Mammalian abasic site base excision repair - Identification of the reaction sequence and rate-determining steps
Mammalian abasic site base excision repair - Identification of the reaction sequence and rate-determining steps
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DOI：
10.1074/jbc.273.33.21203
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发表时间：
1998-08-14
影响因子：
4.8
通讯作者：
Wilson, SH
中科院分区：
文献类型：
--
作者：
Srivastava, DK;Vande Berg, BJ;Wilson, SH
碱基切除修复(BER)是修复基因组DNA中核苷5‘-单磷酸残基损伤的细胞防御机制之一。这种修复途径是由自发的或酶促的N-糖苷键断裂启动的，在双链DNA中产生一个碱性或无嘌呤-脱嘧啶(AP)位点。APⅡ类内切酶、脱氧核糖核酸磷酸(DRP)裂解酶、DNA合成和DNA连接酶活性完全修复AP位点。在哺乳动物细胞核提取物中，BER可由DNA聚合酶β(β-Poll)和DNA连接酶I组成的大分子复合体介导，这两种酶能够贡献四种BER酶活性中的后三种。在本研究中，我们发现AP位点的BER可以用以下纯化的人类蛋白在体外重组：AP内切酶、β-Poll和DNA连接酶I。对BER中各个酶步骤的检测使我们能够确定一条有序的反应途径：在AP内切酶对含有AP位点的DNA链进行5‘’“缺口”切割之后，β-Poll在去除缺口中的5‘-DRP部分之前进行DNA合成。DNA连接酶I严格要求去除(DRP)瓣来封闭产生的缺口，此外，还测量了重组BER系统的催化速率和单个酶的活性。重组的BER系统对AP位点DNA的修复速度慢于AP内切酶、DNA合成和连接的相应速度，这表明在整个重组的BER系统中，这些步骤并不是速率决定的，相反，重组系统中的限速步骤是去除DRP Tie。DRP裂解酶)，这项工作是首次在体外反应中测量误码率。讨论了含DRP中间体在BER调节中的潜在意义。
Base excision repair (BER) is one of the cellular defense mechanisms repairing damage to nucleoside 5'-monophosphate residues in genomic DNA. This repair pathway is initiated by spontaneous or enzymatic N-glycosidic bond cleavage creating an abasic or apurinic-apyrimidinic (AP) site in double-stranded DNA. Class II AP endonuclease, deoxyribonucleotide phosphate (dRP) lyase, DNA synthesis, and DNA ligase activities complete repair of the AP site. In mammalian cell nuclear extract, BER can be mediated by a macromolecular complex containing DNA polymerase beta (beta-pol) and DNA ligase I. These two enzymes are capable of contributing the latter three of the four BER enzymatic activities. In the present study, we found that AP site BER can be reconstituted in vitro using the following purified human proteins:AP endonuclease, beta-pol, and DNA ligase I. Examination of the individual enzymatic steps in BER allowed us to identify an ordered reaction pathway: subsequent to 5' "nicking" of the AP site-containing DNA strand by AP endonuclease, beta-pol performs DNA synthesis prior to removal of the 5'-dRP moiety in the gap. Removal of the (dRP flap is strictly required for DNA ligase I to seal the resulting nick, Additionally, the catalytic rate of the reconstituted BER system and the individual enzymatic activities was measured. The reconstituted BER system performs repair of AP site DNA at a rate that is slower than the respective rates of AP endonuclease, DNA synthesis, and ligation, suggesting that these steps are not rate-determining in the overall reconstituted BER system, Instead, the rate-limiting step in the reconstituted system was found to be removal of dRP tie. dRP lyase), catalyzed by the amino-terminal domain of beta-pol, This work is the first to measure the rate of BER in an in vitro reaction. The potential significance of the dRP-containing intermediate in the regulation of BER is discussed.