Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37237, USA.
Sci Adv. 2022 Apr;8(13):eabm0314. doi: 10.1126/sciadv.abm0314. Epub 2022 Mar 30.
Replication-coupled DNA repair and damage tolerance mechanisms overcome replication stress challenges and complete DNA synthesis. These pathways include fork reversal, translesion synthesis, and repriming by specialized polymerases such as PRIMPOL. Here, we investigated how these pathways are used and regulated in response to varying replication stresses. Blocking lagging-strand priming using a POLα inhibitor slows both leading- and lagging-strand synthesis due in part to RAD51-, HLTF-, and ZRANB3-mediated, but SMARCAL1-independent, fork reversal. ATR is activated, but CHK1 signaling is dampened compared to stalling both the leading and lagging strands with hydroxyurea. Increasing CHK1 activation by overexpressing CLASPIN in POLα-inhibited cells promotes replication elongation through PRIMPOL-dependent repriming. CHK1 phosphorylates PRIMPOL to promote repriming irrespective of the type of replication stress, and this phosphorylation is important for cellular resistance to DNA damage. However, PRIMPOL activation comes at the expense of single-strand gap formation, and constitutive PRIMPOL activity results in reduced cell fitness.
复制偶联的 DNA 修复和损伤容忍机制克服了复制压力的挑战，完成了 DNA 合成。这些途径包括叉反转、跨损伤合成和由特殊聚合酶（如 PRIMPOL）重新引发。在这里，我们研究了这些途径如何在不同的复制压力下被使用和调节。使用 POLα 抑制剂阻断滞后链引发会减缓前导链和滞后链的合成，这部分是由于 RAD51、HLTF 和 ZRANB3 介导的，但与 SMARCAL1 无关的叉反转。ATR 被激活，但与羟脲同时阻断前导链和滞后链相比，CHK1 信号被抑制。在 POLα 抑制剂处理的细胞中过表达 CLASPIN 增加 CHK1 激活，通过 PRIMPOL 依赖性重新引发促进复制延伸。CHK1 磷酸化 PRIMPOL 以促进重新引发，无论复制压力的类型如何，这种磷酸化对细胞抵抗 DNA 损伤很重要。然而，PRIMPOL 的激活是以单链间隙形成的代价为代价的，并且组成型 PRIMPOL 活性导致细胞适应性降低。
