title: Repriming by PrimPol is critical for DNA replication restart downstream of lesions and chain-terminating nucleosides.
abstract: PrimPol is a DNA damage tolerance enzyme possessing both translesion synthesis (TLS) and primase activities. To uncover its potential role in TLS-mediated IgVλ hypermutation and define its interplay with other TLS polymerases, PrimPol(-/-) and PrimPol(-/-)/Polη(-/-)/Polζ (-/-) gene knockouts were generated in avian cells. Loss of PrimPol had no significant impact on the rate of hypermutation or the mutation spectrum of IgVλ. However, PrimPol(-/-) cells were sensitive to methylmethane sulfonate, suggesting that it may bypass abasic sites at the IgVλ segment by repriming DNA synthesis downstream of these sites. PrimPol(-/-) cells were also sensitive to cisplatin and hydroxyurea, indicating that it assists in maintaining / restarting replication at a variety of lesions. To accurately measure the relative contribution of the TLS and primase activities, we examined DNA damage sensitivity in PrimPol(-/-) cells complemented with polymerase or primase-deficient PrimPol. Polymerase-defective, but not primase-deficient, PrimPol suppresses the hypersensitivity of PrimPol(-/-) cells. This indicates that its primase, rather than TLS activity, is pivotal for DNA damage tolerance. Loss of TLS polymerases, Polη and Polζ has an additive effect on the sensitivity of PrimPol(-/-) cells. Moreover, we found that PrimPol and Polη-Polζ redundantly prevented cell death and facilitated unperturbed cell cycle progression. PrimPol(-/-) cells also exhibited increased sensitivity to a wide variety of chain-terminating nucleoside analogs (CTNAs). PrimPol could perform close-coupled repriming downstream of CTNAs and oxidative damage in vitro. Together, these results indicate that PrimPol's repriming activity plays a central role in reinitiating replication downstream from CTNAs and other specific DNA lesions.
authors.affiliation: a Department of Chemistry , Graduate School of Science and Engineering, Tokyo Metropolitan University , Hachioji-shi, Tokyo , Japan.
authors.forename: Kaori
authors.initials: K
authors.lastname: Kobayashi
authors.affiliation: b Genome Damage and Stability Center, School of Life Sciences, University of Sussex , Brighton , UK.
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Guilliam
authors.affiliation: c Department of Radiation Genetics , Graduate School of Medicine, Kyoto University, Yoshidakonoe , Sakyo-ku, Kyoto , Japan.
authors.forename: Masataka
authors.initials: M
authors.lastname: Tsuda
authors.affiliation: d Division of Chemistry, Graduate School of Engineering Science, Osaka University , Toyonaka, Osaka , Japan.
authors.forename: Junpei
authors.initials: J
authors.lastname: Yamamoto
authors.affiliation: b Genome Damage and Stability Center, School of Life Sciences, University of Sussex , Brighton , UK.
authors.forename: Laura J
authors.initials: LJ
authors.lastname: Bailey
authors.affiliation: d Division of Chemistry, Graduate School of Engineering Science, Osaka University , Toyonaka, Osaka , Japan.
authors.forename: Shigenori
authors.initials: S
authors.lastname: Iwai
authors.affiliation: c Department of Radiation Genetics , Graduate School of Medicine, Kyoto University, Yoshidakonoe , Sakyo-ku, Kyoto , Japan.
authors.forename: Shunichi
authors.initials: S
authors.lastname: Takeda
authors.affiliation: b Genome Damage and Stability Center, School of Life Sciences, University of Sussex , Brighton , UK.
authors.forename: Aidan J
authors.initials: AJ
authors.lastname: Doherty
authors.affiliation: a Department of Chemistry , Graduate School of Science and Engineering, Tokyo Metropolitan University , Hachioji-shi, Tokyo , Japan.
authors.forename: Kouji
authors.initials: K
authors.lastname: Hirota
mesh_terms: D000818:Animals; D055162:Biocatalysis / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D002645:Chickens; D002945:Cisplatin / Q000494:pharmacology; D004249:DNA Damage; D019915:DNA Primase / Q000378:metabolism*; D004261:DNA Replication / Q000187:drug effects / Q000528:radiation effects; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000235:genetics; D007146:Immunoglobulin lambda-Chains / Q000235:genetics; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D009705:Nucleosides / Q000378:metabolism*; D027041:Somatic Hypermutation, Immunoglobulin / Q000187:drug effects; D014466:Ultraviolet Rays; D000099270:Y-Family DNA Polymerases
chemical_list: D002945:Cisplatin; D019915:DNA Primase; D004259:DNA-Directed DNA Polymerase; D007135:Immunoglobulin Variable Region; D007146:Immunoglobulin lambda-Chains; D008741:Methyl Methanesulfonate; D009705:Nucleosides; C425929:DNA polymerase zeta; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases
keywords: DT40; chain-terminating nucleoside analogs (CTNAs); primase; primpol; polymerase; replication; repriming; restart