A high-throughput and quantitative method to assess the mutagenic potential of translesion DNA synthesis

Taggart, David; Camerlengo, Terry; Harrison, Jason K.; Sherrer, Shanen M.; Kshetry, Ajay K.; Taylor, John‐Stephen; Huang, Kun; Suo, Zucai

doi:10.1093/nar/gkt141

Cited by 9 publications

(18 citation statements)

References 45 publications

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“…To quantify the types and frequencies of errors induced by oxidative lesion bypass, we utilized a high-throughput short oligonucleotide sequencing assay (HT-SOSA) that was recently developed in our laboratory [25]. Our data support a model wherein the N-terminal domains of Polλ increase the frameshift error rate of Polλ opposite damaged sites by promoting primer/template realignment.…”

Section: Introductionmentioning

confidence: 70%

“…To investigate if the N-terminal domains of human Polλ influence the type or frequency of mutations induced by TLS of an AP site, we used our newly developed HT-SOSA approach [25]. To this end, we generated DNA substrates that possessed a 7-nucleotide gap, and were either undamaged or contained a site-specifically placed AP site (Supplementary Table S2).…”

Section: Resultsmentioning

confidence: 99%

“…HT-SOSA was performed as previously described [25] with the following modifications. A pre-incubated solution containing 100 nM of a polymerase and 100 nM of a 5′-[ 32 P]-labeled, gapped DNA substrate (Supplementary Table S2) was mixed with a solution containing 200 µM of each of the four dNTPs.…”

Section: Methodsmentioning

confidence: 99%

“…DNA sequencing reads were sorted and analyzed as previously described [25]. Briefly, the raw sequence reads that matched the ΦX genome and all sequence reads that contained one or more base calls which were not identified with greater than 99% accuracy (Phred quality score of less than 20) were removed.…”

Section: Methodsmentioning

confidence: 99%

“…After sorting, erroneous sequences were further removed by eliminating all sequence reads that did not perfectly match the reference sequences within the first six nucleotides, which consist of the four nucleotide barcode sequence and two adjacent nucleotides. Each batch of sequence reads (Supplementary Table S3) was then analyzed by using the “The Next-Generation Sequencing Position Counter” software [25] to align the sequences to a defined reference sequence and to quantify the total number of matches, mismatches ( i.e. substitutions), insertions, and deletions generated by the X-family polymerases at each template position.…”

Section: Methodsmentioning

confidence: 99%

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N-terminal domains of human DNA polymerase lambda promote primer realignment during translesion DNA synthesis

et al. 2014

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The X-family DNA polymerases λ (Polλ) and β (Polβ) possess similar 5′-2-deoxyribose-5-phosphatelyase (dRPase) and polymerase domains. Besides these domains, Polλ also possesses a BRCA1 C-terminal (BRCT) domain and a proline-rich domain at its N terminus. However, it is unclear how these non-enzymatic domains contribute to the unique biological functions of Polλ. Here, we used primer extension assays and a newly developed high-throughput short oligonucleotide sequencing assay (HT-SOSA) to compare the efficiency of lesion bypass and fidelity of human Polβ, Polλ and two N-terminal deletion constructs of Polλ during the bypass of either an abasic site or a 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) lesion. We demonstrate that the BRCT domain of Polλ enhances the efficiency of abasic site bypass by approximately 1.6-fold. In contrast, deletion of the N-terminal domains of Polλ did not affect the efficiency of 8-oxodG bypass relative to nucleotide incorporations opposite undamaged dG. HT-SOSA analysis demonstrated that Polλ and Polβ preferentially generated −1 or −2 frameshift mutations when bypassing an abasic site and the single or double base deletion frequency was highly sequence dependent. Interestingly, the BRCT and proline-rich domains of Polλ cooperatively promoted the generation of −2 frameshift mutations when the abasic site was situated within a sequence context that was susceptible to homology-driven primer realignment. Furthermore, both N-terminal domains of Polλ increased the generation of −1 frameshift mutations during 8-oxodG bypass and influenced the frequency of substitution mutations produced by Polλ opposite the 8-oxodG lesion. Overall, our data support a model wherein the BRCT and proline-rich domains of Polλ act cooperatively to promote primer/template realignment between DNA strands of limited sequence homology. This function of the N-terminal domains may facilitate the role of Polλ as a gap-filling polymerase within the non-homologous end joining pathway.

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Section: Introductionmentioning

confidence: 70%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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N-terminal domains of human DNA polymerase lambda promote primer realignment during translesion DNA synthesis

et al. 2014

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Next-generation sequencing-based analysis of the effect of N6-methyldeoxyadenosine modification on DNA replication in human cells

Wang¹,

Sheng²,

Yang³

et al. 2022

Chinese Chemical Letters

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NGS-based analysis of base-substitution signatures created by yeast DNA polymerase eta and zeta on undamaged and abasic DNA templates in vitro

Chen

Sugiyama

2017

DNA Repair

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Translesion synthesis (TLS) is the mechanism in which DNA polymerases (TLS polymerases) bypass unrepaired template damage with high error rates. DNA polymerase η and ζ (Polη and Polζ) are major TLS polymerases that are conserved from yeast to humans. In this study, we quantified frequencies of basesubstitutions by yeast Polη and Polζ on undamaged and abasic templates in vitro. For accurate quantification, we used a next generation sequencing (NGS)-based method where DNA products were directly analyzed by parallel sequencing. On undamaged templates, Polη and Polζ showed distinct basesubstitution profiles, and the substitution frequencies were differently influenced by the template sequence. The base-substitution frequencies were influenced mainly by the adjacent bases both upstream and downstream of the substitution sites. Thus we present the base-substitution signatures of these polymerases in a three-base format. On templates containing abasic sites, Polη created deletions at the lesion in more than 50% of the TLS products, but the formation of the deletions was suppressed by the presence of Polζ. Polζ and Polη cooperatively facilitated the TLS reaction over an abasic site in vitro, suggesting that these two polymerases can cooperate in efficient and high fidelity TLS.

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A high-throughput and quantitative method to assess the mutagenic potential of translesion DNA synthesis

Cited by 9 publications

References 45 publications

N-terminal domains of human DNA polymerase lambda promote primer realignment during translesion DNA synthesis

N-terminal domains of human DNA polymerase lambda promote primer realignment during translesion DNA synthesis

Next-generation sequencing-based analysis of the effect of N6-methyldeoxyadenosine modification on DNA replication in human cells

NGS-based analysis of base-substitution signatures created by yeast DNA polymerase eta and zeta on undamaged and abasic DNA templates in vitro

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