Kim Hyun-Suk, Williamson Elizabeth A, Nickoloff Jac A, Hromas Robert A, Lee Suk-Hee
From the Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202.
the Department of Medicine, University of Florida and Shands Health Care System, Gainesville, Florida 32610, and.
J Biol Chem. 2017 Jan 27;292(4):1414-1425. doi: 10.1074/jbc.M116.745646. Epub 2016 Dec 14.
Stalling at DNA replication forks generates stretches of single-stranded (ss) DNA on both strands that are exposed to nucleolytic degradation, potentially compromising genome stability. One enzyme crucial for DNA replication fork repair and restart of stalled forks in human is Metnase (also known as SETMAR), a chimeric fusion protein consisting of a su(var)3-9, enhancer-of-zeste and trithorax (SET) histone methylase and transposase nuclease domain. We previously showed that Metnase possesses a unique fork cleavage activity necessary for its function in replication restart and that its SET domain is essential for recovery from hydroxyurea-induced DNA damage. However, its exact role in replication restart is unclear. In this study, we show that Metnase associates with exonuclease 1 (Exo1), a 5'-exonuclease crucial for 5'-end resection to mediate DNA processing at stalled forks. Metnase DNA cleavage activity was not required for Exo1 5'-exonuclease activity on the lagging strand daughter DNA, but its DNA binding activity mediated loading of Exo1 onto ssDNA overhangs. Metnase-induced enhancement of Exo1-mediated DNA strand resection required the presence of these overhangs but did not require Metnase's DNA cleavage activity. These results suggest that Metnase enhances Exo1-mediated exonuclease activity on the lagging strand DNA by facilitating Exo1 loading onto a single strand gap at the stalled replication fork.
DNA复制叉停滞会在两条链上产生单链（ss）DNA片段，这些片段会暴露于核酸酶降解作用下，这可能会损害基因组稳定性。在人类中，一种对DNA复制叉修复和停滞复制叉重启至关重要的酶是Metnase（也称为SETMAR），它是一种嵌合融合蛋白，由一个类异染色质蛋白3-9、zeste增强子和三体胸苷（SET）组蛋白甲基转移酶以及转座酶核酸酶结构域组成。我们之前表明，Metnase具有独特的叉状切割活性，这是其在复制重启中发挥功能所必需的，并且其SET结构域对于从羟基脲诱导的DNA损伤中恢复至关重要。然而，其在复制重启中的确切作用尚不清楚。在本研究中，我们表明Metnase与核酸外切酶1（Exo1）相互作用，Exo1是一种5'-核酸外切酶，对5'-末端切除以介导停滞复制叉处的DNA加工至关重要。在滞后链子代DNA上，Exo1的5'-核酸外切酶活性不需要Metnase的DNA切割活性，但其DNA结合活性介导了Exo1加载到单链DNA突出端上。Metnase诱导的Exo1介导的DNA链切除增强需要这些突出端的存在，但不需要Metnase的DNA切割活性。这些结果表明，Metnase通过促进Exo1加载到停滞复制叉处的单链缺口上，增强了Exo1介导的滞后链DNA核酸外切酶活性。
