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Role for DNA double strand end-resection activity of RecBCD in control of aberrant chromosomal replication initiation in Escherichia coli
Nucleic Acids Research ( IF 14.9 ) Pub Date : 2022-08-05 , DOI: 10.1093/nar/gkac670
Sayantan Goswami 1, 2, 3 , Jayaraman Gowrishankar 1, 3
Affiliation  

Replication of the circular bacterial chromosome is initiated from a locus oriC with the aid of an essential protein DnaA. One approach to identify factors acting to prevent aberrant oriC-independent replication initiation in Escherichia coli has been that to obtain mutants which survive loss of DnaA. Here, we show that a ΔrecD mutation, associated with attenuation of RecBCD’s DNA double strand end-resection activity, provokes abnormal replication and rescues ΔdnaA lethality in two situations: (i) in absence of 5′-3′ single-strand DNA exonuclease RecJ, or (ii) when multiple two-ended DNA double strand breaks (DSBs) are generated either by I-SceI endonucleolytic cleavages or by radiomimetic agents phleomycin or bleomycin. One-ended DSBs in the ΔrecD mutant did not rescue ΔdnaA lethality. With two-ended DSBs in the ΔrecD strain, ΔdnaA viability was retained even after linearization of the chromosome. Data from genome-wide DNA copy number determinations in ΔdnaA-rescued cells lead us to propose a model that nuclease-mediated DNA resection activity of RecBCD is critical for prevention of a σ-mode of rolling-circle over-replication when convergent replication forks merge and fuse, as may be expected to occur during normal replication at the chromosomal terminus region or during repair of two-ended DSBs following ‘ends-in’ replication.

中文翻译:

RecBCD DNA双链末端切除活性在控制大肠杆菌异常染色体复制起始中的作用

环状细菌染色体的复制是在必需蛋白 DnaA 的帮助下从基因座 oriC 开始的。一种鉴定阻止大肠杆菌中不依赖于oriC的异常复制起始的因素的方法是获得在DnaA丢失后存活的突变体。在这里,我们表明,与 RecBCD 的 DNA 双链末端切除活性减弱相关的 ΔrecD 突变会在两种情况下引发异常复制并挽救 ΔdnaA 致死性:(i)在没有 5'-3' 单链 DNA 外切核酸酶 RecJ 的情况下, 或 (ii) 当 I-SceI 核酸内切酶或放射模拟剂腐草霉素或博来霉素产生多个双末端 DNA 双链断裂 (DSB) 时。ΔrecD 突变体中的单端 DSB 并没有挽救 ΔdnaA 的杀伤力。在 ΔrecD 菌株中使用两端 DSB,即使在染色体线性化后仍保留了 ΔdnaA 活力。来自 ΔdnaA 拯救细胞中全基因组 DNA 拷贝数测定的数据使我们提出了一个模型,即当聚合复制叉合并时,核酸酶介导的 RecBCD 的 DNA 切除活性对于防止 σ 模式的滚环过度复制至关重要和融合,正如预期在染色体末端区域的正常复制期间或在“末端”复制后修复两端 DSB 期间发生的那样。
更新日期:2022-08-05
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