Crystal structures of human DNA polymerase beta complexed with DNA: Implications for catalytic mechanism, processivity, and fidelity
Crystal structures of human DNA polymerase beta complexed with DNA: Implications for catalytic mechanism, processivity, and fidelity
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DOI：
10.1021/bi952955d
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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
哺乳动物DNA聚合酶β（pol β）是一种小的（39 kDa）DNA间隙填充酶，其包含氨基末端8-kDa结构域和羧基末端31-kDa结构域。在本文报道的工作中，与DNA的平端片段复合的人pol β的晶体结构显示，尽管晶体属于不同的空间群，但DNA仍然以与先前报道的与模板引物和ddCTP复合的大鼠pol β的结构中的DNA相同的方式结合在pal β结合通道中[Pelletier，H.，Sawaya，M. R.，库马尔，A.，威尔逊，S. H、和Kraut，J.（1994）Science 264，1891-1903]。8-kDa结构域是在三个先前观察到的位置相对于31-kDa结构域，这表明8-kDa结构域可能承担只有少量的稳定构象之一。拇指亚结构域在人pol β-DNA二元复合物中比在大鼠pol β-DNA-ddCTP三元复合物中处于更开放的位置，并且核苷酸结合后的闭合拇指可以代表在前稳态动力学研究中观察到的限速构象变化。DNA和一个与DNA相关的pol β分子的8-kDa结构域之间的分子间接触揭示了在远离缺口DNA底物的下游寡核苷酸的8-kDa结构域上的一个似乎合理的结合位点;除了容纳5 ′-PO 4端基的富含赖氨酸的结合口袋外，8-kDa结构域还含有新发现的螺旋-发夹-螺旋（HhH）基序，其以与31-kDa结构域中结构上和顺序上同源的HhH基序相同的方式结合DNA。金属离子促进了HhH基序与DNA的结合，因为HhH基序已经在其他DNA修复酶和DNA聚合酶中被鉴定，所以在pol β中观察到的HhH-DNA相互作用可能适用于广泛的DNA结合蛋白。来自大肠杆菌的内切核酸酶III的HhH基序与pol β的8-kDa结构域的HhH基序之间的序列相似性特别引人注目，因为所有保守残基聚集在一个短序列片段LPGVGXK中，其中LPGV对应于II型β-转角GXK对应于HhH基序的一部分，该基序被认为对DNA结合和两种酶的催化至关重要。这些结果表明，核酸内切酶III和8-kDa的结构域的聚合酶β可能采用类似的模式的DNA结合，并可能有类似的催化机制，为各自的DNA裂解酶的活动。pol β与缺口DNA底物的有效结合的模型需要单链模板中的90度弯曲，这可以增强DNA修复或复制期间的核苷酸选择性。
Mammalian DNA palymerase beta (pol beta) is a small (39 kDa) DNA gap-filling enzyme that comprises an amino-terminal 8-kDa domain and a carboxy-terminal 31-kDa domain. In the work reported here, crystal structures of human poi beta complexed with blunt-ended segments of DNA show that, although the crystals belong to a different space group, the DNA is nevertheless bound in the pal beta binding channel in the same way as the DNA in previously reported structures of rat pol beta complexed with a template-primer and ddCTP [Pelletier, H., Sawaya, M. R., Kumar, A., Wilson, S. H., & Kraut, J. (1994) Science 264, 1891-1903]. The 8-kDa domain is in one of three previously observed positions relative to the 31-kDa domain, suggesting that the 8-kDa domain may assume only a small number of stable conformations. The thumb subdomain is in a more open position in the human poi beta-DNA binary complex than it is in the rat pol beta-DNA-ddCTP ternary complex, and a closing thumb upon nucleotide binding could represent the rate-limiting conformational change that has been observed in pre-steady-state kinetic studies. Intermolecular contacts between the DNA and the 8-kDa domain of a symmetry-related pol beta molecule reveal a plausible binding site on the 8-kDa domain far the downstream oligonucleotide of a gapped-DNA substrate; in addition to a lysine-rich binding pocket that accommodates a 5'-PO4 end group, the 8-kDa domain also contains a newly discovered helix-hairpin-helix (HhH) motif that binds to DNA in the same way as does a structurally and sequentially homologous HhH motif in the 31-kDa domain. DNA binding by both HhH motifs is facilitated by a metal ion, In that HhH motifs have been identified in other DNA repair enzymes and DNA polymerases, the HhH-DNA interactions observed in pol beta may be applicable to a broad range of DNA binding proteins. The sequence similarity between the HhH motif of endonuclease III from Escherichia coli and the HhH motif of the 8-kDa domain of pol beta is particularly striking in that all of the conserved residues are clustered in one short sequence segment, LPGVGXK, where LPGV corresponds to a type II beta-turn (the hairpin turn), and GXK corresponds to a part of the HhH motif that is proposed to be critical for DNA binding and catalysis for both enzymes. These results suggest that endonuclease III and the 8-kDa domain of pol beta may employ a similar mode of DNA binding and may have similar catalytic mechanisms for their respective DNA lyase activities. A model for productive binding of pol beta to a gapped-DNA substrate requires a 90 degrees bend in the single-stranded template, which could enhance nucleotide selectivity during DNA repair or replication.