Catastrophic chromosome fragmentation probes the nucleoid structure and dynamics in Escherichia coli.
Catastrophic chromosome fragmentation probes the nucleoid structure and dynamics in Escherichia coli.
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DOI：
10.1093/nar/gkac865
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发表时间：
2022-10-28
影响因子：
14.9
通讯作者：
Kuzminov, Andrei
中科院分区：
文献类型：
--
作者：
Mahaseth, Tulip;Kuzminov, Andrei
用氰化物（CN）和过氧化氢（HP）组合处理的大肠杆菌细胞屈服于灾难性染色体断裂（CCF），在脉冲场凝胶中可检测到每个基因组当量>100个双链断裂。在这里，我们表明，CN + HP诱导的双链断裂是独立的复制和均匀地发生在染色体上，因此，我们使用CCF探测的类核结构，通过测量从沉淀的类核释放的DNA。CCF从类核中释放的染色体DNA令人惊讶地少，这表明：（i）类核是单个DNA-蛋白质复合物，仅具有有限的无蛋白质DNA片段，以及（ii）CN + HP诱导的断裂发生在这些未固定的DNA片段内，而不是在与中心支架的DNA附着处。缺乏单个核相关蛋白（NAP）的突变体在CCF期间释放更多的DNA，这与NAP将染色体锚定到中心支架一致（Dps也直接减少了双链断裂的数量）。最后，一旦ATP产生恢复，就会释放出更多的断裂DNA，其中约三分之二的ATP依赖性DNA释放是由于转录，这表明转录复合物作为滑轮移动DNA环。除了NAP之外，双链断裂的重组修复也抑制CCF释放DNA，有助于动态和复杂的类核结构。目前公认的细菌类核的结构是通过测量染色体遭受100次双链DNA断裂后游离DNA的释放来测试的。
Escherichia coli cells treated with a combination of cyanide (CN) and hydrogen peroxide (HP) succumb to catastrophic chromosome fragmentation (CCF), detectable in pulsed-field gels as >100 double-strand breaks per genome equivalent. Here we show that CN + HP-induced double-strand breaks are independent of replication and occur uniformly over the chromosome,—therefore we used CCF to probe the nucleoid structure by measuring DNA release from precipitated nucleoids. CCF releases surprisingly little chromosomal DNA from the nucleoid suggesting that: (i) the nucleoid is a single DNA-protein complex with only limited stretches of protein-free DNA and (ii) CN + HP-induced breaks happen within these unsecured DNA stretches, rather than at DNA attachments to the central scaffold. Mutants lacking individual nucleoid-associated proteins (NAPs) release more DNA during CCF, consistent with NAPs anchoring chromosome to the central scaffold (Dps also reduces the number of double-strand breaks directly). Finally, significantly more broken DNA is released once ATP production is restored, with about two-thirds of this ATP-dependent DNA release being due to transcription, suggesting that transcription complexes act as pulleys to move DNA loops. In addition to NAPs, recombinational repair of double-strand breaks also inhibits DNA release by CCF, contributing to a dynamic and complex nucleoid structure. The currently accepted structure of bacterial nucleoid is tested by measuring release of free DNA after the chromosome suffers a hundred of double-strand DNA breaks.
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DOI：
10.1073/pnas.130192397
发表时间：
2000-06-20
影响因子：
11.1
作者：
Amundsen, SK;Taylor, AF;Smith, GR
通讯作者：
Smith, GR
影响因子：
3.6
作者：
Cabrera, JE;Jin, DJ
通讯作者：
Jin, DJ
影响因子：
4.8
作者：
Amado, Luciana;Kuzminov, Andrei
通讯作者：
Kuzminov, Andrei
DOI：
10.1007/bf00282458
发表时间：
1991-08-01
期刊：
MOLECULAR AND GENERAL GENETICS
影响因子：
--
作者：
BRCICKOSTIC, K;SALAJSMIC, E;TRGOVCEVIC, Z
通讯作者：
TRGOVCEVIC, Z
影响因子：
3.6
作者：
Rotman E;Khan SR;Kouzminova E;Kuzminov A
通讯作者：
Kuzminov A