Structural and biochemical investigation of the role in proofreading of a β hairpin loop found in the exonuclease domain of a replicative DNA polymerase of the B family
Structural and biochemical investigation of the role in proofreading of a β hairpin loop found in the exonuclease domain of a replicative DNA polymerase of the B family
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DOI：
10.1074/jbc.m605675200
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发表时间：
2007-01-12
影响因子：
4.8
通讯作者：
Doublie, Sylvie
中科院分区：
文献类型：
--
作者：
Hogg, Matthew;Aller, Pierre;Doublie, Sylvie
复制型DNA聚合酶，如来自噬菌体T4和RB 69的B家族聚合酶所例示的，不仅复制DNA，而且具有校正错误掺入的核苷酸的能力。由于这两种活性存在于不同的蛋白质结构域中，因此聚合酶必须采用允许引物链在两个活性位点之间有效切换的机制，以实现快速准确的复制。先前的突变和结构研究表明，位于家族B聚合酶的核酸外切酶结构域中的β发夹结构可能在核苷酸错误掺入的情况下在活性位点转换中起重要作用。我们发现，删除RB 69 gp 43中的β发夹环既不影响聚合酶也不影响核酸外切酶活性。然而，具有错配引物末端的单结合事件研究表明，β发夹在引物DNA结合于核酸外切酶活性位点期间，但在校正的引物返回聚合酶活性位点时，在维持聚合酶/DNA相互作用的稳定性方面发挥作用。此外，缺失变体显示出与脱碱基位点相对的核苷酸的更稳定的掺入。此外，在掺入与模板呋喃相对的A的β发夹缺失变体的2.4埃晶体结构中，晶体不对称单元中的所有四个分子在聚合酶活性位点中具有DNA，尽管由于错误掺入而存在DNA扭曲，证实了在不存在β发夹环的情况下引物链不稳定地结合在核酸外切酶活性位点内。
Replicative DNA polymerases, as exemplified by the B family polymerases from bacteriophages T4 and RB69, not only replicate DNA but also have the ability to proofread misincorporated nucleotides. Because the two activities reside in separate protein domains, polymerases must employ a mechanism that allows for efficient switching of the primer strand between the two active sites to achieve fast and accurate replication. Prior mutational and structural studies suggested that a beta hairpin structure located in the exonuclease domain of family B polymerases might play an important role in active site switching in the event of a nucleotide misincorporation. We show that deleting the beta hairpin loop in RB69 gp43 affects neither polymerase nor exonuclease activities. Single binding event studies with mismatched primer termini, however, show that the beta hairpin plays a role in maintaining the stability of the polymerase/DNA interactions during the binding of the primer DNA in the exonuclease active site but not on the return of the corrected primer to the polymerase active site. In addition, the deletion variant showed a more stable incorporation of a nucleotide opposite an abasic site. Moreover, in the 2.4 angstrom crystal structure of the beta hairpin deletion variant incorporating an A opposite a templating furan, all four molecules in the crystal asymmetric unit have DNA in the polymerase active site, despite the presence of DNA distortions because of the misincorporation, confirming that the primer strand is not stably bound within the exonuclease active site in the absence of the beta hairpin loop.