title: Analysis of CPD ultraviolet lesion bypass in chicken DT40 cells: polymerase η and PCNA ubiquitylation play identical roles.
abstract: Translesion synthesis (TLS) provides a mechanism of copying damaged templates during DNA replication. This potentially mutagenic process may operate either at the replication fork or at post-replicative gaps. We used the example of T-T cyclobutane pyrimidine dimer (CPD) bypass to determine the influence of polymerase recruitment via PCNA ubiquitylation versus the REV1 protein on the efficiency and mutagenic outcome of TLS. Using mutant chicken DT40 cell lines we show that, on this numerically most important UV lesion, defects in polymerase η or in PCNA ubiquitylation similarly result in the long-term failure of lesion bypass with persistent strand gaps opposite the lesion, and the elevation of mutations amongst successful TLS events. Our data suggest that PCNA ubiquitylation promotes CPD bypass mainly by recruiting polymerase η, resulting in the majority of CPD lesions bypassed in an error-free manner. In contrast, we find that polymerase ζ is responsible for the majority of CPD-dependent mutations, but has no essential function in the completion of bypass. These findings point to a hierarchy of access of the different TLS polymerases to the lesion, suggesting a temporal order of their recruitment. The similarity of REV1 and REV3 mutant phenotypes confirms that the involvement of polymerase ζ in TLS is largely determined by its recruitment to DNA by REV1. Our data demonstrate the influence of the TLS polymerase recruitment mechanism on the success and accuracy of bypass.
authors.affiliation: Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: Agnes
authors.initials: A
authors.lastname: Varga
authors.forename: Adam P
authors.initials: AP
authors.lastname: Marcus
authors.forename: Masayuki
authors.initials: M
authors.lastname: Himoto
authors.forename: Shigenori
authors.initials: S
authors.lastname: Iwai
authors.forename: Dávid
authors.initials: D
authors.lastname: Szüts
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002645:Chickens; D004249:DNA Damage / Q000528:radiation effects; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009713:Nucleotidyltransferases / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D011740:Pyrimidine Dimers / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects*; D016196:S Phase; D054875:Ubiquitination; D014466:Ultraviolet Rays / Q000009:adverse effects*; D000099270:Y-Family DNA Polymerases
chemical_list: D004259:DNA-Directed DNA Polymerase; D009713:Nucleotidyltransferases; D018809:Proliferating Cell Nuclear Antigen; D011740:Pyrimidine Dimers; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases