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首页> 美国卫生研究院文献>PLoS Genetics >DNA polymerase IV primarily operates outside of DNA replication forks in Escherichia coli
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DNA polymerase IV primarily operates outside of DNA replication forks in Escherichia coli

机译：DNA聚合酶IV主要在大肠杆菌中的DNA复制叉之外运行

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摘要

In Escherichia coli, damage to the chromosomal DNA induces the SOS response, setting in motion a series of different DNA repair and damage tolerance pathways. DNA polymerase IV (pol IV) is one of three specialised DNA polymerases called into action during the SOS response to help cells tolerate certain types of DNA damage. The canonical view in the field is that pol IV primarily acts at replisomes that have stalled on the damaged DNA template. However, the results of several studies indicate that pol IV also acts on other substrates, including single-stranded DNA gaps left behind replisomes that re-initiate replication downstream of a lesion, stalled transcription complexes and recombination intermediates. In this study, we use single-molecule time-lapse microscopy to directly visualize fluorescently labelled pol IV in live cells. We treat cells with the DNA-damaging antibiotic ciprofloxacin, Methylmethane sulfonate (MMS) or ultraviolet light and measure changes in pol IV concentrations and cellular locations through time. We observe that only 5–10% of foci induced by DNA damage form close to replisomes, suggesting that pol IV predominantly carries out non-replisomal functions. The minority of foci that do form close to replisomes exhibit a broad distribution of colocalisation distances, consistent with a significant proportion of pol IV molecules carrying out postreplicative TLS in gaps behind the replisome. Interestingly, the proportion of pol IV foci that form close to replisomes drops dramatically in the period 90–180 min after treatment, despite pol IV concentrations remaining relatively constant. In an SOS-constitutive mutant that expresses high levels of pol IV, few foci are observed in the absence of damage, indicating that within cells access of pol IV to DNA is dependent on the presence of damage, as opposed to concentration-driven competition for binding sites.
机译：在大肠杆菌中，对染色体DNA的损伤会诱导SOS反应，从而引发一系列不同的DNA修复和损伤耐受途径。 DNA聚合酶IV（pol IV）是三种特殊的DNA聚合酶之一，在SOS应答期间被称为起作用，以帮助细胞耐受某些类型的DNA损伤。该领域的典型观点是pol IV主要作用于停滞在受损DNA模板上的复制体。但是，多项研究的结果表明，pol IV也作用于其他底物，包括留在复制体后的单链DNA缺口，这些复制子可重新启动病变下游的复制，停滞的转录复合物和重组中间体。在这项研究中，我们使用单分子延时显微镜直接观察活细胞中荧光标记的pol IV。我们用破坏DNA的抗生素环丙沙星，甲烷磺酸甲酯（MMS）或紫外线处理细胞，并测量pol IV浓度和细胞位置随时间的变化。我们观察到，由DNA损伤诱导的病灶中只有5–10％形成接近复制体，这表明pol IV主要执行非复制体功能。确实形成接近复制体的少数病灶表现出广泛的共定位距离分布，这与在复制体后面的间隙中进行复制后TLS的大部分pol IV分子一致。有趣的是，尽管pol IV浓度保持相对恒定，但在治疗后90-180分钟内，形成接近复制体的pol IV病灶的比例急剧下降。在表达高水平pol IV的SOS组成型突变体中，在没有损伤的情况下观察到很少的病灶，这表明pol IV对DNA的进入取决于损伤的存在，而不是浓度驱动的竞争。结合位点。

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