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DNA polymerases ? and Rev1 mediate error-prone bypass of non-B DNA structures.

ABSTRACT: DNA polymerase ? (Pol ?) and Rev1 are key players in translesion DNA synthesis. The error-prone Pol ? can also participate in replication of undamaged DNA when the normal replisome is impaired. Here we define the nature of the replication disturbances that trigger the recruitment of error-prone polymerases in the absence of DNA damage and describe the specific roles of Rev1 and Pol ? in handling these disturbances. We show that Pol ?/Rev1-dependent mutations occur at sites of replication stalling at short repeated sequences capable of forming hairpin structures. The Rev1 deoxycytidyl transferase can take over the stalled replicative polymerase and incorporate an additional 'C' at the hairpin base. Full hairpin bypass often involves template-switching DNA synthesis, subsequent realignment generating multiply mismatched primer termini and extension of these termini by Pol ?. The postreplicative pathway dependent on polyubiquitylation of proliferating cell nuclear antigen provides a backup mechanism for accurate bypass of these sequences that is primarily used when the Pol ?/Rev1-dependent pathway is inactive. The results emphasize the pivotal role of noncanonical DNA structures in mutagenesis and reveal the long-sought-after mechanism of complex mutations that represent a unique signature of Pol ?.

SUBMITTER: Northam MR

PROVIDER: S-EPMC3874155 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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DNA polymerases ζ and Rev1 mediate error-prone bypass of non-B DNA structures.

Northam Matthew R MR Moore Elizabeth A EA Mertz Tony M TM Binz Sara K SK Stith Carrie M CM Stepchenkova Elena I EI Wendt Kathern L KL Burgers Peter M J PM Shcherbakova Polina V PV

Nucleic acids research 20130918 1

DNA polymerase ζ (Pol ζ) and Rev1 are key players in translesion DNA synthesis. The error-prone Pol ζ can also participate in replication of undamaged DNA when the normal replisome is impaired. Here we define the nature of the replication disturbances that trigger the recruitment of error-prone polymerases in the absence of DNA damage and describe the specific roles of Rev1 and Pol ζ in handling these disturbances. We show that Pol ζ/Rev1-dependent mutations occur at sites of replication stallin ...[more]

PMID: 24049079

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Project description:Translesion synthesis (TLS) of the N(2)-2'-deoxyguanosine (dG-N(2)-IQ) adduct of the carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) was investigated in human embryonic kidney 293T cells by replicating plasmid constructs in which the adduct was individually placed at each guanine (G1, G2, or G3) of the NarI sequence (5'-CG1G2CG3CC-3'). TLS efficiency was 38%, 29%, and 25% for the dG-N(2)-IQ located at G1, G2, and G3, respectively, which suggests that dG-N(2)-IQ is bypassed more efficiently by one or more DNA polymerases at G1 than at either G2 or G3. TLS efficiency was decreased 8-35% in cells with knockdown of pol ?, pol ?, pol ?, pol ?, or Rev1. Up to 75% reduction in TLS occurred when pol ?, pol ?, and Rev1 were simultaneously knocked down, suggesting that these three polymerases play important roles in dG-N(2)-IQ bypass. Mutation frequencies (MFs) of dG-N(2)-IQ at G1, G2, and G3 were 23%, 17%, and 11%, respectively, exhibiting a completely reverse trend of the previously reported MF of the C8-dG adduct of IQ (dG-C8-IQ), which is most mutagenic at G3 ( ( 2015 ) Nucleic Acids Res. 43 , 8340 - 8351 ). The major type of mutation induced by dG-N(2)-IQ was targeted G ? T, as was reported for dG-C8-IQ. In each site, knockdown of pol ? resulted in an increase in MF, whereas MF was reduced when pol ?, pol ?, pol ?, or Rev1 was knocked down. The reduction in MF was most pronounced when pol ?, pol ?, and Rev1 were simultaneously knocked down and especially when the adduct was located at G3, where MF was reduced by 90%. We conclude that pol ? predominantly performs error-free TLS of the dG-N(2)-IQ adduct, whereas pols ?, pol ?, and Rev1 cooperatively carry out the error-prone TLS. However, in vitro experiments using yeast pol ? and ? showed that the former was inefficient in full-length primer extension on dG-N(2)-IQ templates, whereas the latter was efficient in both error-free and error-prone extensions. We believe that the observed differences between the in vitro experiments using purified DNA polymerases, and the cellular results may arise from several factors including the crucial roles played by the accessory proteins in TLS.

Dataset Information

DNA polymerases ? and Rev1 mediate error-prone bypass of non-B DNA structures.

Publications

DNA polymerases ζ and Rev1 mediate error-prone bypass of non-B DNA structures.

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