Mendonca V M, Klepin H D, Matson S W
Department of Biology, University of North Carolina at Chapel Hill 27599.
J Bacteriol. 1995 Mar;177(5):1326-35. doi: 10.1128/jb.177.5.1326-1335.1995.
DNA helicases play pivotal roles in homologous recombination and recombinational DNA repair. They are involved in both the generation of recombinogenic single-stranded DNA ends and branch migration of synapsed Holliday junctions. Escherichia coli helicases II (uvrD), IV (helD), and RecQ (recQ) have all been implicated in the presynaptic stage of recombination in the RecF pathway. To probe for functional redundancy among these helicases, mutant strains containing single, double, and triple deletions in the helD, uvrD, and recQ genes were constructed and examined for conjugational recombination efficiency and DNA repair proficiency. We were unable to construct a strain harboring a delta recQ delta uvrD double deletion in a recBC sbcB(C) background (RecF pathway), suggesting that a delta recQ deletion mutation was lethal to the cell in a recBC sbcB(C) delta D background. However, we were able to construct a triple delta recQ delta uvrD Delta helD mutant in the recBC sbcB(C) background. This may be due to the increased mutator frequency in delta uvrD mutants which may have resulted in the fortuitous accumulation of a suppressor mutation(s). The triple helicase mutant recBC sbcB(C) delta uvrD delta recQ delta helD severely deficient in Hfr-mediated conjugational recombination and in the repair of methylmethane sulfonate-induced DNA damage. This suggests that the presence of at least one helicase--helicase II, RecQ helicase, or helicase IV--is essential for homologous recombination and recombinational DNA repair in a recBC sbcB(C) background. The triple helicase mutant was recombination and repair proficient in a rec+ background. Genetic analysis of the various double mutants unmasked additional functional redundancies with regard to conjugational recombination and DNA repair, suggesting that mechanisms of recombination depend both on the DNA substrates and on the genotype of the cell.
DNA解旋酶在同源重组和重组性DNA修复中发挥着关键作用。它们参与了重组单链DNA末端的产生以及联会霍利迪连接体的分支迁移。大肠杆菌解旋酶II（uvrD）、IV（helD）和RecQ（recQ）都与RecF途径中重组的突触前阶段有关。为了探究这些解旋酶之间的功能冗余性，构建了在helD、uvrD和recQ基因中含有单缺失、双缺失和三缺失的突变菌株，并检测其接合重组效率和DNA修复能力。我们无法在recBC sbcB(C)背景（RecF途径）中构建一个携带ΔrecQΔuvrD双缺失的菌株，这表明在recBC sbcB(C)ΔD背景下，ΔrecQ缺失突变对细胞是致死的。然而，我们能够在recBC sbcB(C)背景中构建一个三缺失ΔrecQΔuvrDΔhelD突变体。这可能是由于ΔuvrD突变体中诱变频率增加，这可能导致了抑制突变的偶然积累。三解旋酶突变体recBC sbcB(C)ΔuvrDΔrecQΔhelD在Hfr介导的接合重组以及甲磺酸甲酯诱导的DNA损伤修复方面严重缺陷。这表明在recBC sbcB(C)背景下，至少存在一种解旋酶——解旋酶II、RecQ解旋酶或解旋酶IV——对于同源重组和重组性DNA修复是必不可少的。三解旋酶突变体在rec+背景下具有重组和修复能力。对各种双突变体的遗传分析揭示了在接合重组和DNA修复方面的额外功能冗余，这表明重组机制既取决于DNA底物，也取决于细胞的基因型。
