Hirota Kouji, Yoshikiyo Kazunori, Guilbaud Guillaume, Tsurimoto Toshiki, Murai Junko, Tsuda Masataka, Phillips Lara G, Narita Takeo, Nishihara Kana, Kobayashi Kaori, Yamada Kouich, Nakamura Jun, Pommier Yves, Lehmann Alan, Sale Julian E, Takeda Shunichi
Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshidakonoe, Sakyo-ku, Kyoto 606-8501, Japan Department of Chemistry, GraduateSchool of Science and Engineering, Tokyo Metropolitan University, Minami-Osawa, Hachioji- shi, Tokyo 192-0397, Japan.
Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshidakonoe, Sakyo-ku, Kyoto 606-8501, Japan.
Nucleic Acids Res. 2015 Feb 18;43(3):1671-83. doi: 10.1093/nar/gkv023. Epub 2015 Jan 27.
The replicative DNA polymerase Polδ consists of a catalytic subunit POLD1/p125 and three regulatory subunits POLD2/p50, POLD3/p66 and POLD4/p12. The ortholog of POLD3 in Saccharomyces cerevisiae, Pol32, is required for a significant proportion of spontaneous and UV-induced mutagenesis through its additional role in translesion synthesis (TLS) as a subunit of DNA polymerase ζ. Remarkably, chicken DT40 B lymphocytes deficient in POLD3 are viable and able to replicate undamaged genomic DNA with normal kinetics. Like its counterpart in yeast, POLD3 is required for fully effective TLS, its loss resulting in hypersensitivity to a variety of DNA damaging agents, a diminished ability to maintain replication fork progression after UV irradiation and a significant decrease in abasic site-induced mutagenesis in the immunoglobulin loci. However, these defects appear to be largely independent of Polζ, suggesting that POLD3 makes a significant contribution to TLS independently of Polζ in DT40 cells. Indeed, combining polη, polζ and pold3 mutations results in synthetic lethality. Additionally, we show in vitro that POLD3 promotes extension beyond an abasic by the Polδ holoenzyme suggesting that while POLD3 is not required for normal replication, it may help Polδ to complete abasic site bypass independently of canonical TLS polymerases.
复制性DNA聚合酶Polδ由一个催化亚基POLD1/p125和三个调节亚基POLD2/p50、POLD3/p66及POLD4/p12组成。酿酒酵母中POLD3的直系同源物Pol32，因其作为DNA聚合酶ζ的一个亚基在跨损伤合成（TLS）中的额外作用，对于相当一部分自发和紫外线诱导的诱变是必需的。值得注意的是，缺乏POLD3的鸡DT40 B淋巴细胞是有活力的，并且能够以正常动力学复制未受损的基因组DNA。与酵母中的对应物一样，POLD3对于完全有效的TLS是必需的，其缺失导致对多种DNA损伤剂高度敏感，紫外线照射后维持复制叉进展的能力减弱，以及免疫球蛋白基因座中无碱基位点诱导的诱变显著减少。然而，这些缺陷似乎在很大程度上与Polζ无关，这表明在DT40细胞中，POLD3独立于Polζ对TLS有重大贡献。事实上，将polη、polζ和pold3突变结合会导致合成致死。此外，我们在体外表明，POLD3促进Polδ全酶在无碱基位点后的延伸，这表明虽然正常复制不需要POLD3，但它可能帮助Polδ独立于经典TLS聚合酶完成无碱基位点的绕过。
