DNA polymerase β and flap endonuclease 1 enzymatic specificities sustain DNA synthesis for long patch base excision repair
DNA polymerase β and flap endonuclease 1 enzymatic specificities sustain DNA synthesis for long patch base excision repair
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DOI：
10.1074/jbc.m412922200
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发表时间：
2005-02-04
影响因子：
4.8
通讯作者：
Wilson, SH
中科院分区：
文献类型：
--
作者：
Liu, Y;Beard, WA;Wilson, SH
DNA聚合酶β（pol β）和瓣状核酸内切酶1（FEN 1）是pol β介导的长补丁碱基切除修复（LP-BER）中的关键参与者。有人提出，这种类型的LP-BER是通过FEN 1去除由pol β链置换DNA合成产生的2至11个核苷酸瓣来实现的。为了理解这些酶在LP-BER过程中如何合作，我们通过利用各种BER中间体来表征纯化的人pol β DNA合成，所述BER中间体包括单核苷酸缺口DNA、切口DNA和具有各种长度的瓣的切口DNA，所述瓣均具有5 '末端四氢呋喃（THF）残基。我们观察到切口DNA和切口-THF瓣DNA是pol β介导的DNA合成的不良底物;然而，DNA合成被纯化的人FEN 1强烈刺激。FEN 1没有改善pol β底物结合。刺激需要FEN 1切割活性，表明FEN 1消除了pol β DNA合成的障碍。此外，FEN 1切割切口和切口-THF瓣DNA导致一个核苷酸缺口的DNA分子，这是一个理想的底物pol β。这项研究表明，pol β与FEN 1合作，通过“打了就跑”的机制，包括交替短缺口生产FEN 1和缺口填充pol β，而不是通过协调形成和去除链移位皮瓣，以消除DNA损伤。
DNA polymerase beta (pol beta) and flap endonuclease 1 (FEN1) are key players in pol beta-mediated long-patch base excision repair (LP-BER). It was proposed that this type of LP-BER is accomplished through FEN1 removal of a 2- to 11-nucleotide flap created by pol beta strand displacement DNA synthesis. To understand how these enzymes might cooperate during LP-BER, we characterized purified human pol beta DNA synthesis by utilizing various BER intermediates, including single-nucleotide-gapped DNA, nicked DNA, and nicked DNA with various lengths of flaps all with a 5'-terminal tetrahydrofuran (THF) residue. We observed that nicked DNA and nicked-THF flap DNA were poor substrates for pol beta-mediated DNA synthesis; yet, DNA synthesis was strongly stimulated by purified human FEN1. FEN1 did not improve pol beta substrate binding. FEN1 cleavage activity was required for the stimulation, suggesting that FEN1 removed a barrier to pol beta DNA synthesis. In addition, FEN1 cleavage on both nicked and nicked-THF flap DNA resulted in a one-nucleotide gapped DNA molecule that was an ideal substrate for pol beta. This study demonstrates that pol beta cooperates with FEN1 to remove DNA damage via a "Hit and Run" mechanism, involving alternating short gap production by FEN1 and gap filling by pol beta, rather than through coordinated formation and removal of a strand-displaced flap.