De Haro Leyma P, Wray Justin, Williamson Elizabeth A, Durant Stephen T, Corwin Lori, Gentry Amanda C, Osheroff Neil, Lee Suk-Hee, Hromas Robert, Nickoloff Jac A
Department of Molecular Genetics and Microbiology, Division of Hematology-Oncology, Cancer Research and Treatment Center, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA.
Nucleic Acids Res. 2010 Sep;38(17):5681-91. doi: 10.1093/nar/gkq339. Epub 2010 May 10.
Metnase is a human protein with methylase (SET) and nuclease domains that is widely expressed, especially in proliferating tissues. Metnase promotes non-homologous end-joining (NHEJ), and knockdown causes mild hypersensitivity to ionizing radiation. Metnase also promotes plasmid and viral DNA integration, and topoisomerase IIα (TopoIIα)-dependent chromosome decatenation. NHEJ factors have been implicated in the replication stress response, and TopoIIα has been proposed to relax positive supercoils in front of replication forks. Here we show that Metnase promotes cell proliferation, but it does not alter cell cycle distributions, or replication fork progression. However, Metnase knockdown sensitizes cells to replication stress and confers a marked defect in restart of stalled replication forks. Metnase promotes resolution of phosphorylated histone H2AX, a marker of DNA double-strand breaks at collapsed forks, and it co-immunoprecipitates with PCNA and RAD9, a member of the PCNA-like RAD9-HUS1-RAD1 intra-S checkpoint complex. Metnase also promotes TopoIIα-mediated relaxation of positively supercoiled DNA. Metnase is not required for RAD51 focus formation after replication stress, but Metnase knockdown cells show increased RAD51 foci in the presence or absence of replication stress. These results establish Metnase as a key factor that promotes restart of stalled replication forks, and implicate Metnase in the repair of collapsed forks.
金属酶是一种具有甲基化酶（SET）和核酸酶结构域的人类蛋白质，广泛表达，尤其在增殖组织中。金属酶促进非同源末端连接（NHEJ），敲低该蛋白会导致对电离辐射轻度敏感。金属酶还促进质粒和病毒DNA整合以及拓扑异构酶IIα（TopoIIα）依赖性的染色体解连环。NHEJ因子与复制应激反应有关，并且有人提出TopoIIα可缓解复制叉前方的正超螺旋。在此我们表明，金属酶促进细胞增殖，但不改变细胞周期分布或复制叉进展。然而，敲低金属酶会使细胞对复制应激敏感，并在停滞的复制叉重新启动方面产生明显缺陷。金属酶促进磷酸化组蛋白H2AX的消退，H2AX是塌陷叉处DNA双链断裂的标志物，并且它与增殖细胞核抗原（PCNA）以及PCNA样的RAD9-HUS1-RAD1 S期内检查点复合物的成员RAD9共免疫沉淀。金属酶还促进TopoIIα介导的正超螺旋DNA的松弛。复制应激后RAD51焦点形成不需要金属酶，但在有或无复制应激的情况下，敲低金属酶的细胞显示RAD51焦点增加。这些结果确立了金属酶是促进停滞的复制叉重新启动的关键因素，并表明金属酶参与塌陷叉的修复。
