A structural basis for metal ion mutagenicity and nucleotide selectivity in human DNA polymerase beta
A structural basis for metal ion mutagenicity and nucleotide selectivity in human DNA polymerase beta
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DOI：
10.1021/bi9529566
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发表时间：
1996-10-01
期刊：
影响因子：
2.9
通讯作者：
Kraut, J
中科院分区：
文献类型：
--
作者：
Pelletier, H;Sawaya, MR;Kraut, J
当人DNA聚合酶β（pol β）的晶体与DNA复合时[Pelletier，H.，Sawaya，M. R.，Wolfle，W.，威尔逊，S。H、& Kraut，J.（1996）Biochemistry 35，12742-12761]在dATP和Mn 2+存在下浸泡，X射线结构分析表明，核苷酸转移到引物3 ′-OH直接发生在晶体中，即使DNA在活性位点处是平端的。在类似的晶体浸泡条件下，当Mn 2+被Mg 2+取代时，没有证据表明发生了反应，Mg 2+被认为是体内大多数聚合酶所利用的二价金属离子。这些结果表明，Mn 2+可能表现为一种方式。其对聚合酶的诱变作用是通过在催化位点促进比Mg 2+更大的反应性，从而允许核苷酸基转移反应在很少或不考虑来自模板的指令的情况下发生。当pol β-DNA共晶体在dATP和Zn 2+的存在下浸泡时，也观察到非模板定向的核苷酸转移，但反应产物的不同之处在于掺入的核苷酸的糖部分出现扭曲或以其他方式裂解，这与Zn 2+可以作为聚合酶抑制剂而不是作为诱变剂的报道一致[Sirover，M.一、& Loeb，L. A.（1976）Science 194，1434-1436]。尽管当晶体在dATP和其他金属离子如Ca 2+、Co 2+、Cr 3+或Ni 2+存在下浸泡时没有观察到反应，但X射线结构分析表明，这些金属离子以与Mg 2+不同的方式与核苷酸的三磷酸部分配位。此外，除了Mg 2+之外，所有测试的金属离子都促进天冬氨酸192侧链位置的变化，天冬氨酸192是三个高度保守的活性位点羧酸残基之一。用除dATP以外的核苷酸（即dCTP、dCTP、dTTP、ATP、ddATP、ddCTP、AZT-TP和dATP α S）进行的浸泡实验揭示了核苷酸的三磷酸和糖部分在pol β上的非碱基特异性结合位点，表明了核苷酸选择性的可能机制，其中三磷酸-糖结合先于检查与模板的正确碱基配对。
When crystals of human DNA polymerase beta (pol beta) complexed with DNA [Pelletier, H., Sawaya, M. R., Wolfle, W., Wilson, S. H., & Kraut, J. (1996) Biochemistry 35, 12742-12761] are soaked in the presence of dATP and Mn2+, X-ray structural analysis shows that nucleotidyl transfer to the primer 3'-OH takes place directly in the crystals, even though the DNA is blunt-ended at the active site. Under similar crystal-soaking conditions, there is no evidence for a reaction when Mn2+ is replaced by Mg2+ which is thought to be the divalent metal ion utilized by most polymerases in vivo. These results suggest that one way Mn2+ may manifest. its mutagenic effect on polymerases is by promoting greater reactivity than Mg2+ at the catalytic site, thereby allowing the nucleotidyl transfer reaction to take place with Little or no regard to instructions from a template. Non-template-directed nucleotidyl transfer is also observed when pol beta-DNA cocrystals are soaked in the presence of dATP and Zn2+, but the reaction products differ in that the sugar moiety of the incorporated nucleotide appears distorted or otherwise cleaved, in agreement with reports that Zn2+ may act as a polymerase inhibitor rather than as a mutagen [Sirover, M. A., & Loeb, L. A. (1976) Science 194, 1434-1436]. Although no reaction is observed when crystals are soaked in the presence of dATP and other metal ions such as Ca2+, Co2+, Cr3+, Or Ni2+, X-ray structural analyses show that these metal ions coordinate the triphosphate; moiety of the nucleotide in a manner that differs from that observed with Mg2+. In addition, all metal ions tested, with the exception of Mg2+, promote a change in die side-chain position of aspartic acid 192, which is one of three highly conserved active-site carboxylate residues. Soaking experiments with nucleotides other than dATP (namely, dCTP, dCTP, dTTP, ATP, ddATP, ddCTP, AZT-TP, and dATP alpha S) reveal a non-base-specific binding site on pol beta for the triphosphate and sugar moieties of a nucleotide, suggesting a possible mechanism for nucleotide selectivity whereby triphosphate-sugar binding precedes a check for correct base pairing with the template.