Mangiameli Sarah M, Merrikh Christopher N, Wiggins Paul A, Merrikh Houra
Department of Physics, University of Washington, Seattle, United States.
Department of Microbiology, University of Washington, Seattle, United States.
Elife. 2017 Jan 16;6:e19848. doi: 10.7554/eLife.19848.
The canonical model of DNA replication describes a highly-processive and largely continuous process by which the genome is duplicated. This continuous model is based upon reconstitution and ensemble experiments. Here, we characterize the replisome-complex stoichiometry and dynamics with single-molecule resolution in bacterial cells. Strikingly, the stoichiometries of the replicative helicase, DNA polymerase, and clamp loader complexes are consistent with the presence of only one active replisome in a significant fraction of cells (>40%). Furthermore, many of the observed complexes have short lifetimes (<8 min), suggesting that replisome disassembly is quite prevalent, possibly occurring several times per cell cycle. The instability of the replisome complex is conflict-induced: transcription inhibition stabilizes these complexes, restoring the second replisome in many of the cells. Our results suggest that, in contrast to the canonical model, DNA replication is a largely discontinuous process due to pervasive replication-transcription conflicts.
DNA复制的经典模型描述了一个高度连续且主要为连续的过程，通过该过程基因组得以复制。这个连续模型是基于重组和整体实验得出的。在此，我们以单分子分辨率在细菌细胞中表征了复制体复合物的化学计量和动力学。令人惊讶的是，复制性解旋酶、DNA聚合酶和钳位装载器复合物的化学计量与相当一部分细胞（>40%）中仅存在一个活性复制体的情况一致。此外，许多观察到的复合物寿命较短（<8分钟），这表明复制体的解体相当普遍，可能在每个细胞周期中发生多次。复制体复合物的不稳定性是由冲突诱导的：转录抑制可稳定这些复合物，在许多细胞中恢复第二个复制体。我们的结果表明，与经典模型相反，由于普遍存在的复制 - 转录冲突，DNA复制在很大程度上是一个不连续的过程。
