DNA replication in all organisms must overcome nucleoprotein blocks to complete genome duplication. Accessory replicative helicases in Escherichia coli, Rep and UvrD, help remove these blocks and aid the re-initiation of replication. Mechanistic details of Rep function have emerged from recent live cell studies; however, the division of UvrD functions between its activities in DNA repair and role as an accessory helicase remain unclear in live cells. By integrating super-resolved single-molecule fluorescence microscopy with biochemical analysis, we find that UvrD self-associates into tetrameric assemblies and, unlike Rep, is not recruited to a specific replisome protein despite being found at approximately 80% of replication forks. Instead, its colocation with forks is likely due to the very high frequency of replication blocks composed of DNA-bound proteins, including RNA polymerase and factors involved in repairing DNA damage. Deleting rep and DNA repair factor genes mutS and uvrA, and inhibiting transcription through RNA polymerase mutation and antibiotic inhibition, indicates that the level of UvrD at the fork is dependent on UvrD's function. Our findings show that UvrD is recruited to sites of nucleoprotein blocks via different mechanisms to Rep and plays a multi-faceted role in ensuring successful DNA replication.

中文翻译：

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由于频繁的复制块，四聚体 UvrD 解旋酶位于大肠杆菌复制体中

所有生物体中的DNA复制必须克服核蛋白阻断才能完成基因组复制。大肠杆菌、Rep 和 UvrD中的辅助复制解旋酶有助于去除这些阻断并帮助重新启动复制。最近的活细胞研究揭示了 Rep 功能的机制细节；然而，在活细胞中，UvrD 在 DNA 修复活性和辅助解旋酶作用之间的功能划分仍不清楚。通过将超分辨单分子荧光显微镜与生化分析相结合，我们发现 UvrD 自关联成四聚体组装体，并且与 Rep 不同的是，尽管在大约 80% 的复制叉中发现了 UvrD，但它并未被招募到特定的复制体蛋白上。相反，它与叉的共置可能是由于由 DNA 结合蛋白组成的复制块的频率非常高，包括 RNA 聚合酶和参与修复 DNA 损伤的因子。删除rep和DNA修复因子基因mutS和uvrA，并通过RNA聚合酶突变和抗生素抑制来抑制转录，表明分叉处的UvrD水平取决于UvrD的功能。我们的研究结果表明，UvrD 通过与 Rep 不同的机制被招募到核蛋白块位点，并在确保 DNA 成功复制方面发挥多方面的作用。