Effects of single-strand DNases ExoI, RecJ, ExoVII, and SbcCD on homologous recombination of recBCD+ strains of Escherichia coli and roles of SbcB15 and XonA2 exol mutant enzymes
Effects of single-strand DNases ExoI, RecJ, ExoVII, and SbcCD on homologous recombination of recBCD+ strains of Escherichia coli and roles of SbcB15 and XonA2 exol mutant enzymes
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DOI：
10.1128/jb.01052-07
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发表时间：
2008-01-01
影响因子：
3.2
通讯作者：
Wackernagel, Wilfried
中科院分区：
文献类型：
--
作者：
Thoms, Brigitte;Borchers, Inka;Wackernagel, Wilfried
为了评估单链 DNase (ssDNase) 在 recBCD(+) 背景下对重组的贡献，我们研究了 31 个菌株，其具有外切核酸酶 I (Delta xon)、外切核酸酶 VII (xseA)、RecJ DNase (reef) 和 SbcCD DNase (sbcCD) 的无效等位基因的所有组合以及外切核酸酶 I 突变等位基因 xonA2 和sbcB15。 xse recJ sbcCD Axon 和 xse recJ sbcCD sbcB15 四重突变体对冷敏感，而具有 xonA2 的四重突变体则不然。紫外线敏感性随着 ssDNase 缺陷而增加。大多数三重和四重突变体是高度敏感的。 ssDNase 的缺失几乎不影响 P1 转导重组体的形成，并且在一些情况下接合重组体的产量下降（高达 94%）。具有 sbcB15 的菌株通常与野生型相似。我们确定 sbcB15 突变引起核酸外切酶基序 III 中的 A183V 交换，并确定 xonA2 作为终止密码子，消除了末端 8 个氨基酸。纯化酶的比活性分别为野生型 Xon (Xon(+)) 的 1.6% (SbcB15) 和 0.9% (XonA2)，但活性谱发生了改变。在凝胶转移测定中，SbcB15 与 3' DNA 突出端相对稳定地结合，提供针对 Xon(+) 的保护。除了在 RecBCD 途径中的突触后作用外，核酸外切酶 I 和 RecJ 还被认为具有 DNA 末端平淡化的突触前作用。在缀合过程中可能特别需要钝化，以产生带有突出端 RecBCD 靶标的 DNA，以启动重组。有证据表明，已知 SbcB15 蛋白可激活 recBC 菌株中的 RecF 途径，它独立于 RecF 参与 recBCD(+) 细胞中的重组。 SbcB15 的 DNA 末端结合还可以解释具有 sbcB15 的菌株的其他特定表型。
To assess the contributions of single-strand DNases (ssDNases) to recombination in a recBCD(+) background, we studied 31 strains with all combinations of null alleles of exonuclease I (Delta xon), exonuclease VII (xseA), RecJ DNase (reef), and SbcCD DNase (sbcCD) and exonuclease I mutant alleles xonA2 and sbcB15. The xse recJ sbcCD Axon and xse recJ sbcCD sbcB15 quadruple mutants were cold sensitive, while the quadruple mutant with xonA2 was not. UV sensitivity increased with ssDNase deficiencies. Most triple and quadruple mutants were highly sensitive. The absence of ssDNases hardly affected P1 transductional recombinant formation, and conjugational recombinant production was decreased (as much as 94%) in several cases. Strains with sbcB15 were generally like the wild type. We determined that the sbcB15 mutation caused an A183V exchange in exonuclease motif III and identified xonA2 as a stop codon eliminating the terminal 8 amino acids. Purified enzymes had 1.6% (SbcB15) and 0.9% (XonA2) of the specific activity of wild-type Xon (Xon(+)), respectively, with altered activity profiles. In gel shift assays, SbcB15 associated relatively stably with 3' DNA overhangs, giving protection against Xon(+). In addition to their postsynaptic roles in the RecBCD pathway, exonuclease I and RecJ are proposed to have presynaptic roles of DNA end blunting. Blunting may be specifically required during conjugation to make DNAs with overhangs RecBCD targets for initiation of recombination. Evidence is provided that SbcB15 protein, known to activate the RecF pathway in recBC strains, contributes independently of RecF to recombination in recBCD(+) cells. DNA end binding by SbcB15 can also explain other specific phenotypes of strains with sbcB15.