Bertram Jeffrey G, Bloom Linda B, O'Donnell Mike, Goodman Myron F
Departments of Biological Sciences and Chemistry, Hedco Molecular Biology Laboratories, University of Southern California, University Park, Los Angeles, California 90089-1340, USA.
J Biol Chem. 2004 Aug 6;279(32):33047-50. doi: 10.1074/jbc.C400265200. Epub 2004 Jun 20.
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.
在大肠杆菌中，复制叉常常在未受损或受损的模板位点处停滞。随后，通过用三种损伤诱导的DNA聚合酶II、IV或V之一取代与DNA通过β滑动夹结合的DNA聚合酶III来恢复DNA合成。β夹的主要作用是将通常弱结合的聚合酶拴系到DNA上，从而提高它们的持续合成能力。与其他大肠杆菌聚合酶相比，DNA聚合酶IV结合dNTP底物的亲和力约低10倍，如果不加以控制，这可能会阻碍其在体内合成DNA的能力。在此，我们报道了β夹的一种新特性，当它与DNA聚合酶IV结合时，会导致dNTP结合亲和力大幅增加，同时提高在正常和瞬时滑动错配引物/模板末端的核苷酸掺入效率。导致单核苷酸缺失的引物-模板DNA滑动是DNA聚合酶IV保真度的生物学标志，其负责在应激反应中提高细胞适应性。我们表明，DNA聚合酶IV与dNTP结合亲和力的增加是DNA聚合酶IV与β夹相互作用的固有特性，而不是DNA聚合酶IV与DNA结合增加的间接结果。
