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Evaluation and modulation of DNA lesion bypass in an SV40 large T antigen‐based in vitro replication system

DNA damage removal by nucleotide excision repair (NER) and replicative bypass via translesion synthesis (TLS) and template switch (TSw) are important in ensuring genome stability. In this study, we tested the applicability of an SV40 large T antigen‐based replication system for the simultaneous exam...

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Autores principales: Szeltner, Zoltán, Póti, Ádám, Harami, Gábor M., Kovács, Mihály, Szüts, Dávid
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley and Sons Inc. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8016126/
https://www.ncbi.nlm.nih.gov/pubmed/33512058
http://dx.doi.org/10.1002/2211-5463.13099
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author Szeltner, Zoltán
Póti, Ádám
Harami, Gábor M.
Kovács, Mihály
Szüts, Dávid
author_facet Szeltner, Zoltán
Póti, Ádám
Harami, Gábor M.
Kovács, Mihály
Szüts, Dávid
author_sort Szeltner, Zoltán
collection PubMed
description DNA damage removal by nucleotide excision repair (NER) and replicative bypass via translesion synthesis (TLS) and template switch (TSw) are important in ensuring genome stability. In this study, we tested the applicability of an SV40 large T antigen‐based replication system for the simultaneous examination of these damage tolerance processes. Using both Sanger and next‐generation sequencing combined with lesion‐specific qPCR and replication efficiency studies, we demonstrate that this system works well for studying NER and TLS, especially its one‐polymerase branch, while it is less suited to investigations of homology‐related repair processes, such as TSw. Cis‐syn cyclobutane pyrimidine dimer photoproducts were replicated with equal efficiency to lesion‐free plasmids in vitro, and the majority of TLS on this lesion could be inhibited by a peptide (PIR) specific for the polη‐PCNA interaction interface. TLS on 6–4 pyrimidine–pyrimidone photoproduct proved to be inefficient and was slightly facilitated by PIR as well as by a recombinant ubiquitin‐binding zinc finger domain of polη in HeLa extract, possibly by promoting polymerase exchange. Supplementation of the extract with recombinant PCNA variants indicated the dependence of TLS on PCNA ubiquitylation. In contrast to active TLS and NER, we found no evidence of successful TSw in cellular extracts. The established methods can promote in vitro investigations of replicative DNA damage bypass.
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spelling pubmed-80161262021-04-02 Evaluation and modulation of DNA lesion bypass in an SV40 large T antigen‐based in vitro replication system Szeltner, Zoltán Póti, Ádám Harami, Gábor M. Kovács, Mihály Szüts, Dávid FEBS Open Bio Research Articles DNA damage removal by nucleotide excision repair (NER) and replicative bypass via translesion synthesis (TLS) and template switch (TSw) are important in ensuring genome stability. In this study, we tested the applicability of an SV40 large T antigen‐based replication system for the simultaneous examination of these damage tolerance processes. Using both Sanger and next‐generation sequencing combined with lesion‐specific qPCR and replication efficiency studies, we demonstrate that this system works well for studying NER and TLS, especially its one‐polymerase branch, while it is less suited to investigations of homology‐related repair processes, such as TSw. Cis‐syn cyclobutane pyrimidine dimer photoproducts were replicated with equal efficiency to lesion‐free plasmids in vitro, and the majority of TLS on this lesion could be inhibited by a peptide (PIR) specific for the polη‐PCNA interaction interface. TLS on 6–4 pyrimidine–pyrimidone photoproduct proved to be inefficient and was slightly facilitated by PIR as well as by a recombinant ubiquitin‐binding zinc finger domain of polη in HeLa extract, possibly by promoting polymerase exchange. Supplementation of the extract with recombinant PCNA variants indicated the dependence of TLS on PCNA ubiquitylation. In contrast to active TLS and NER, we found no evidence of successful TSw in cellular extracts. The established methods can promote in vitro investigations of replicative DNA damage bypass. John Wiley and Sons Inc. 2021-02-25 /pmc/articles/PMC8016126/ /pubmed/33512058 http://dx.doi.org/10.1002/2211-5463.13099 Text en © 2021 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Articles
Szeltner, Zoltán
Póti, Ádám
Harami, Gábor M.
Kovács, Mihály
Szüts, Dávid
Evaluation and modulation of DNA lesion bypass in an SV40 large T antigen‐based in vitro replication system
title Evaluation and modulation of DNA lesion bypass in an SV40 large T antigen‐based in vitro replication system
title_full Evaluation and modulation of DNA lesion bypass in an SV40 large T antigen‐based in vitro replication system
title_fullStr Evaluation and modulation of DNA lesion bypass in an SV40 large T antigen‐based in vitro replication system
title_full_unstemmed Evaluation and modulation of DNA lesion bypass in an SV40 large T antigen‐based in vitro replication system
title_short Evaluation and modulation of DNA lesion bypass in an SV40 large T antigen‐based in vitro replication system
title_sort evaluation and modulation of dna lesion bypass in an sv40 large t antigen‐based in vitro replication system
topic Research Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8016126/
https://www.ncbi.nlm.nih.gov/pubmed/33512058
http://dx.doi.org/10.1002/2211-5463.13099
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