DNA synthesis and dRPase activities of polymerase beta are both essential for single-nucleotide patch base excision repair in mammalian cell extracts.
DNA synthesis and dRPase activities of polymerase beta are both essential for single-nucleotide patch base excision repair in mammalian cell extracts.
复制标题
DNA 合成和聚合酶 β 的 dRPase 活性对于哺乳动物细胞提取物中的单核苷酸补丁碱基切除修复都是必需的。
DOI：
10.1021/bi002064s
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发表时间：
2001
期刊：
影响因子：
2.9
通讯作者：
Dianov,GL
中科院分区：
文献类型：
--
作者：
Podlutsky,AJ;Dianova,II;Wilson,SH;Bohr,VA;Dianov,GL
在哺乳动物细胞中，DNA中大多数碱基的改变是通过单核苷酸补片碱基切除修复机制进行的。碱基切除修复是由DNA糖基化酶启动的，它去除受损的碱基并产生一个碱基位点（AP位点）。该AP位点被AP内切酶活性进一步加工，该酶活性切割AP位点附近的磷酸二酯键，并产生含有3 ' -OH和5 ' -糖磷酸端的链断裂。在哺乳动物细胞中，5 ' -糖磷酸被DNA聚合酶β (Pol β)的AP裂解酶活性去除。同样的酶也会填补空隙，DNA末端最终通过DNA连接酶重新连接起来。我们测量了正常小鼠和Pol β-null小鼠细胞提取物中含有单个AP位点的寡核苷酸底物的修复，结果表明，Pol β-null提取物中减少的修复可以通过添加纯化的Pol β来补充。通过这种互补实验，我们证明了没有dRPase活性的突变Pol β能够刺激长斑块BER。突变体Pol β缺乏DNA合成，但dRPase活性正常，不刺激Pol β-null细胞的修复。然而，在我们测量仅通过单核苷酸补丁BER完成的碱基切除修复的条件下，无论是单独的dRPase还是Pol β的DNA合成突变体，还是两者一起，都无法弥补修复缺陷。这些数据表明，dRPase和Pol β的DNA合成活性是偶联的，并且Pol β的这两种功能在短片段BER中都是必需的，并且不能被其他细胞酶有效地取代。
In mammalian cells the majority of altered bases in DNA are processed through a single-nucleotide patch base excision repair mechanism. Base excision repair is initiated by a DNA glycosylase that removes a damaged base and generates an abasic site (AP site). This AP site is further processed by an AP endonuclease activity that incises the phosphodiester bond adjacent to the AP site and generates a strand break containing 3‘-OH and 5‘-sugar phosphate ends. In mammalian cells, the 5‘-sugar phosphate is removed by the AP lyase activity of DNA polymerase β (Pol β). The same enzyme also fills the gap, and the DNA ends are finally rejoined by DNA ligase. We measured repair of oligonucleotide substrates containing a single AP site in cell extracts prepared from normal and Pol β-null mouse cells and show that the reduced repair in Pol β-null extracts can be complemented by addition of purified Pol β. Using this complementation assay, we demonstrate that mutated Pol β without dRPase activity is able to stimulate long patch BER. Mutant Pol β deficient in DNA synthesis, but with normal dRPase activity, does not stimulate repair in Pol β-null cells. However, under conditions where we measure base excision repair accomplished exclusively through a single-nucleotide patch BER, neither dRPase nor DNA synthesis mutants of Pol β alone, or the two together, were able to complement the repair defect. These data suggest that the dRPase and DNA synthesis activities of Pol β are coupled and that both of these Pol β functions are essential during short patch BER and cannot be efficiently substituted by other cellular enzymes.