Institute of Genetics, University of Nottingham, Queen's Medical Centre, Nottingham, United Kingdom.
Genetics. 2010 May;185(1):23-37. doi: 10.1534/genetics.110.114413. Epub 2010 Feb 15.
RuvABC and RecG are thought to provide alternative pathways for the late stages of recombination in Escherichia coli. Inactivation of both blocks the recovery of recombinants in genetic crosses. RuvABC resolves Holliday junctions, with RuvAB driving branch migration and RuvC catalyzing junction cleavage. RecG also drives branch migration, but no nuclease has been identified that might act with RecG to cleave junctions, apart from RusA, which is not normally expressed. We searched for an alternative nuclease using a synthetic lethality assay to screen for mutations causing inviability in the absence of RuvC, on the premise that a strain without any ability to cut junctions might be inviable. All the mutations identified mapped to polA, dam, or uvrD. None of these genes encodes a nuclease that cleaves Holliday junctions. Probing the reason for the inviability using the RusA Holliday junction resolvase provided strong evidence in each case that the RecG pathway is very ineffective at removing junctions and indicated that a nuclease component most probably does not exist. It also revealed new suppressors of recG, which were located to the ssb gene. Taken together with the results from the synthetic lethality assays, the properties of the mutant SSB proteins provide evidence that, rather than promoting recombination, a major function of RecG is to curb potentially pathological replication initiated via PriA protein at sites remote from oriC.
RuvABC 和 RecG 被认为为大肠杆菌中重组的后期阶段提供了替代途径。这两种途径的失活都会阻止遗传交叉中重组体的恢复。RuvABC 可以解决 Holliday 连接，其中 RuvAB 驱动分支迁移，RuvC 催化连接断裂。RecG 也驱动分支迁移，但除了通常不表达的 RusA 之外，尚未鉴定出可能与 RecG 一起切割连接的核酸酶。我们使用合成致死性测定法搜索替代核酸酶，以筛选在没有 RuvC 的情况下导致生存能力丧失的突变，前提是没有任何切割连接能力的菌株可能无法生存。鉴定出的所有突变都映射到 polA、dam 或 uvrD。这些基因都没有编码能够切割 Holliday 连接的核酸酶。使用 RusA Holliday 连接解决酶探测生存能力丧失的原因，在每种情况下都提供了强有力的证据，表明 RecG 途径非常有效地去除连接，并且表明极不可能存在核酸酶成分。它还揭示了 recG 的新抑制剂，这些抑制剂位于 ssb 基因上。与合成致死性测定的结果一起，突变 SSB 蛋白的特性提供了证据表明，与其促进重组，RecG 的主要功能是遏制通过 PriA 蛋白在远离 oriC 的位点起始的潜在病理复制。
