Department of Cell Biology and Physiology, Washington University School of Medicine, 660 S. Euclid Avenue, St. Louis, MO 63110, USA.
Nucleic Acids Res. 2013 Nov;41(20):9325-38. doi: 10.1093/nar/gkt672. Epub 2013 Aug 11.
Exo1-mediated resection of DNA double-strand break ends generates 3' single-stranded DNA overhangs required for homology-based DNA repair and activation of the ATR-dependent checkpoint. Despite its critical importance in inducing the overall DNA damage response, the mechanisms and regulation of the Exo1 resection pathway remain incompletely understood. Here, we identify the ring-shaped DNA clamp PCNA as a new factor in the Exo1 resection pathway. Using mammalian cells, Xenopus nuclear extracts and purified proteins, we show that after DNA damage, PCNA loads onto double-strand breaks and promotes Exo1 damage association through direct interaction with Exo1. By tethering Exo1 to the DNA substrate, PCNA confers processivity to Exo1 in resection. This role of PCNA in DNA resection is analogous to its function in DNA replication where PCNA serves as a processivity co-factor for DNA polymerases.
Exo1 介导的 DNA 双链断裂末端的切除产生同源性 DNA 修复和激活 ATR 依赖性检查点所需的 3'单链 DNA 突出端。尽管它在诱导整体 DNA 损伤反应中至关重要，但 Exo1 切除途径的机制和调控仍不完全清楚。在这里，我们鉴定了环形 DNA 夹 PCNA 为 Exo1 切除途径中的一个新因子。使用哺乳动物细胞、非洲爪蟾核提取物和纯化蛋白，我们表明，在 DNA 损伤后，PCNA 加载到双链断裂上，并通过与 Exo1 的直接相互作用促进 Exo1 损伤关联。通过将 Exo1 束缚在 DNA 底物上，PCNA 赋予 Exo1 在切除过程中的连续性。PCNA 在 DNA 切除中的这种作用类似于其在 DNA 复制中的功能，在 DNA 复制中，PCNA 作为 DNA 聚合酶的连续性辅助因子。
