Division of Cancer Cell Biology, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045, USA.
Nucleic Acids Res. 2021 May 21;49(9):5003-5016. doi: 10.1093/nar/gkab269.
DNA ligase 1 (LIG1) is known as the major DNA ligase responsible for Okazaki fragment joining. Recent studies have implicated LIG3 complexed with XRCC1 as an alternative player in Okazaki fragment joining in cases where LIG1 is not functional, although the underlying mechanisms are largely unknown. Here, using a cell-free system derived from Xenopus egg extracts, we demonstrated the essential role of PARP1-HPF1 in LIG3-dependent Okazaki fragment joining. We found that Okazaki fragments were eventually ligated even in the absence of LIG1, employing in its place LIG3-XRCC1, which was recruited onto chromatin. Concomitantly, LIG1 deficiency induces ADP-ribosylation of histone H3 in a PARP1-HPF1-dependent manner. The depletion of PARP1 or HPF1 resulted in a failure to recruit LIG3 onto chromatin and a subsequent failure in Okazaki fragment joining in LIG1-depleted extracts. Importantly, Okazaki fragments were not ligated at all when LIG1 and XRCC1 were co-depleted. Our results suggest that a unique form of ADP-ribosylation signaling promotes the recruitment of LIG3 on chromatin and its mediation of Okazaki fragment joining as a backup system for LIG1 perturbation.
DNA 连接酶 1（LIG1）是负责连接冈崎片段的主要 DNA 连接酶。最近的研究表明，LIG3 与 XRCC1 形成复合物是 LIG1 失活时冈崎片段连接的替代因子，尽管其潜在机制在很大程度上尚不清楚。在这里，我们使用源自非洲爪蟾卵提取物的无细胞系统，证明了 PARP1-HPF1 在 LIG3 依赖性冈崎片段连接中的重要作用。我们发现，即使没有 LIG1，也可以利用替代的 LIG3-XRCC1 将冈崎片段最终连接起来，而 LIG3-XRCC1 被招募到染色质上。同时，LIG1 缺陷以 PARP1-HPF1 依赖的方式诱导组蛋白 H3 的 ADP-ribosylation。PARP1 或 HPF1 的耗竭导致 LIG3 无法招募到染色质上，从而导致 LIG1 耗尽提取物中的冈崎片段连接失败。重要的是，当 LIG1 和 XRCC1 被共同耗尽时，冈崎片段根本没有连接。我们的结果表明，一种独特的 ADP-ribosylation 信号促进了 LIG3 在染色质上的募集及其作为 LIG1 扰动的备份系统介导冈崎片段连接。
