DNA polymerase III protein, HolC, helps resolve replication/transcription conflicts.
DNA polymerase III protein, HolC, helps resolve replication/transcription conflicts.
复制标题
DOI：
10.15698/mic2021.06.753
复制
发表时间：
2021-05-06
期刊：
影响因子：
--
通讯作者：
Lovett ST
中科院分区：
文献类型：
--
作者：
Lovett ST
在大肠杆菌中，DNA复制是由蛋白质组装催化的，DNA聚合酶III全酶。该复合体包括聚合酶和校正亚基、持续合成性钳和钳装载复合体。holC基因编码核心夹装载复合物的辅助蛋白（称为χ），并且是全酶中唯一与单链DNA结合蛋白SSB结合的蛋白。虽然突变体表现出生长障碍、遗传不稳定性和对DNA损伤剂的敏感性，但HolC对生存能力不是必需的。在这项研究中，我们在holCΔ菌株中分离自发抑制突变体，并通过全基因组测序鉴定这些突变体。一些抑制基因是RNA聚合酶的等位基因，这表明holC突变株和sspA（严格饥饿蛋白）的转录存在问题。使用条件holC质粒，我们研究了影响转录延长和终止的因素对holC突变体表型的协同或抑制作用。RpoA（α）、RpoB（β）和RpoC（β '）RNA聚合酶全酶等位基因可部分抑制HolC的缺失。相反，转录因子DksA和NusA的突变增强了holC突变体的活力。HolC突变体表现出增强的敏感性双环霉素，一个特定的抑制剂Rho依赖性终止。双环霉素还逆转了rpoA、rpoC和sspA对holC的抑制。高度表达的rrnA操纵子的倒置加剧了holC突变体的生长缺陷。我们建议，转录复合物块复制holC突变体和Rho依赖的转录终止和DksA功能是特别重要的，以维持生存能力和染色体的完整性。
In Escherichia coli, DNA replication is catalyzed by an assembly of proteins, the DNA polymerase III holoenzyme. This complex includes the polymerase and proofreading subunits, the processivity clamp and clamp loader complex. The holC gene encodes an accessory protein (known as χ) to the core clamp loader complex and is the only protein of the holoenzyme that binds to single-strand DNA binding protein, SSB. HolC is not essential for viability although mutants show growth impairment, genetic instability and sensitivity to DNA damaging agents. In this study we isolate spontaneous suppressor mutants in a holCΔ strain and identify these by whole genome sequencing. Some suppressors are alleles of RNA polymerase, suggesting that transcription is problematic for holC mutant strains, and of sspA, stringent starvation protein. Using a conditional holC plasmid, we examine factors affecting transcription elongation and termination for synergistic or suppressive effects on holC mutant phenotypes. Alleles of RpoA (α), RpoB (β) and RpoC (β') RNA polymerase holoenzyme can partially suppress loss of HolC. In contrast, mutations in transcription factors DksA and NusA enhanced the inviability of holC mutants. HolC mutants showed enhanced sensitivity to bicyclomycin, a specific inhibitor of Rho-dependent termination. Bicyclomycin also reverses suppression of holC by rpoA, rpoC and sspA. An inversion of the highly expressed rrnA operon exacerbates the growth defects of holC mutants. We propose that transcription complexes block replication in holC mutants and Rho-dependent transcriptional termination and DksA function are particularly important to sustain viability and chromosome integrity.