Increased dNTP binding affinity reveals a nonprocessive role for Escherichia coli β clamp with DNA polymerase IV
Increased dNTP binding affinity reveals a nonprocessive role for Escherichia coli β clamp with DNA polymerase IV
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DOI：
10.1074/jbc.c400265200
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发表时间：
2004-08-06
影响因子：
4.8
通讯作者：
Goodman, MF
中科院分区：
文献类型：
--
作者：
Bertram, JG;Bloom, LB;Goodman, MF
在大肠杆菌中，复制叉经常在未受损或受损的模板位点停止。随后DNA合成的恢复是通过用三种损伤诱导的DNA聚合酶II、IV或v中的一种取代DNA聚合酶III（通过β滑动钳与DNA结合）来实现的。β滑动钳的主要作用是将通常弱结合的聚合酶系在DNA上，从而增加其加工能力。与其他大肠杆菌聚合酶相比，DNA聚合酶IV结合dNTP底物的亲和力低约10倍，如果不加以控制，可能会阻碍其在体内合成DNA的能力。在这里，我们报告了β钳的一个新特性，当它与DNA聚合酶IV结合时，会导致dNTP结合亲和力的大幅增加，从而同时提高核苷酸在正常和瞬时滑移错配引物/模板端结合的效率。引物模板DNA滑移导致单核苷酸缺失是DNA聚合酶IV不贞的生物学标志，负责增强细胞适应性以应对压力。我们表明，DNA聚合酶IV- dntp结合亲和力的增加是DNA聚合酶IV- β钳相互作用的内在特性，而不是DNA聚合酶IV与DNA结合增加的间接结果。
Replication forks often stall at undamaged or damaged template sites in Escherichia coli. Subsequent resumption of DNA synthesis occurs by replacing DNA polymerase III, which is bound to DNA by the beta-sliding clamp, with one of three damage-induced DNA polymerases II, IV, or V. The principal role of the beta clamp is to tether the normally weakly bound polmerases to DNA thereby increasing their processivities. DNA polymerase IV binds dNTP substrates with about 10-fold lower affinity compared with the other E. coli polymerases, which if left unchecked could hinder its ability to synthesize DNA in vivo. Here we report a new property for the beta clamp, which when bound to DNA polymerase IV results in a large increase in dNTP binding affinity that concomitantly increases the efficiency of nucleotide incorporation at normal and transiently slipped mispaired primer/template ends. Primer-template DNA slippage resulting in single nucleotide deletions is a biological hallmark of DNA polymerase IV infidelity responsible for enhancing cell fitness in response to stress. We show that the increased DNA polymerase IV-dNTP binding affinity is an intrinsic property of the DNA polymerase IV-beta clamp interaction and not an indirect consequence of an increased binding of DNA polymerase IV to DNA.