BsiPolηcont-AuaaaccuugugcaguuguaTTgaaguuauguccagaucuuTT   gcuucgcuuucaucucuuaTT   uaaaccuggugcagucguaTTuacaacugcacaagguuuaTTaagaucuggacauaacuucTTuaagagaugaaagcgaagcTTuacgacugcaccagguuuaTTpartialsignificantsignificantno effectsiPolι-AsiPolι-5gccucauacagugagauuaTTaaguguccacaguugguauTTuaaucucacuguaugaggcTTauaccaacuguggacacuuTTno effectno effectsiPolκ-AsiPolκ-BsiPolκ-CsiPolκcont-BgagaaaauuaacaaaauuaTTgaauaaaccaaauggacaaTT cuguuaccauuaaguugaaTT   gagaaaaguaacaagcuuaTTuaauuuuguuaauuuucucTTuuguccauuugguuuauucTTuucaacuuaaugguaacagTTuaagcuuguuacuuuucucTTpartialpartialconsiderablelittle to no effect(a)Small letters mean ribonucleotides, and large letters mean deoxyribonucleotides.(b)Mismatched bases are illustrated in bold and italic letters.


### 2.2. HeLa Cell Culture and siRNA Treatment

HeLa cells were maintained in monolayers in Dulbecco’s modified Eagle’s medium (D’MEM) supplemented with 10% fetal calf serum (FCS) (“normal” medium), trypsinized and seeded into culture dishes (2 × 105 cells/ϕ 60 mm dish). About 6–8 hours later, the cells were treated with micelles of siRNA and OligofectAMINE (Invitrogen), formed according to the manufacturer’s protocol, except Opti-MEM was replaced with D’MEM. The siRNA concentration used in RT-PCR analysis and western blot analysis was 5 nM.


### 2.3. UV Irradiation and Translesion Replication

Forty hours after siRNA addition, HeLa cells were exposed to UV light (10 J/m2) from a germicidal lamp (Toshiba GL15) at 0.6 J/m2 per s. After 30 minutes in culture, the medium was changed to labeling medium consisting of D’MEM supplemented with 10% FCS and 10 μCi/mL of [U-14C]thymidine (Moravek MC267, 470 mCi/mmol). UV-irradiated cells were pulse-labelled for 1 hour, while nonirradiated cells were labelled for 30 minutes. The medium was changed to normal medium, and the cells were chased for 5 hours. These cells were harvested by trypsinisation and examined by ASDG [34].


### 2.4. Alkaline Sucrose Density Gradient Centrifugation (ASDG)

Cells (about 1 × 105 in 50 μL PBS) were gently layered onto 50 μL of 1% sucrose in PBS, which was overlaid on 100 μL of lysis solution (0.6 M KOH, 2.0 M  KCl, 10 mM EDTA, and 1% N-lauroylsarcosine), which was placed on top of a 4.35 mL alkaline 5–20% sucrose gradient (0.3 M KOH, 2.0 M  KCl, 1 mM EDTA, and 0.1% N-lauroylsarcosine) with 0.4 mL of alkaline 80% sucrose as a cushion at the bottom. The gradient was centrifuged at 6,000 rpm (4,320 × g) for 15.6 hours at 15°C in a Beckman SW 50.1 rotor. The gradient was fractionated onto 30 circles of no. 17 paper (Whatman). The paper circles were dried, immersed in cold 5% trichloroacetic acid for 10 minutes, washed 3 times with ethanol and once with acetone, and dried; radioactivity was then measured. As a molecular weight marker, [14C]-labelled T4 DNA phage particles were placed on the lysis layer and sedimented in a parallel run. The approximate fragment length of each fraction was estimated on the basis of the position of the T4 DNA marker and that of E. coli DNA and adjusted by sucrose density curve [34]. Average fragment length (in megabase(Mb)) of each profile is shown in Figures as fragment length of the median fraction [35]. (Median fraction is the middle fraction that separates the higher half of the profile from the lower half.)


### 2.5. RT-PCR

Total RNA was isolated using an RNeasy spin column (QIAGEN). Oneμg of total RNA was treated with DNase I (Invitrogen), reverse-transcribed using SuperScript II (Invitrogen) with random hexamers or PrimeScript II (TaKaRa) with oligo(dT) primers, followed by treatment with E. coli RNase H (Invitrogen). The PCR mixture contained cDNA templates, dNTPs, rTaq Pol (TOYOBO), anti-Taq (TOYOBO), and appropriate primers. Primers to assess knockdown of each gene were as follows: Rev3, 5′-GGAACGTCAACAGGAGCAAC-3′ and 5′-GGAGCAAATCCAACACCTGC-3′; Rev7, 5′-TGCTGTCCATCAGCTCAGAC-3′ and 5′-AGAGCACTTGGAATCAGGGC-3′; Rev1, 5′-CTCCTGCAGAGAAACCCCTG-3′ and 5′-ACAAGCACTTATGGCACAGCT-3′; Polη, 5′-CCCAGGCAACTACCCAAAAC-3′ and 5′-GGGCTCAGTTCCTGTACTTTG-3′; Polι, 5′-ATGATCAAGTGTTGCCCACAC-3′ and 5′-ACATGACCCGACACAGTCAC-3′; Polκ, 5′-AGACAAGAATACCGCCAGCC-3′ and 5′-AGGAAGGATTATTGCACTTGCC-3′; GAPDH, 5′-ACCACAGTCCATGCCATCAC-3′ and 5′-TCCACCACCCTGTTGCTGTA-3′. All PCR reactions were carried out for 27 cycles, with the exception of GAPDH (25 cycles). PCR products were subjected to a MultiNA microtip electrophoresis DNA/RNA analyzer (Shimadzu Biotech).


### 2.6. Western Blotting

Cells were rinsed with PBS, lysed with 1 × SDS-PAGE loading buffer and collected by scraping. Cell lysate was boiled for 10 minutes, sonicated for 30 seconds and centrifuged at 20,000 × g for 10 minutes. The supernatant was used for western blotting after measuring protein content by Bradford method. The supernatant (60μg protein) was separated on 5–20% (Rev7, Rev1 and Polι) or 3–10% (the rest) SDS-polyacrylamide gels (Wako) and transferred onto Immobilon-PSQ membranes (Millipore). The membranes were incubated with antibodies to bypass-polymerases (Rev1, Santa Cruz Biotechnology sc-48806; Rev7, BD Transduction Laboratories 612266; Polη, Santa Cruz Biotechnology sc-17770; Polι, Abnova H00011201-M01; Polκ, a kind gift from Dr. Tomoo Ogi, Nagasaki University), and then with horseradish peroxidase-conjugated secondary antibodies (anti mouse IgG, DAKO PO-0447; anti-rabit IgG, DAKO PO-0448). Antibodies were diluted with CanGetSignal (TOYOBO) to enhance immunoreactivity. The signals were developed with ECL-Plus (GE Healthcare), and blots were stripped for reprobing with anti α-tubulin antibody (Sigma T5168).


## 2.1. Design of siRNAs

All siRNAs duplexes were synthesized by JBioS (Japan) (Table1). We designed the sequences for siRNA according to the JBioS guide (http://www.JBioS.co.jp/RNAiselect.htm). Selection of target sequence for each siRNA proceeds as follows: first, an AAG or AAC sequence is found at least 75 nucleotides (nt) downstream from the start codon; the AAG (or AAC) and the following 18 nt sequence (N18) are picked up. Ideally, the 2 nt following the N18 are TT, TN, NT, or AA. GC content of N18 is recommended to be about 40% to 50%. If GC content is less than 30%, or greater than 70%, avoid the sequence. The N18 had better be AT-rich in the 3′ half and especially at the 3′ end (2 nt).Table 1
siRNAs used.
siRNASense strand(a),(b)Antisense strand(a),(b)Prevention of UV-TLS (result)siRev3-AgcuuuacaugagauacaaaTTuuuguaucucauguaaagcTTsignificantsiRev3-BgacaguuuucagucaagauTTaucuugacugaaaacugucTTsignificantsiRev3-CgagguaugauccugauauuTTaauaucaggaucauaccucTTsignificantsiRev3-DguauugacuuaugucggauTTauccgacauaagucaauacTTsignificantsiRev3cont-A(6nt mismatches)gguuugaguaauguacgauTTaucguacauuacucaaaccTTno effectsiRev3cont-B (4nt mismatches)ggauugaguuauguacgauTTaucguacauaacucaauccTTno effectsiRev3cont-C (2nt mismatches)guauugaguuauguccgauTTaucggacauaacucaauacTTno effectsiRev7-AgauccaggucaucaaggauTTauccuugaugaccuggaucTTpartialsiRev7-BgaugcagcuuuacguggaaTTuuccacguaaagcugcaucTTsignificantsiRev7-CcacugucugucucaaauacTTguauuugagacagacagugTTsignificantsiRev7cont-AgaugcagguuuacgucgaaTTuucgacguaaaccugcaucTTno effectsiRev1-AsiRev1-BsiRev1-CsiRev1-DsiRev1cont-EcacauauuauugccacaaaTTgaagauugaaacggaaaauTTccuucagacugcaauuuuaTTgugugaauugacugaguuuTTccuucacaccgcaacguuaTTuuuguggcaauaauaugugTTauuuuccguuucaaucuucTTuaaaauugcagucugaaggTTaaacucagucaauucacacTTuaacguugcggugugaaggTTsignificantsignificantsignificantsignificantlittle to no effectsiPolη-4siPolη-AsiPolη-BsiPolηcont-AuaaaccuugugcaguuguaTTgaaguuauguccagaucuuTT   gcuucgcuuucaucucuuaTT   uaaaccuggugcagucguaTTuacaacugcacaagguuuaTTaagaucuggacauaacuucTTuaagagaugaaagcgaagcTTuacgacugcaccagguuuaTTpartialsignificantsignificantno effectsiPolι-AsiPolι-5gccucauacagugagauuaTTaaguguccacaguugguauTTuaaucucacuguaugaggcTTauaccaacuguggacacuuTTno effectno effectsiPolκ-AsiPolκ-BsiPolκ-CsiPolκcont-BgagaaaauuaacaaaauuaTTgaauaaaccaaauggacaaTT cuguuaccauuaaguugaaTT   gagaaaaguaacaagcuuaTTuaauuuuguuaauuuucucTTuuguccauuugguuuauucTTuucaacuuaaugguaacagTTuaagcuuguuacuuuucucTTpartialpartialconsiderablelittle to no effect(a)Small letters mean ribonucleotides, and large letters mean deoxyribonucleotides.(b)Mismatched bases are illustrated in bold and italic letters.


## 2.2. HeLa Cell Culture and siRNA Treatment

HeLa cells were maintained in monolayers in Dulbecco’s modified Eagle’s medium (D’MEM) supplemented with 10% fetal calf serum (FCS) (“normal” medium), trypsinized and seeded into culture dishes (2 × 105 cells/ϕ 60 mm dish). About 6–8 hours later, the cells were treated with micelles of siRNA and OligofectAMINE (Invitrogen), formed according to the manufacturer’s protocol, except Opti-MEM was replaced with D’MEM. The siRNA concentration used in RT-PCR analysis and western blot analysis was 5 nM.


## 2.3. UV Irradiation and Translesion Replication

Forty hours after siRNA addition, HeLa cells were exposed to UV light (10 J/m2) from a germicidal lamp (Toshiba GL15) at 0.6 J/m2 per s. After 30 minutes in culture, the medium was changed to labeling medium consisting of D’MEM supplemented with 10% FCS and 10 μCi/mL of [U-14C]thymidine (Moravek MC267, 470 mCi/mmol). UV-irradiated cells were pulse-labelled for 1 hour, while nonirradiated cells were labelled for 30 minutes. The medium was changed to normal medium, and the cells were chased for 5 hours. These cells were harvested by trypsinisation and examined by ASDG [34].


## 2.4. Alkaline Sucrose Density Gradient Centrifugation (ASDG)

Cells (about 1 × 105 in 50 μL PBS) were gently layered onto 50 μL of 1% sucrose in PBS, which was overlaid on 100 μL of lysis solution (0.6 M KOH, 2.0 M  KCl, 10 mM EDTA, and 1% N-lauroylsarcosine), which was placed on top of a 4.35 mL alkaline 5–20% sucrose gradient (0.3 M KOH, 2.0 M  KCl, 1 mM EDTA, and 0.1% N-lauroylsarcosine) with 0.4 mL of alkaline 80% sucrose as a cushion at the bottom. The gradient was centrifuged at 6,000 rpm (4,320 × g) for 15.6 hours at 15°C in a Beckman SW 50.1 rotor. The gradient was fractionated onto 30 circles of no. 17 paper (Whatman). The paper circles were dried, immersed in cold 5% trichloroacetic acid for 10 minutes, washed 3 times with ethanol and once with acetone, and dried; radioactivity was then measured. As a molecular weight marker, [14C]-labelled T4 DNA phage particles were placed on the lysis layer and sedimented in a parallel run. The approximate fragment length of each fraction was estimated on the basis of the position of the T4 DNA marker and that of E. coli DNA and adjusted by sucrose density curve [34]. Average fragment length (in megabase(Mb)) of each profile is shown in Figures as fragment length of the median fraction [35]. (Median fraction is the middle fraction that separates the higher half of the profile from the lower half.)


## 2.5. RT-PCR

Total RNA was isolated using an RNeasy spin column (QIAGEN). Oneμg of total RNA was treated with DNase I (Invitrogen), reverse-transcribed using SuperScript II (Invitrogen) with random hexamers or PrimeScript II (TaKaRa) with oligo(dT) primers, followed by treatment with E. coli RNase H (Invitrogen). The PCR mixture contained cDNA templates, dNTPs, rTaq Pol (TOYOBO), anti-Taq (TOYOBO), and appropriate primers. Primers to assess knockdown of each gene were as follows: Rev3, 5′-GGAACGTCAACAGGAGCAAC-3′ and 5′-GGAGCAAATCCAACACCTGC-3′; Rev7, 5′-TGCTGTCCATCAGCTCAGAC-3′ and 5′-AGAGCACTTGGAATCAGGGC-3′; Rev1, 5′-CTCCTGCAGAGAAACCCCTG-3′ and 5′-ACAAGCACTTATGGCACAGCT-3′; Polη, 5′-CCCAGGCAACTACCCAAAAC-3′ and 5′-GGGCTCAGTTCCTGTACTTTG-3′; Polι, 5′-ATGATCAAGTGTTGCCCACAC-3′ and 5′-ACATGACCCGACACAGTCAC-3′; Polκ, 5′-AGACAAGAATACCGCCAGCC-3′ and 5′-AGGAAGGATTATTGCACTTGCC-3′; GAPDH, 5′-ACCACAGTCCATGCCATCAC-3′ and 5′-TCCACCACCCTGTTGCTGTA-3′. All PCR reactions were carried out for 27 cycles, with the exception of GAPDH (25 cycles). PCR products were subjected to a MultiNA microtip electrophoresis DNA/RNA analyzer (Shimadzu Biotech).


## 2.6. Western Blotting

Cells were rinsed with PBS, lysed with 1 × SDS-PAGE loading buffer and collected by scraping. Cell lysate was boiled for 10 minutes, sonicated for 30 seconds and centrifuged at 20,000 × g for 10 minutes. The supernatant was used for western blotting after measuring protein content by Bradford method. The supernatant (60μg protein) was separated on 5–20% (Rev7, Rev1 and Polι) or 3–10% (the rest) SDS-polyacrylamide gels (Wako) and transferred onto Immobilon-PSQ membranes (Millipore). The membranes were incubated with antibodies to bypass-polymerases (Rev1, Santa Cruz Biotechnology sc-48806; Rev7, BD Transduction Laboratories 612266; Polη, Santa Cruz Biotechnology sc-17770; Polι, Abnova H00011201-M01; Polκ, a kind gift from Dr. Tomoo Ogi, Nagasaki University), and then with horseradish peroxidase-conjugated secondary antibodies (anti mouse IgG, DAKO PO-0447; anti-rabit IgG, DAKO PO-0448). Antibodies were diluted with CanGetSignal (TOYOBO) to enhance immunoreactivity. The signals were developed with ECL-Plus (GE Healthcare), and blots were stripped for reprobing with anti α-tubulin antibody (Sigma T5168).


## 3. Results

### 3.1. Effect of siRNAs to Rev3 or Rev7 on HeLa UV-TLS

We selected 4 target sites from the human Rev3 mRNA sequence (NCBI locus: NM_002912), according to the JBioS guide. All the Rev3 siRNAs (Table1) effectively knocked down expression of Rev3 (Figure 1(a)) and, at 5 nM, abolished UV-TLS in HeLa cells (Figure 1(b)). Replication products immediately after UV irradiation were sedimented as a sharp peak, illustrated by a thin line in Figure 1(b), slightly larger in size than the T4 phage DNA marker. When only Oligofectamine-treated cells were chased in normal medium for 5 hours, the products joined to form larger DNA with lengths in the order of megabases (Mb), illustrated by a thick line. In Rev3 siRNA-transfected cells, the products remain in smaller size, as depicted by a thin line with open marks, demonstrating these siRNAs prevent UV-TLS (Figure 1(b)).Efficient knockdown by Rev3 siRNAs and their effects on UV-induced TLS in HeLa cells (ASDG profiles of replication products). (a) Efficiency of knockdown on Rev3 expression (RT-PCR analysis); (b) Effects of various Rev3 siRNAs; (c) Effects of Rev3 control siRNAs; (d) Dose-response of siRev3-D; (e) Effect of various Rev3 siRNAs (no UV control). Twenty-four hours after Rev3 siRNA transfection, total RNA was isolated and Rev3 RNA was quantified by RT-PCR. Results were shown in MultiNA gel images and the expression level was presented under the panel (a). Forty hours after Rev3 siRNA transfection, cells were UV-irradiated (10 J/m2), incubated in normal medium for 30 minutes, pulse-labelled with 10 μCi/mL of [14C]thymidine for 1 hour, washed twice with PBS, and incubated for 5 hours at 37°C in normal medium (b, c, d). siRev3cont-A, 6 nt mismatches; siRev3cont-B, 4 nt mismatches; siRev3cont-C, 2 nt mismatches (c). Forty hours after Rev3 siRNA transfection, cells were not UV-irradiated, pulse-labelled with 10 μCi/mL of [14C]thymidine for 30 minutes, washed twice with PBS, and incubated at 37°C in normal medium for 1 hour (e). Some of these profiles overlap (c, d, e). Sedimentation is from right to left. The arrow indicates the position of T4 phage DNA (166 kb, i.e., approximately 5.5 × 107 Da/single strand). Labelled E.coli DNA (approximately 4 Mb) sedimented near the bottom (fractions 3–6) (4). Average fragment length (in Mb) of each profile is shown in square brackets as fragment length of the median fraction.
(a)
RT-PCR(b)
Effects of various siRNAs (UV)(c)
Effects of control siRNAs (UV)(d)
Dose-response (siRev3-D)(e)
Effect of various siRNAs (noUV)We assessed how many mismatched nucleotides (nt) are necessary at minimum for the negative control siRNA (Figure1(c)) and found that siRev3cont-C, designed from siRev3-D with 2 nt mismatches, did not prevent UV-TLS, indicating that these Rev3 siRNAs degrade Rev3 mRNA with high specificity. The dose-response profile of siRev3-D, shows that only 1 nM siRNA sufficiently inhibited UV-TLS (Figure 1(d)). The siRev3-D siRNA had no effect on normal replication (Figure 1(e)).We selected 3 target sites from the human Rev7 mRNA sequence (NM_006341) (Table1), and found that all siRNAs effectively reduced Rev7 expression (Figure 2(a)); 5 nM siRev7-B or siRev7-C completely abolished UV-TLS (siRev7-A elicited only partial prevention) (Figure 2(b)). The siRev7cont-A, designed from siRev7-B with 2 nt mismatches, had no effect (ibid). The dose-response profile of siRev7-B shows that 1 nM siRNA was sufficient to inhibit UV-TLS (Figure 2(c)); Rev7 siRNAs had no effect on normal replication (Figure 2(d)).Efficient knockdown by Rev7 siRNAs and their effects on UV-induced TLS in HeLa cells (ASDG profiles of replication products). (a) Efficiency of knockdown on Rev7 expression (RT-PCR analysis and western blot analysis); (b) Effects of various Rev7 siRNAs; (c) Dose-response of siRev7-B; (d) Effect of various Rev7 siRNAs (no UV control). Twenty-four hours after Rev7 siRNA transfection, total RNA was isolated and Rev7 RNA was quantified by RT-PCR. Results were shown in MultiNA gel images and the expression level was presented under the panel (a). Forty hours after Rev7 siRNA transfection, whole cell extracts were prepared and Rev7 protein was quantified by western blot analysis (a). Forty hours after Rev7 siRNA transfection, cells were UV-irradiated (10 J/m2), incubated in normal medium for 30 minutes, pulse-labelled with 10 μCi/mL of [14C]thymidine for 1 hour, washed twice with PBS, and incubated for 5 hours at 37°C in normal medium (b, c). Forty hours after Rev7 siRNA transfection, cells were not UV-irradiated, pulse-labelled with 10 μCi/mL of [14C]thymidine for 30 minutes, washed twice with PBS, and incubated at 37°C in normal medium for 1 hour (d). Some of these profiles overlap (c, d). Sedimentation is from right to left. The arrow indicates the position of T4 phage DNA (166 kb, i.e., approximately 5.5 × 107 Da/single strand). Average fragment length (in Mb) of each profile is shown in square brackets.
(a)
RT-PCR and western blotting(b)
Effects of various siRNAs (UV)(c)
Dose-response (siRev7-B)(d)
Effect of various siRNAs (noUV)


### 3.2. SiRNAs to Rev1 Significantly Abrogated UV-TLS

For knockdown ofRev1 expression, we selected 4 sites from the human Rev1 mRNA sequence (NM_016316) (Table 1) (Figure 3(a)). The siRNAs (5 nM) targeted to these sites abolished UV-TLS in HeLa cells (Figure 3(b)). The siRev1cont-E, designed from siRev1-C with 4 nt mismatches, had little to no effect. The dose-response profile of siRev1-C shows that 1 nM of the siRNA was enough to inhibit UV-TLS (Figure 3(c)); siRev1-C or siRev1-D siRNA had no effect on normal replication (Figure 3(d)).Efficient knockdown by Rev1 siRNAs and their effects on UV-induced TLS in HeLa cells (ASDG profiles of replication products). (a) Efficiency of knockdown on Rev1 expression RT-PCR analysis and western blot analysis); (b) Effects of various Rev1 siRNAs on UV-TLS; (c) Dose-response of siRev1-C on UV-TLS; (d) Effect of various siRNAs (no UV control). Twenty-four hours after Rev1 siRNA transfection, total RNA was isolated and Rev1 RNA was quantified by RT-PCR. Results were shown in MultiNA gel images and the expression level was presented under the panel (a). Forty hours after Rev1 siRNA transfection, whole cell extracts were prepared and Rev1 protein was quantified by western blot analysis (a). Forty hours after Rev1 siRNA transfection, cells were UV-irradiated (10 J/m2), incubated in normal medium for 30 minutes, pulse-labelled with 10 μCi/mL of [14C]thymidine for 1 hour, then washed twice with PBS, and incubated for 5 hours at 37°C in normal medium (b, c). Forty hours after Rev1 siRNA transfection, cells were not UV-irradiated, pulse-labelled with 10 μCi/mL of [14C]thymidine for 30 minutes, washed twice with PBS, and incubated at 37°C in normal medium for 1 hour (d). Some of these profiles overlap (b, c). Sedimentation is from right to left. The arrow indicates the position of T4 phage DNA (166 kb, i.e., approximately 5.5 × 107 Da/single strand). Average fragment length (in Mb) of each profile is shown in square brackets.
(a)
RT-PCR and western blotting(b)
Effects of various siRNAs (UV)(c)
Dose-response (siRev1-C)(d)
Effect of various siRNAs (noUV)


### 3.3. SiRNAs to Polη Prevented UV-TLS to a Great Extent

First, we tested the siRNA described by Choi and Pfeifer [37], siPolη-4, which partially inhibited UV-TLS (Figure 4(a)). The negative control, siPolηcont-A, was designed from siPolη-4 with 2 nt mismatches, had no effect (ibid). We added 2 Polη siRNAs of new design (human Polη mRNA sequence: NM_006502) (Table 1), and these siRNAs also effectively knocked down Polη expression (Figure 4(b)). These latter siRNAs at 5 nM abolished UV-TLS to a greater extent than siPolη-4 (Figure 4(c)). The Polη siRNAs had no effect on normal replication (Figure 4(d)).Efficient knockdown by Polη siRNAs and the effects on UV-induced TLS in HeLa cells (ASDG profiles of replication products). (a) Dose-response of siPolη-4 on UV-TLS, Efficiency of knockdown on Polη expression; (b) RT-PCR analysis and western blot analysis, (c) Effects of various Polη siRNAs on UV-TLS, (d) Effect of various Polη siRNAs (no UV control). Twenty-four hours after Polη siRNA transfection, total RNA was isolated and Polη RNA was quantified by RT-PCR. Results were shown in MultiNA gel images and the expression level was presented under the panel (b). Forty hours after Polη siRNA transfection, whole cell extracts were prepared and Polη protein was quantified by western blot analysis (b). Forty hours after Polη siRNA transfection, cells were UV-irradiated (10 J/m2), incubated in normal medium for 30 minutes, pulse-labelled with 10 μCi/mL of [14C]thymidine for 1 hour, then washed twice with PBS, and incubated for 5 hours at 37°C in normal medium (a, c). Forty hours after Polη siRNA transfection, cells were not UV-irradiated, pulse-labelled with 10 μCi/mL of [14C]thymidine for 30 minutes, washed twice with PBS, and incubated at 37°C in normal medium for 1 hour (d). Some of these profiles overlap (d). Sedimentation is from right to left. The arrow indicates the position of T4 phage DNA (166 kb, i.e., approximately 5.5 × 107 Da/single strand). Average fragment length (in Mb) of each profile is shown in square brackets.
(a)
dose-response (siPolη-4)(b)
RT-PCR and western blotting(c)
Effects of various siRNAs (UV)(d)
Effect of various siRNAs (noUV)


### 3.4. Polκ siRNAs Delayed UV-TLS, While siRNAs to Polι Did Not

Although Polι siRNAs efficiently prevented Polι expression (Figure 5(a)), we could not detect these effects on ASDG profiles (Figure 5(b)). The siPolι-5 was reported by Choi et al. [38], and the target sequence of siPolι-A was 4 nt downstream from the one reported by Machida et al. [39] (human Polι mRNA sequence: NM_007195).Efficient knockdown by Polι siRNAs and the effects on UV-induced TLS in HeLa cells (ASDG profiles of replication products). (a)  Efficiency of knockdown on Polι expression RT-PCR analysis and western blot analysis); (b) Effects of two Polι siRNAs on UV-TLS. Twenty-four hours after Polι siRNA transfection, total RNA was isolated and Polι RNA was quantified by RT-PCR. Results were shown in MultiNA gel images and the expression level was presented under the panel (a). Forty hours after Polι siRNA transfection, whole cell extracts were prepared and Polι protein was quantified by western blot analysis (a). Forty hours after Polι siRNA transfection, cells were UV-irradiated (10 J/m2), incubated in normal medium for 30 minutes, pulse-labelled with 10 μCi/mL of [14C]thymidine for 1 hour, then washed twice with PBS, and incubated for 5 hours at 37°C in normal medium (b). Sedimentation is from right to left. The arrow indicates the position of T4 phage DNA (166 kb, i.e., approximately 5.5 × 107 Da/single strand). Average fragment length (in Mb) of each profile is shown in square brackets.
(a)
RT-PCR and western blotting(b)
Effects of various siRNAs (UV)We prepared 3 siRNAs for knockdown of Polκ mRNA (NM_016218) (Figure 6(a)). In contrast to siPolι, these Polκ siRNAs (5 nM) delayed UV-TLS in HeLa cells (Figure 6(b)). The siPolκcont-B, from siPolκ-A with 3 nt mismatches, had little to no effect. The dose-response profile of siPolκ-A shows that the molecule sufficiently inhibited UV-TLS at 1 nM (Figure 6(c)). The Polκ siRNAs had no effect on normal replication (Figure 6(d)).Efficient knockdown by Polκ siRNAs and the effects on UV-induced TLS in HeLa cells (ASDG profiles of replication products). (a)  Efficiency of knockdown on Polκ expression RT-PCR analysis and western blot analysis), (b) Effects of various Polκ siRNAs on UV-TLS, (c) Dose-response of siPolκ-A on UV-TLS, (d) Effect of various Polκ siRNAs (no UV control). Twenty-four hours after Polκ siRNA transfection, total RNA was isolated and Polκ RNA was quantified by RT-PCR. Results were shown in MultiNA gel images and the expression level was presented under the panel (a). Forty hours after Polκ siRNA transfection, whole cell extracts were prepared, and Polκ protein was quantified by western blot analysis (a). Forty hours after Polκ siRNA transfection, cells were UV-irradiated (10 J/m2), incubated in normal medium for 30 minutes, pulse-labelled with 10 μCi/mL of [14C]thymidine for 1 hour, then washed twice with PBS, and incubated for 5 hours at 37°C in normal medium (b, c). Forty hours after Polκ siRNA transfection, cells were not UV-irradiated, pulse-labelled with 10 μCi/mL of [14C]thymidine for 30 minutes, washed twice with PBS, and incubated at 37°C in normal medium for 1 hour (d). Some of these profiles overlap (c, d). Sedimentation is from right to left. The arrow indicates the position of T4 phage DNA (166 kb, i.e., approximately 5.5 × 107 Da/single strand). Average fragment length (in Mb) of each profile is shown in square brackets.
(a)
RT-PCR and western blotting(b)
Effects of various siRNAs (UV)(c)
dose-response (siPolκ-A)(d)
Effect of various siRNAs (noUV)


## 3.1. Effect of siRNAs to Rev3 or Rev7 on HeLa UV-TLS

We selected 4 target sites from the human Rev3 mRNA sequence (NCBI locus: NM_002912), according to the JBioS guide. All the Rev3 siRNAs (Table1) effectively knocked down expression of Rev3 (Figure 1(a)) and, at 5 nM, abolished UV-TLS in HeLa cells (Figure 1(b)). Replication products immediately after UV irradiation were sedimented as a sharp peak, illustrated by a thin line in Figure 1(b), slightly larger in size than the T4 phage DNA marker. When only Oligofectamine-treated cells were chased in normal medium for 5 hours, the products joined to form larger DNA with lengths in the order of megabases (Mb), illustrated by a thick line. In Rev3 siRNA-transfected cells, the products remain in smaller size, as depicted by a thin line with open marks, demonstrating these siRNAs prevent UV-TLS (Figure 1(b)).Efficient knockdown by Rev3 siRNAs and their effects on UV-induced TLS in HeLa cells (ASDG profiles of replication products). (a) Efficiency of knockdown on Rev3 expression (RT-PCR analysis); (b) Effects of various Rev3 siRNAs; (c) Effects of Rev3 control siRNAs; (d) Dose-response of siRev3-D; (e) Effect of various Rev3 siRNAs (no UV control). Twenty-four hours after Rev3 siRNA transfection, total RNA was isolated and Rev3 RNA was quantified by RT-PCR. Results were shown in MultiNA gel images and the expression level was presented under the panel (a). Forty hours after Rev3 siRNA transfection, cells were UV-irradiated (10 J/m2), incubated in normal medium for 30 minutes, pulse-labelled with 10 μCi/mL of [14C]thymidine for 1 hour, washed twice with PBS, and incubated for 5 hours at 37°C in normal medium (b, c, d). siRev3cont-A, 6 nt mismatches; siRev3cont-B, 4 nt mismatches; siRev3cont-C, 2 nt mismatches (c). Forty hours after Rev3 siRNA transfection, cells were not UV-irradiated, pulse-labelled with 10 μCi/mL of [14C]thymidine for 30 minutes, washed twice with PBS, and incubated at 37°C in normal medium for 1 hour (e). Some of these profiles overlap (c, d, e). Sedimentation is from right to left. The arrow indicates the position of T4 phage DNA (166 kb, i.e., approximately 5.5 × 107 Da/single strand). Labelled E.coli DNA (approximately 4 Mb) sedimented near the bottom (fractions 3–6) (4). Average fragment length (in Mb) of each profile is shown in square brackets as fragment length of the median fraction.
(a)
RT-PCR(b)
Effects of various siRNAs (UV)(c)
Effects of control siRNAs (UV)(d)
Dose-response (siRev3-D)(e)
Effect of various siRNAs (noUV)We assessed how many mismatched nucleotides (nt) are necessary at minimum for the negative control siRNA (Figure1(c)) and found that siRev3cont-C, designed from siRev3-D with 2 nt mismatches, did not prevent UV-TLS, indicating that these Rev3 siRNAs degrade Rev3 mRNA with high specificity. The dose-response profile of siRev3-D, shows that only 1 nM siRNA sufficiently inhibited UV-TLS (Figure 1(d)). The siRev3-D siRNA had no effect on normal replication (Figure 1(e)).We selected 3 target sites from the human Rev7 mRNA sequence (NM_006341) (Table1), and found that all siRNAs effectively reduced Rev7 expression (Figure 2(a)); 5 nM siRev7-B or siRev7-C completely abolished UV-TLS (siRev7-A elicited only partial prevention) (Figure 2(b)). The siRev7cont-A, designed from siRev7-B with 2 nt mismatches, had no effect (ibid). The dose-response profile of siRev7-B shows that 1 nM siRNA was sufficient to inhibit UV-TLS (Figure 2(c)); Rev7 siRNAs had no effect on normal replication (Figure 2(d)).Efficient knockdown by Rev7 siRNAs and their effects on UV-induced TLS in HeLa cells (ASDG profiles of replication products). (a) Efficiency of knockdown on Rev7 expression (RT-PCR analysis and western blot analysis); (b) Effects of various Rev7 siRNAs; (c) Dose-response of siRev7-B; (d) Effect of various Rev7 siRNAs (no UV control). Twenty-four hours after Rev7 siRNA transfection, total RNA was isolated and Rev7 RNA was quantified by RT-PCR. Results were shown in MultiNA gel images and the expression level was presented under the panel (a). Forty hours after Rev7 siRNA transfection, whole cell extracts were prepared and Rev7 protein was quantified by western blot analysis (a). Forty hours after Rev7 siRNA transfection, cells were UV-irradiated (10 J/m2), incubated in normal medium for 30 minutes, pulse-labelled with 10 μCi/mL of [14C]thymidine for 1 hour, washed twice with PBS, and incubated for 5 hours at 37°C in normal medium (b, c). Forty hours after Rev7 siRNA transfection, cells were not UV-irradiated, pulse-labelled with 10 μCi/mL of [14C]thymidine for 30 minutes, washed twice with PBS, and incubated at 37°C in normal medium for 1 hour (d). Some of these profiles overlap (c, d). Sedimentation is from right to left. The arrow indicates the position of T4 phage DNA (166 kb, i.e., approximately 5.5 × 107 Da/single strand). Average fragment length (in Mb) of each profile is shown in square brackets.
(a)
RT-PCR and western blotting(b)
Effects of various siRNAs (UV)(c)
Dose-response (siRev7-B)(d)
Effect of various siRNAs (noUV)


## 3.2. SiRNAs to Rev1 Significantly Abrogated UV-TLS

For knockdown ofRev1 expression, we selected 4 sites from the human Rev1 mRNA sequence (NM_016316) (Table 1) (Figure 3(a)). The siRNAs (5 nM) targeted to these sites abolished UV-TLS in HeLa cells (Figure 3(b)). The siRev1cont-E, designed from siRev1-C with 4 nt mismatches, had little to no effect. The dose-response profile of siRev1-C shows that 1 nM of the siRNA was enough to inhibit UV-TLS (Figure 3(c)); siRev1-C or siRev1-D siRNA had no effect on normal replication (Figure 3(d)).Efficient knockdown by Rev1 siRNAs and their effects on UV-induced TLS in HeLa cells (ASDG profiles of replication products). (a) Efficiency of knockdown on Rev1 expression RT-PCR analysis and western blot analysis); (b) Effects of various Rev1 siRNAs on UV-TLS; (c) Dose-response of siRev1-C on UV-TLS; (d) Effect of various siRNAs (no UV control). Twenty-four hours after Rev1 siRNA transfection, total RNA was isolated and Rev1 RNA was quantified by RT-PCR. Results were shown in MultiNA gel images and the expression level was presented under the panel (a). Forty hours after Rev1 siRNA transfection, whole cell extracts were prepared and Rev1 protein was quantified by western blot analysis (a). Forty hours after Rev1 siRNA transfection, cells were UV-irradiated (10 J/m2), incubated in normal medium for 30 minutes, pulse-labelled with 10 μCi/mL of [14C]thymidine for 1 hour, then washed twice with PBS, and incubated for 5 hours at 37°C in normal medium (b, c). Forty hours after Rev1 siRNA transfection, cells were not UV-irradiated, pulse-labelled with 10 μCi/mL of [14C]thymidine for 30 minutes, washed twice with PBS, and incubated at 37°C in normal medium for 1 hour (d). Some of these profiles overlap (b, c). Sedimentation is from right to left. The arrow indicates the position of T4 phage DNA (166 kb, i.e., approximately 5.5 × 107 Da/single strand). Average fragment length (in Mb) of each profile is shown in square brackets.
(a)
RT-PCR and western blotting(b)
Effects of various siRNAs (UV)(c)
Dose-response (siRev1-C)(d)
Effect of various siRNAs (noUV)


## 3.3. SiRNAs to Polη Prevented UV-TLS to a Great Extent

First, we tested the siRNA described by Choi and Pfeifer [37], siPolη-4, which partially inhibited UV-TLS (Figure 4(a)). The negative control, siPolηcont-A, was designed from siPolη-4 with 2 nt mismatches, had no effect (ibid). We added 2 Polη siRNAs of new design (human Polη mRNA sequence: NM_006502) (Table 1), and these siRNAs also effectively knocked down Polη expression (Figure 4(b)). These latter siRNAs at 5 nM abolished UV-TLS to a greater extent than siPolη-4 (Figure 4(c)). The Polη siRNAs had no effect on normal replication (Figure 4(d)).Efficient knockdown by Polη siRNAs and the effects on UV-induced TLS in HeLa cells (ASDG profiles of replication products). (a) Dose-response of siPolη-4 on UV-TLS, Efficiency of knockdown on Polη expression; (b) RT-PCR analysis and western blot analysis, (c) Effects of various Polη siRNAs on UV-TLS, (d) Effect of various Polη siRNAs (no UV control). Twenty-four hours after Polη siRNA transfection, total RNA was isolated and Polη RNA was quantified by RT-PCR. Results were shown in MultiNA gel images and the expression level was presented under the panel (b). Forty hours after Polη siRNA transfection, whole cell extracts were prepared and Polη protein was quantified by western blot analysis (b). Forty hours after Polη siRNA transfection, cells were UV-irradiated (10 J/m2), incubated in normal medium for 30 minutes, pulse-labelled with 10 μCi/mL of [14C]thymidine for 1 hour, then washed twice with PBS, and incubated for 5 hours at 37°C in normal medium (a, c). Forty hours after Polη siRNA transfection, cells were not UV-irradiated, pulse-labelled with 10 μCi/mL of [14C]thymidine for 30 minutes, washed twice with PBS, and incubated at 37°C in normal medium for 1 hour (d). Some of these profiles overlap (d). Sedimentation is from right to left. The arrow indicates the position of T4 phage DNA (166 kb, i.e., approximately 5.5 × 107 Da/single strand). Average fragment length (in Mb) of each profile is shown in square brackets.
(a)
dose-response (siPolη-4)(b)
RT-PCR and western blotting(c)
Effects of various siRNAs (UV)(d)
Effect of various siRNAs (noUV)


## 3.4. Polκ siRNAs Delayed UV-TLS, While siRNAs to Polι Did Not

Although Polι siRNAs efficiently prevented Polι expression (Figure 5(a)), we could not detect these effects on ASDG profiles (Figure 5(b)). The siPolι-5 was reported by Choi et al. [38], and the target sequence of siPolι-A was 4 nt downstream from the one reported by Machida et al. [39] (human Polι mRNA sequence: NM_007195).Efficient knockdown by Polι siRNAs and the effects on UV-induced TLS in HeLa cells (ASDG profiles of replication products). (a)  Efficiency of knockdown on Polι expression RT-PCR analysis and western blot analysis); (b) Effects of two Polι siRNAs on UV-TLS. Twenty-four hours after Polι siRNA transfection, total RNA was isolated and Polι RNA was quantified by RT-PCR. Results were shown in MultiNA gel images and the expression level was presented under the panel (a). Forty hours after Polι siRNA transfection, whole cell extracts were prepared and Polι protein was quantified by western blot analysis (a). Forty hours after Polι siRNA transfection, cells were UV-irradiated (10 J/m2), incubated in normal medium for 30 minutes, pulse-labelled with 10 μCi/mL of [14C]thymidine for 1 hour, then washed twice with PBS, and incubated for 5 hours at 37°C in normal medium (b). Sedimentation is from right to left. The arrow indicates the position of T4 phage DNA (166 kb, i.e., approximately 5.5 × 107 Da/single strand). Average fragment length (in Mb) of each profile is shown in square brackets.
(a)
RT-PCR and western blotting(b)
Effects of various siRNAs (UV)We prepared 3 siRNAs for knockdown of Polκ mRNA (NM_016218) (Figure 6(a)). In contrast to siPolι, these Polκ siRNAs (5 nM) delayed UV-TLS in HeLa cells (Figure 6(b)). The siPolκcont-B, from siPolκ-A with 3 nt mismatches, had little to no effect. The dose-response profile of siPolκ-A shows that the molecule sufficiently inhibited UV-TLS at 1 nM (Figure 6(c)). The Polκ siRNAs had no effect on normal replication (Figure 6(d)).Efficient knockdown by Polκ siRNAs and the effects on UV-induced TLS in HeLa cells (ASDG profiles of replication products). (a)  Efficiency of knockdown on Polκ expression RT-PCR analysis and western blot analysis), (b) Effects of various Polκ siRNAs on UV-TLS, (c) Dose-response of siPolκ-A on UV-TLS, (d) Effect of various Polκ siRNAs (no UV control). Twenty-four hours after Polκ siRNA transfection, total RNA was isolated and Polκ RNA was quantified by RT-PCR. Results were shown in MultiNA gel images and the expression level was presented under the panel (a). Forty hours after Polκ siRNA transfection, whole cell extracts were prepared, and Polκ protein was quantified by western blot analysis (a). Forty hours after Polκ siRNA transfection, cells were UV-irradiated (10 J/m2), incubated in normal medium for 30 minutes, pulse-labelled with 10 μCi/mL of [14C]thymidine for 1 hour, then washed twice with PBS, and incubated for 5 hours at 37°C in normal medium (b, c). Forty hours after Polκ siRNA transfection, cells were not UV-irradiated, pulse-labelled with 10 μCi/mL of [14C]thymidine for 30 minutes, washed twice with PBS, and incubated at 37°C in normal medium for 1 hour (d). Some of these profiles overlap (c, d). Sedimentation is from right to left. The arrow indicates the position of T4 phage DNA (166 kb, i.e., approximately 5.5 × 107 Da/single strand). Average fragment length (in Mb) of each profile is shown in square brackets.
(a)
RT-PCR and western blotting(b)
Effects of various siRNAs (UV)(c)
dose-response (siPolκ-A)(d)
Effect of various siRNAs (noUV)


## 4. Discussion

We verified the involvement of multiple bypass polymerases in UV-TLS in HeLa cells using original siRNAs and ASDG technique, which is consistent with the recent model of 2 polymerase mechanisms [40, 41]. Rev3 and Rev7, which comprise Polζ, were confirmed to participate in mutagenic UV-TLS. Also, Rev1 was suggested to play an important role in human TLS, although in avian DT40 cells, Rev1 may have a distinct role [42]. We were surprised to find that siRNAs against Polη prevented TLS to a great extent. TLS was delayed in Polκ siRNA-transfected cells, but not in siPolι-transfected cells.We anticipated a limited participation of Polη, because UV-TLS in HeLa cells is very slow (i.e., inefficient) and caffeine-sensitive [35]. However, siRNAs against Polη, particularly siPolη-A and siPolη-B, prevented TLS to a great extent. Since both Rev3 and Rev7 siRNAs also significantly abolished UV-TLS, these results suggest that the Polζ-dependent TLS pathway and the Polη-dependent process are not mutually exclusive but overlapped.Enzymology of yeast Polζ revealed that this polymerase is too faithful to insert nucleotides opposite a CPD, although it efficiently extends from a matched or mismatched 3′ end [5, 6]. Therefore, we assumed that mutagenic (error-prone) TLS proceeded through the insertion by Polι or Polκ of mismatched nucleotides opposite UV photoproducts, followed by extension by Polζ. Our data showed, however, that siPolι had no effect, and siPolκ partially prevented TLS. These results suggest that in some cases, Polη, and to a lesser extent Polκ, may insert nucleotide(s) opposite UV photoproducts, followed by extension by Polζ.Polη is capable of bypassing a CPD without aid of other TLS polymerases. Both yeast and human Polη, however, incorporate wrong nucleotide at a fairly high rate and can extend these mismatched primer termini with only a frequency of ~10−2 to 10−3 relative to extension from matched primer termini [6, 43]. Plausibly, Polη dissociates from there and the proof-reading exonuclease of Polδ remove the wrong nucleotide [44]. To the primer termini, Polη is recruited again and incorporates a new nucleotide. This cycle is repeated until Polη incorporates a correct nucleotide. We suppose that disruption or malfunction of this cooperation renders mismatched primer termini accessible to Polζ.Recently, Yoon et al. published 2 papers describing the effects of siRNA knockdown on the efficiency of TLS at TT-CPD [45] or (6-4)TT PP [46] on duplex plasmids in human cells. They also reported the effects of siRNA knockdown on mutation frequencies in the λ phage cII gene lysogenized in mouse cells expressing a (6-4)PP photolyase [45] or CPD photolyase [46]. The results of this tremendous and detailed study demonstrated that Pols η, κ, and ζ contribute to CPD bypass, wherein Pols κ and ζ promote mutagenic TLS and Polη executes error-free bypasses (Polι siRNA had no effect) [45]. As for (6-4)PP bypass, Pols η and ι provide alternate pathways for mutagenic TLS, and Polζ acts in a predominantly error-free manner (Polκ siRNA had no effect) [46].The participation of Polsκ or ι in CPD bypass was similarly demonstrated by our results and those of Yoon et al. [45]. Because (6-4)PP is a minor photoproduct, which is removed predominantly by NER, and because HeLa cells possess high NER activity (unpublished observation), it is reasonable to conclude that our phenomena observed in HeLa cells by ASDG are largely attributable to CPD, although we have not yet determined the extent of remaining (6-4)PP.We may also conclude that Rev1 is indispensable for TLS across CPD. Thus far, it is unknown if Rev1 is equally involved in TLS across CPD and (6-4)PP, or if it exhibits some preference. Nelson et al. [25] demonstrated that Rev1p participates in UV-TLS across (6-4)PP, based on yeast transfected with a (6-4)PP-carrying plasmid; only slight differences were observed with a CPD-carrying plasmid.In vitro lesion-bypass assay showed that Polη alone accomplishes bypass across TT-CPD as above (i.e., both insertion and extension) [8, 9]. However, Yoon et al. presented complex results showing involvement of multiple bypass polymerases. They used SV-untransformed XP-A and XP-V cells, but did not include SV-untransformed normal fibroblasts, wherein we detected quick and caffeine-insensitive UV-TLS [35, 36]. It is possible that the kind of damage, as well as cell status (normal, transformed, or cancerous) may determine the participation of bypass polymerase(s).We have presented the first apparent evidence that Polκ participates in UV-TLS. Polκ knockout mouse embryonic cells are known to be UV sensitive [47], but the mechanism had not yet been determined. Polκ is also thought to play a part in the repair-synthesis step of NER [48, 49]. From the results of lesion-bypass assays, human Polκ was suggested to be unable to bypass CPD or (6-4)PP. Because the outcomes of such in vitro assays depend on the assay conditions [12], these results must be validated in vivo, such as by ASDG analysis.


## 5. Conclusions

Using siRNAs originally designed and ASDG technique, we verified the participation of multiple bypass polymerases in UV-induced TLS in HeLa cells, which is the consistent with recent model of 2 polymerase mechanisms. UV-TLS was largely abolished by siRNAs to Rev3 or Rev7, suggesting that these 2 proteins, which constitute Polζ, play a primary role in mutagenic TLS. Rev1-targeted siRNAs also significantly abolished UV-TLS, consistent with prior suggestions that Rev1 is indispensable in mammalian mutagenic TLS. Unexpectedly, siRNAs to Polη prevented TLS to a great extent, implying that the Polη- and Polζ-dependent processes do not alternate but overlap. Polκ siRNAs, but not siRNAs to Polι, delayed TLS; this is the first apparent evidence for the participation of Polκ in UV-TLS.



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*Source: 750296-2010-12-01.xml*