Tang M, Shen X, Frank E G, O'Donnell M, Woodgate R, Goodman M F
Department of Biological Sciences, Hedco Molecular Biology Laboratories, University of Southern California, Los Angeles, CA 90089-1340, USA.
Proc Natl Acad Sci U S A. 1999 Aug 3;96(16):8919-24. doi: 10.1073/pnas.96.16.8919.
The damage-inducible UmuD' and UmuC proteins are required for most SOS mutagenesis in Escherichia coli. Our recent assay to reconstitute this process in vitro, using a native UmuD'(2)C complex, revealed that the highly purified preparation contained DNA polymerase activity. Here we eliminate the possibility that this activity is caused by a contaminating DNA polymerase and show that it is intrinsic to UmuD'(2)C. E. coli dinB has recently been shown to have DNA polymerase activity (pol IV). We suggest that UmuD'(2)C, the fifth DNA polymerase discovered in E. coli, be designated as E. coli pol V. In the presence of RecA, beta sliding clamp, gamma clamp loading complex, and E. coli single-stranded binding protein (SSB), pol V's polymerase activity is highly "error prone" at both damaged and undamaged DNA template sites, catalyzing efficient bypass of abasic lesions that would otherwise severely inhibit replication by pol III holoenzyme complex (HE). Pol V bypasses a site-directed abasic lesion with an efficiency about 100- to 150-fold higher than pol III HE. In accordance with the "A-rule," dAMP is preferentially incorporated opposite the lesion. A pol V mutant, UmuD'(2)C104 (D101N), has no measurable lesion bypass activity. A kinetic analysis shows that addition of increasing amounts of pol III to a fixed level of pol V inhibits lesion bypass, demonstrating that both enzymes compete for free 3'-OH template-primer ends. We show, however, that despite competition for primer-3'-ends, pol V and pol III HE can nevertheless interact synergistically to stimulate synthesis downstream from a template lesion.
损伤诱导型UmuD'和UmuC蛋白是大肠杆菌中大多数SOS诱变所必需的。我们最近利用天然的UmuD'(2)C复合物在体外重建这一过程的实验表明，高度纯化的制剂含有DNA聚合酶活性。在这里，我们排除了这种活性是由污染的DNA聚合酶引起的可能性，并表明它是UmuD'(2)C所固有的。最近发现大肠杆菌dinB具有DNA聚合酶活性（pol IV）。我们建议将在大肠杆菌中发现的第五种DNA聚合酶UmuD'(2)C命名为大肠杆菌pol V。在RecA、β滑动夹、γ夹加载复合物和大肠杆菌单链结合蛋白（SSB）存在的情况下，pol V的聚合酶活性在受损和未受损的DNA模板位点都具有高度的“易错性”，催化无碱基损伤的有效跨越，否则这些损伤会严重抑制pol III全酶复合物（HE）的复制。Pol V跨越定点无碱基损伤的效率比pol III HE高约100至150倍。根据“A规则”，dAMP优先掺入损伤位点的对面。一个pol V突变体UmuD'(2)C104（D101N）没有可测量的损伤跨越活性。动力学分析表明，向固定水平的pol V中添加越来越多的pol III会抑制损伤跨越，这表明两种酶竞争游离的3'-OH模板-引物末端。然而，我们表明，尽管竞争引物-3'-末端，但pol V和pol III HE仍能协同相互作用，刺激模板损伤下游的合成。
