Human Pol ζ purified with accessory subunits is active in translesion DNA synthesis and complements Pol. in cisplatin bypass
Human Pol ζ purified with accessory subunits is active in translesion DNA synthesis and complements Pol. in cisplatin bypass
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DOI：
10.1073/pnas.1324001111
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发表时间：
2014-02-25
影响因子：
11.1
通讯作者：
Yang, Wei
中科院分区：
文献类型：
--
作者：
Lee, Young-Sam;Gregory, Mark T.;Yang, Wei
DNA聚合酶zeta（Pol zeta）是真核生物B家族的DNA聚合酶，其特异于跨损伤合成并且对于正常胚胎发生是必需的。Pol zeta至少由催化亚基Rev 3和辅助亚基Rev 7组成。哺乳动物Rev 3含有> 3，000个残基，是酵母同源物的两倍大。迄今为止，没有脊椎动物Pol zeta已被纯化用于生物化学表征。在这里，我们报告了一系列人Rev 3缺失构建体在HEK 293细胞中表达的纯化和最低催化活性的人Pol zeta变体的鉴定。利用活性Pol zeta变体的标记形式，我们分离了人Pol zeta的另外两个辅助亚基，PolD 2和PolD 3。纯化的四亚基Pol zeta 4（Rev 3-Rev 7-PolD 2-PolD 3）在绕过1，2-链内d（GpG）-顺铂交联方面比两亚基Pol zeta 2（Rev 3-Rev 7）更有效且更有进行性。我们表明，顺铂病变的完全旁路需要Pol eta在3'鸟嘌呤对面插入dCTP，Pol zeta 4延伸引物。
DNA polymerase zeta (Pol zeta) is a eukaryotic B-family DNA polymerase that specializes in translesion synthesis and is essential for normal embryogenesis. At a minimum, Pol zeta consists of a catalytic subunit Rev3 and an accessory subunit Rev7. Mammalian Rev3 contains >3,000 residues and is twice as large as the yeast homolog. To date, no vertebrate Pol zeta has been purified for biochemical characterization. Here we report purification of a series of human Rev3 deletion constructs expressed in HEK293 cells and identification of a minimally catalytically active human Pol zeta variant. With a tagged form of an active Pol zeta variant, we isolated two additional accessory subunits of human Pol zeta, PolD2 and PolD3. The purified four-subunit Pol zeta 4 (Rev3-Rev7-PolD2-PolD3) is much more efficient and more processive at bypassing a 1,2-intrastrand d(GpG)-cisplatin cross-link than the two-subunit Pol zeta 2 (Rev3-Rev7). We show that complete bypass of cisplatin lesions requires Pol eta to insert dCTP opposite the 3' guanine and Pol zeta 4 to extend the primers.