The base substitution fidelity of DNA polymerase β-dependent single nucleotide base excision repair
The base substitution fidelity of DNA polymerase β-dependent single nucleotide base excision repair
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DOI：
10.1074/jbc.c300170200
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发表时间：
2003-07-11
影响因子：
4.8
通讯作者：
Kunkel, TA
中科院分区：
文献类型：
--
作者：
Matsuda, T;Berg, BJV;Kunkel, TA
通过碱基切除修复（BER）从哺乳动物基因组中去除受损的DNA碱基。单核苷酸BER需要几种酶活性，包括DNA聚合酶和5 '，2'-脱氧核糖-5-磷酸裂解酶。这两种活性都是四种人类DNA聚合酶所固有的，它们在间隙填充DNA合成期间的碱基取代错误率变化超过10，000倍。这表明BER保真度可以以酶依赖性方式在宽范围内变化。为了研究这种可能性，在这里，我们描述了一种测定方法来测量用纯化的酶重建的BER反应的保真度。当人尿嘧啶DNA糖基化酶、AP核酸内切酶、DNA聚合酶β和DNA连接酶1取代尿嘧啶相对模板A或G时，碱基取代错误率小于或等于0.3至小于或等于2.8 × 10 - 4。当反应中包含过量的不正确的dNTP或当野生型DNA聚合酶β被以较低保真度填充单核苷酸空位的DNA聚合酶β变体取代时，BER错误率较高。在这些条件下，聚合酶β依赖性BER的碱基取代保真度比单独由聚合酶β进行的单核苷酸空位填充高3-8倍。因此，BER反应中的其他蛋白质可以增强DNA聚合酶β在单核苷酸BER期间的碱基取代保真度。
Damaged DNA bases are removed from mammalian genomes by base excision repair (BER). Single nucleotide BER requires several enzymatic activities, including DNA polymerase and 5',2'-deoxyribose-5-phosphate lyase. Both activities are intrinsic to four human DNA polymerases whose base substitution error rate during gap-filling DNA synthesis varies by more than 10,000-fold. This suggests that BER fidelity could vary over a wide range in an enzyme dependent manner. To investigate this possibility, here we describe an assay to measure the fidelity of BER reactions reconstituted with purified enzymes. When human uracil DNA glycosylase, AP endonuclease, DNA polymerase beta, and DNA ligase 1 replace uracil opposite template A or G, base substitution error rates are less than or equal to0.3 to less than or equal to2.8 x 10(-4). BER error rates are higher when excess incorrect dNTPs are included in the reaction or when wild type DNA polymerase beta is replaced by DNA polymerase beta variants that fill single nucleotide gaps with lower fidelity. Under these conditions, the base substitution fidelity of polymerase beta-dependent BER is 3-8-fold higher than is single nucleotide gap filling by polymerase beta alone. Thus other proteins in the BER reaction may enhance the base substitution fidelity of DNA polymerase beta during single nucleotide BER.