title: PCNA ubiquitination is important, but not essential for translesion DNA synthesis in mammalian cells.
abstract: Translesion DNA synthesis (TLS) is a DNA damage tolerance mechanism in which specialized low-fidelity DNA polymerases bypass replication-blocking lesions, and it is usually associated with mutagenesis. In Saccharomyces cerevisiae a key event in TLS is the monoubiquitination of PCNA, which enables recruitment of the specialized polymerases to the damaged site through their ubiquitin-binding domain. In mammals, however, there is a debate on the requirement for ubiquitinated PCNA (PCNA-Ub) in TLS. We show that UV-induced Rpa foci, indicative of single-stranded DNA (ssDNA) regions caused by UV, accumulate faster and disappear more slowly in Pcna(K164R/K164R) cells, which are resistant to PCNA ubiquitination, compared to Pcna(+/+) cells, consistent with a TLS defect. Direct analysis of TLS in these cells, using gapped plasmids with site-specific lesions, showed that TLS is strongly reduced across UV lesions and the cisplatin-induced intrastrand GG crosslink. A similar effect was obtained in cells lacking Rad18, the E3 ubiquitin ligase which monoubiquitinates PCNA. Consistently, cells lacking Usp1, the enzyme that de-ubiquitinates PCNA exhibited increased TLS across a UV lesion and the cisplatin adduct. In contrast, cells lacking the Rad5-homologs Shprh and Hltf, which polyubiquitinate PCNA, exhibited normal TLS. Knocking down the expression of the TLS genes Rev3L, PolH, or Rev1 in Pcna(K164R/K164R) mouse embryo fibroblasts caused each an increased sensitivity to UV radiation, indicating the existence of TLS pathways that are independent of PCNA-Ub. Taken together these results indicate that PCNA-Ub is required for maximal TLS. However, TLS polymerases can be recruited to damaged DNA also in the absence of PCNA-Ub, and perform TLS, albeit at a significantly lower efficiency and altered mutagenic specificity.
authors.affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel.
authors.forename: Ayal
authors.initials: A
authors.lastname: Hendel
authors.forename: Peter H L
authors.initials: PH
authors.lastname: Krijger
authors.forename: Noam
authors.initials: N
authors.lastname: Diamant
authors.forename: Zohar
authors.initials: Z
authors.lastname: Goren
authors.forename: Petra
authors.initials: P
authors.lastname: Langerak
authors.forename: Jungmin
authors.initials: J
authors.lastname: Kim
authors.forename: Thomas
authors.initials: T
authors.lastname: Reissner
authors.forename: Kyoo-young
authors.initials: KY
authors.lastname: Lee
authors.forename: Nicholas E
authors.initials: NE
authors.lastname: Geacintov
authors.forename: Thomas
authors.initials: T
authors.lastname: Carell
authors.forename: Kyungjae
authors.initials: K
authors.lastname: Myung
authors.forename: Satoshi
authors.initials: S
authors.lastname: Tateishi
authors.forename: Alan
authors.initials: A
authors.lastname: D'Andrea
authors.forename: Heinz
authors.initials: H
authors.lastname: Jacobs
authors.forename: Zvi
authors.initials: Z
authors.lastname: Livneh
mesh_terms: D000818:Animals; D002945:Cisplatin / Q000494:pharmacology; D004247:DNA / Q000096:biosynthesis* / Q000187:drug effects / Q000235:genetics; D004249:DNA Damage; D004260:DNA Repair; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D016296:Mutagenesis; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism*; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism; D054875:Ubiquitination; D014466:Ultraviolet Rays
chemical_list: D004277:DNA, Single-Stranded; D018809:Proliferating Cell Nuclear Antigen; D025801:Ubiquitin; D004247:DNA; D002945:Cisplatin