Tomida Junya, Takata Kei-ichi, Lange Sabine S, Schibler Andria C, Yousefzadeh Matthew J, Bhetawal Sarita, Dent Sharon Y R, Wood Richard D
Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center Science Park, Smithville, TX 78957, USA.
Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center Science Park, Smithville, TX 78957, USA The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, TX 77030, USA.
Nucleic Acids Res. 2015 Jan;43(2):1000-11. doi: 10.1093/nar/gku1385. Epub 2015 Jan 7.
DNA polymerase zeta (pol ζ) is exceptionally important for controlling mutagenesis and genetic instability. REV3L comprises the catalytic subunit, while REV7 (MAD2L2) is considered an accessory subunit. However, it has not been established that the role of REV7 in DNA damage tolerance is necessarily connected with mammalian pol ζ, and there is accumulating evidence that REV7 and REV3L have independent functions. Analysis of pol ζ has been hampered by difficulties in expression of REV3L in mammalian cells, and lack of a functional complementation system. Here, we report that REV7 interacts with full-length REV3L in vivo and we identify a new conserved REV7 interaction site in human REV3L (residues 1993-2003), distinct from the known binding site (residues 1877-1887). Mutation of both REV7-binding sites eliminates the REV3L-REV7 interaction. In vivo complementation shows that both REV7-binding sites in REV3L are necessary for preventing spontaneous chromosome breaks and conferring resistance to UV radiation and cisplatin. This demonstrates a damage-specific function of REV7 in pol ζ, in contrast to the distinct roles of REV3L and REV7 in primary cell viability and embryogenesis.
DNA聚合酶ζ（pol ζ）对于控制诱变和遗传不稳定性异常重要。REV3L构成催化亚基，而REV7（MAD2L2）被认为是辅助亚基。然而，尚未确定REV7在DNA损伤耐受中的作用必然与哺乳动物pol ζ相关，并且有越来越多的证据表明REV7和REV3L具有独立的功能。由于REV3L在哺乳动物细胞中表达困难以及缺乏功能性互补系统，对pol ζ的分析受到了阻碍。在此，我们报告REV7在体内与全长REV3L相互作用，并且我们在人REV3L中鉴定出一个新的保守REV7相互作用位点（第1993 - 2003位氨基酸残基），与已知结合位点（第1877 - 1887位氨基酸残基）不同。两个REV7结合位点的突变消除了REV3L - REV7相互作用。体内互补表明，REV3L中的两个REV7结合位点对于防止自发染色体断裂以及赋予对紫外线辐射和顺铂的抗性都是必需的。这证明了REV7在pol ζ中具有损伤特异性功能，这与REV3L和REV7在原代细胞活力和胚胎发生中的不同作用形成对比。
