Division of Protein & Nucleic Acid Chemistry, Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
Nucleic Acids Res. 2024 Jan 11;52(1):243-258. doi: 10.1093/nar/gkad1054.
The primase/polymerase PRIMPOL restarts DNA synthesis when replication is arrested by template impediments. However, we do not have a comprehensive view of how PRIMPOL-dependent repriming integrates with the main pathways of damage tolerance, REV1-dependent 'on-the-fly' lesion bypass at the fork and PCNA ubiquitination-dependent post-replicative gap filling. Guided by genome-wide CRISPR/Cas9 screens to survey the genetic interactions of PRIMPOL in a non-transformed and p53-proficient human cell line, we find that PRIMPOL is needed for cell survival following loss of the Y-family polymerases REV1 and POLη in a lesion-dependent manner, while it plays a broader role in promoting survival of cells lacking PCNA K164-dependent post-replicative gap filling. Thus, while REV1- and PCNA K164R-bypass provide two layers of protection to ensure effective damage tolerance, PRIMPOL is required to maximise the effectiveness of the interaction between them. We propose this is through the restriction of post-replicative gap length provided by PRIMPOL-dependent repriming.
当复制被模板阻碍而停滞时，引物酶/聚合酶 PRIMPOL 会重新启动 DNA 合成。然而，我们对于 PRIMPOL 依赖性重新引发如何与损伤容忍的主要途径（REV1 依赖性“实时”叉突处的损伤绕过以及 PCNA 泛素化依赖性复制后缺口填充）整合，还没有全面的认识。在非转化和 p53 功能正常的人细胞系中，通过全基因组 CRISPR/Cas9 筛选来调查 PRIMPOL 的遗传相互作用，我们发现，在依赖损伤的情况下，当 Y 家族聚合酶 REV1 和 POLη 丧失时，PRIMPOL 对于细胞存活是必需的，而在促进缺乏 PCNA K164 依赖性复制后缺口填充的细胞存活方面，它则发挥着更广泛的作用。因此，虽然 REV1 和 PCNA K164R 旁路提供了两层保护以确保有效的损伤容忍，但 PRIMPOL 对于最大限度地提高它们之间相互作用的有效性是必需的。我们提出，这是通过 PRIMPOL 依赖性重新引发限制复制后缺口的长度来实现的。
