title: Roles of PCNA ubiquitination and TLS polymerases κ and η in the bypass of methyl methanesulfonate-induced DNA damage.
abstract: Translesion synthesis (TLS) provides a highly conserved mechanism that enables DNA synthesis on a damaged template. TLS is performed by specialized DNA polymerases of which polymerase (Pol) κ is important for the cellular response to DNA damage induced by benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), ultraviolet (UV) light and the alkylating agent methyl methanesulfonate (MMS). As TLS polymerases are intrinsically error-prone, tight regulation of their activity is required. One level of control is provided by ubiquitination of the homotrimeric DNA clamp PCNA at lysine residue 164 (PCNA-Ub). We here show that Polκ can function independently of PCNA modification and that Polη can function as a backup during TLS of MMS-induced lesions. Compared to cell lines deficient for PCNA modification (Pcna(K164R)) or Polκ, double mutant cell lines display hypersensitivity to MMS but not to BPDE or UV-C. Double mutant cells also displayed delayed post-replicative TLS, accumulate higher levels of replication stress and delayed S-phase progression. Furthermore, we show that Polη and Polκ are redundant in the DNA damage bypass of MMS-induced DNA damage. Taken together, we provide evidence for PCNA-Ub-independent activation of Polκ and establish Polη as an important backup polymerase in the absence of Polκ in response to MMS-induced DNA damage.
authors.affiliation: Division of Biological Stress Responses, The Netherlands Cancer Institute, Amsterdam, The Netherlands.
authors.forename: Niek
authors.initials: N
authors.lastname: Wit
authors.affiliation: Division of Biological Stress Responses, The Netherlands Cancer Institute, Amsterdam, The Netherlands.
authors.forename: Olimpia Alessandra
authors.initials: OA
authors.lastname: Buoninfante
authors.affiliation: Division of Biological Stress Responses, The Netherlands Cancer Institute, Amsterdam, The Netherlands.
authors.forename: Paul C M
authors.initials: PC
authors.lastname: van den Berk
authors.affiliation: Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Jacob G
authors.initials: JG
authors.lastname: Jansen
authors.affiliation: Division of Biological Stress Responses, The Netherlands Cancer Institute, Amsterdam, The Netherlands.
authors.forename: Marc A
authors.initials: MA
authors.lastname: Hogenbirk
authors.affiliation: Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Niels
authors.initials: N
authors.lastname: de Wind
authors.affiliation: Division of Biological Stress Responses, The Netherlands Cancer Institute, Amsterdam, The Netherlands h.jacobs@nki.nl.
authors.forename: Heinz
authors.initials: H
authors.lastname: Jacobs
mesh_terms: D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins / Q000378:metabolism; D002470:Cell Survival; D002478:Cells, Cultured; D000071877:Checkpoint Kinase 1; D004249:DNA Damage; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000502:physiology*; D008741:Methyl Methanesulfonate / Q000633:toxicity; D018345:Mice, Knockout; D009154:Mutation; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism; D016196:S Phase; D054875:Ubiquitination; D000099270:Y-Family DNA Polymerases
chemical_list: D064007:Ataxia Telangiectasia Mutated Proteins; D000071877:Checkpoint Kinase 1; D004259:DNA-Directed DNA Polymerase; D008741:Methyl Methanesulfonate; D018809:Proliferating Cell Nuclear Antigen; D011494:Protein Kinases; C576897:Atr protein, mouse; C498613:Polk protein, mouse; D000099270:Y-Family DNA Polymerases; C112338:Rad30 protein