Replication fork reversal in DNA polymerase III mutants of Escherichia coli:: a role for the β clamp
Replication fork reversal in DNA polymerase III mutants of Escherichia coli:: a role for the β clamp
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DOI：
10.1046/j.1365-2958.2002.02962.x
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发表时间：
2002-06-01
影响因子：
3.6
通讯作者：
Michel, B
中科院分区：
文献类型：
--
作者：
Grompone, G;Seigneur, M;Michel, B
某些复制突变导致大肠杆菌中的特定反应，称为复制叉逆转：在阻断的叉处，新生链的退火和模板链的配对形成四向连接。RuvABC催化的这种Holliday连接的解析在recBC背景下引起染色体双链断裂（DSB），因此产生了对重组蛋白RecBC的活力的需要。在目前的工作中，两个突变体进行了测试复制叉逆转：dnaEts突变体和dnaNts突变体，影响的α（聚合酶）和β（持续合成能力钳）亚基的DNA聚合酶III全酶分别。在dnaEts recB菌株中，由dnaEts突变引起的RuvABC依赖性DSB发生在37 ℃或42 ℃，表明DNA聚合酶α亚基部分或完全失活后发生复制叉逆转。DSB的形成不依赖于RecA、RecQ和PriA的解旋酶功能。在dnaNts recB突变体中，由dnaNts突变引起的RuvABC依赖性DSB仅在半容许温度37 ℃下发生，表明在β钳的剩余活性足以维持生存力的条件下发生复制叉逆转。相比之下，dnaNts突变在42摄氏度下不会引起染色体断裂，在这个温度下，DnaN完全失活，dnaNts突变体也不能存活。我们建议，DNA聚合酶III β钳的残留活性是需要在dnaNts突变体的复制叉逆转。
Certain replication mutations lead in Escherichia coli to a specific reaction named replication fork reversal: at blocked forks, annealing of the nascent strands and pairing of the template strands form a four-way junction. RuvABC-catalysed resolution of this Holliday junction causes chromosome double-strand breaks (DSBs) in a recBC context and therefore creates a requirement for the recombination proteins RecBC for viability. In the present work, two mutants were tested for replication fork reversal: a dnaEts mutant and a dnaNts mutant, affected in the alpha (polymerase) and beta (processivity clamp) subunits of DNA polymerase III holoenzyme respectively. In the dnaEts recB strain, RuvABC-dependent DSBs caused by the dnaEts mutation occurred at 37degreesC or 42degreesC, indicating the occurrence of replication fork reversal upon partial or complete inactivation of the DNA polymerase alpha subunit. DSB formation was independent of RecA, RecQ and the helicase function of PriA. In the dnaNts recB mutant, RuvABC-dependent DSB caused by the dnaNts mutation occurred only at semi-permissive temperature, 37degreesC, indicating the occurrence of replication fork reversal in conditions in which the remaining activity of the beta clamp is sufficient for viability. In contrast, the dnaNts mutation did not cause chromosome breakage at 42degreesC, a temperature at which DnaN is totally inactive and the dnaNts mutant is inviable. We propose that a residual activity of the DNA polymerase III beta clamp is required for replication fork reversal in the dnaNts mutant.