Division of Protein and Nucleic Acid Chemistry, Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK.
Division of Protein and Nucleic Acid Chemistry, Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK.
J Mol Biol. 2019 May 3;431(10):2040-2049. doi: 10.1016/j.jmb.2019.03.011. Epub 2019 Mar 17.
Leading-strand polymerase stalling at DNA damage impairs replication fork progression. Using biochemical approaches, we show this arises due to both slower template unwinding following helicase-polymerase uncoupling and establishment of prolonged stalled fork structures. Fork slowing and stalling occur at structurally distinct lesions, are always associated with continued lagging-strand synthesis, are observed when either Pol ε or Pol δ stalls at leading-strand damage, and do not require specific helicase-polymerase coupling factors. Hence, the key trigger for these replisome-intrinsic responses is cessation of leading-strand polymerization, revealing this as a crucial driver of normal replication fork rates. We propose that this helps balance the need for sufficient uncoupling to activate the DNA replication checkpoint with excessive destabilizing single-stranded DNA exposure in eukaryotes.
DNA 损伤导致的领头链聚合酶停滞会损害复制叉的推进。通过生化方法，我们发现这是由于解旋酶-聚合酶解偶联后模板的解旋速度减慢以及长时间的停滞叉结构的建立所致。叉的减速和停滞发生在结构上不同的损伤处，总是与持续的滞后链合成相关，当 Pol ε 或 Pol δ 在领头链损伤处停滞时都会观察到，并且不需要特定的解旋酶-聚合酶偶联因子。因此，这些复制体内在反应的关键触发因素是领头链聚合的停止，这揭示了它是正常复制叉速率的关键驱动因素。我们提出，这有助于平衡激活 DNA 复制检查点所需的足够解偶联与真核生物中过多的破坏单链 DNA 暴露之间的需求。
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