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生物
DNA损伤
dnaB解旋酶
解旋酶
DNA复制
DNA
DNA聚合酶
分子生物学
DNA修复
复制蛋白A
细胞生物学
细菌圆形染色体
遗传学
基因
核糖核酸
DNA结合蛋白
转录因子
作者
Megan S. Behrmann,Michael A. Trakselis
出处
期刊:Methods in Enzymology
日期:2022-01-01
卷期号:: 125-142
被引量:4
标识
DOI:10.1016/bs.mie.2022.02.021
摘要
The genome of prokaryotes can be damaged by a variety of endogenous and exogenous factors, including reactive oxygen species, UV exposure, and antibiotics. To better understand these repair processes and the impact they may have on DNA replication, normal genome maintenance processes can be perturbed by removing or editing associated genes and monitoring DNA repair outcomes. In particular, the replisome activities of DNA unwinding by the helicase and DNA synthesis by the polymerase must be tightly coupled to prevent any appreciable single strand DNA (ssDNA) from accumulating and amplifying genomic stress. If decoupled, vulnerable ssDNA would persist, likely leading to double strand breaks (DSBs) or requiring replication restart mechanisms downstream of a stall. In either case, free 3'-OH strands would exist, resulting from ssDNA gaps in the leading strand or complete DSBs. Terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick end labeling (TUNEL) can enzymatically label ssDNA ends with bromo-deoxy uridine triphosphate (BrdU) to detect free 3′-OH DNA ends in the E. coli genome. Labeled DNA ends can be detected and quantified using fluorescence microscopy or flow cytometry. This methodology is useful in applications where in situ investigation of DNA damage and repair are of interest, including effects from enzyme mutations or deletions and exposure to various environmental conditions.
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