Robust linear DNA degradation supports replication-initiation-defective mutants in Escherichia coli.
Robust linear DNA degradation supports replication-initiation-defective mutants in Escherichia coli.
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DOI：
10.1093/g3journal/jkac228
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发表时间：
2022-11-04
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RecBCD解旋酶/核酸酶通过重组修复或线性DNA降解支持复制叉的进展，解释了recBC突变体合成致死性与复制延伸缺陷。由于复制起始缺陷使染色体没有复制叉，因此它们应该对recBCD状态不敏感。令人惊讶的是，我们发现半允许温度下的大肠杆菌 dnaA46(Ts) 和 dnaC2(Ts) 起始突变体也是recBC-colethal。有趣的是，dnaA46 recBC 致死抑制因子表明问题是启动不足，而 dnaC2 recBC 抑制因子则表明问题是启动过度。使用遗传和物理方法，我们研究了 dnaA46 recBC 合成致死性，以了解 RecBCD 参与复制起始的可能性。过量产生的 DnaA46 突变蛋白会干扰 dnaA+ 细胞的生长，而 dnaA46 recBC 突变体的残余活力取决于辅助复制解旋酶 Rep，表明 DnaA46 突变蛋白抑制复制叉。 dnaA46 突变体依赖于 RecBCD 的线性 DNA 降解，而不是重组修复。同时，dnaA46 缺陷也与霍利迪连接体移动缺陷相互作用，表明受抑制的分叉发生逆转。然而，与所有已知的recBC-colethals（其染色体片段化）相比，dnaA46 recBC 突变体不产生染色体片段化，表明其受抑制的复制叉是稳定的。物理测量证实 dnaA46 突变体的复制抑制在伸长和起始水平上转变为半允许温度，而 RecBCD 逐渐恢复伸长，然后恢复起始。我们提出，RecBCD 催化的受抑制复制叉的重置允许复制从染色体 DNA 中取代“粘性”DnaA46(Ts) 蛋白，从而聚集足够的 DnaA 以进行新的起始。
RecBCD helicase/nuclease supports replication fork progress via recombinational repair or linear DNA degradation, explaining recBC mutant synthetic lethality with replication elongation defects. Since replication initiation defects leave chromosomes without replication forks, these should be insensitive to the recBCD status. Surprisingly, we found that both Escherichia coli dnaA46(Ts) and dnaC2(Ts) initiation mutants at semi-permissive temperatures are also recBC-colethal. Interestingly, dnaA46 recBC lethality suppressors suggest underinitiation as the problem, while dnaC2 recBC suppressors signal overintiation. Using genetic and physical approaches, we studied the dnaA46 recBC synthetic lethality, for the possibility that RecBCD participates in replication initiation. Overproduced DnaA46 mutant protein interferes with growth of dnaA+ cells, while the residual viability of the dnaA46 recBC mutant depends on the auxiliary replicative helicase Rep, suggesting replication fork inhibition by the DnaA46 mutant protein. The dnaA46 mutant depends on linear DNA degradation by RecBCD, rather than on recombinational repair. At the same time, the dnaA46 defect also interacts with Holliday junction-moving defects, suggesting reversal of inhibited forks. However, in contrast to all known recBC-colethals, which fragment their chromosomes, the dnaA46 recBC mutant develops no chromosome fragmentation, indicating that its inhibited replication forks are stable. Physical measurements confirm replication inhibition in the dnaA46 mutant shifted to semi-permissive temperatures, both at the level of elongation and initiation, while RecBCD gradually restores elongation and then initiation. We propose that RecBCD-catalyzed resetting of inhibited replication forks allows replication to displace the “sticky” DnaA46(Ts) protein from the chromosomal DNA, mustering enough DnaA for new initiations.
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DOI：
10.1073/pnas.83.15.5558
发表时间：
1986-08-01
影响因子：
11.1
作者：
AMUNDSEN, SK;TAYLOR, AF;SMITH, GR
通讯作者：
SMITH, GR
影响因子：
64.8
作者：
Cox, MM;Goodman, MF;Marians, KJ
通讯作者：
Marians, KJ
影响因子：
64.5
作者：
BRAUN, RE;ODAY, K;WRIGHT, A
通讯作者：
WRIGHT, A
影响因子：
4
作者：
COX, MM
通讯作者：
COX, MM
DOI：
10.1073/pnas.80.19.5817
发表时间：
1983-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
FULLER, RS;KORNBERG, A
通讯作者：
KORNBERG, A