Sharma Neelam, Speed Michael C, Allen Christopher P, Maranon David G, Williamson Elizabeth, Singh Sudha, Hromas Robert, Nickoloff Jac A
Department of Environmental and Radiological Health Sciences, Colorado State University, 1618 Campus Delivery, Fort Collins, CO 80523-1618, USA.
Department of Microbiology, Immunology, and Pathology, Colorado State University, 1601Campus Delivery, Fort Collins, CO 80523-1601, USA.
NAR Cancer. 2020 Jun;2(2):zcaa008. doi: 10.1093/narcan/zcaa008. Epub 2020 Jun 8.
Accurate DNA replication and segregation are critical for maintaining genome integrity and suppressing cancer. Metnase and EEPD1 are DNA damage response (DDR) proteins frequently dysregulated in cancer and implicated in cancer etiology and tumor response to genotoxic chemo- and radiotherapy. Here, we examine the DDR in human cell lines with CRISPR/Cas9 knockout of Metnase or EEPD1. The knockout cell lines exhibit slightly slower growth rates, significant hypersensitivity to replication stress, increased genome instability and distinct alterations in DDR signaling. Metnase and EEPD1 are structure-specific nucleases. EEPD1 is recruited to and cleaves stalled forks to initiate fork restart by homologous recombination. Here, we demonstrate that Metnase is also recruited to stalled forks where it appears to dimethylate histone H3 lysine 36 (H3K36me2), raising the possibility that H3K36me2 promotes DDR factor recruitment or limits nucleosome eviction to protect forks from nucleolytic attack. We show that stalled forks are cleaved normally in the absence of Metnase, an important and novel result because a prior study indicated that Metnase nuclease is important for timely fork restart. A double knockout was as sensitive to etoposide as either single knockout, suggesting a degree of epistasis between Metnase and EEPD1. We propose that EEPD1 initiates fork restart by cleaving stalled forks, and that Metnase may promote fork restart by processing homologous recombination intermediates and/or inducing H3K36me2 to recruit DDR factors. By accelerating fork restart, Metnase and EEPD1 reduce the chance that stalled replication forks will adopt toxic or genome-destabilizing structures, preventing genome instability and cancer. Metnase and EEPD1 are overexpressed in some cancers and thus may also promote resistance to genotoxic therapeutics.
准确的DNA复制和分离对于维持基因组完整性和抑制癌症至关重要。金属酶（Metnase）和EEPD1是DNA损伤反应（DDR）蛋白，在癌症中经常失调，并与癌症病因和肿瘤对基因毒性化学疗法及放射疗法的反应有关。在这里，我们用CRISPR/Cas9敲除金属酶或EEPD1来检测人类细胞系中的DDR。敲除细胞系的生长速度略慢，对复制应激显著敏感，基因组不稳定性增加，DDR信号传导有明显改变。金属酶和EEPD1是结构特异性核酸酶。EEPD1被招募到停滞的复制叉并切割它们，以通过同源重组启动复制叉重新启动。在这里，我们证明金属酶也被招募到停滞的复制叉，在那里它似乎使组蛋白H3赖氨酸36（H3K36me2）发生二甲基化，这增加了H3K36me2促进DDR因子招募或限制核小体去除以保护复制叉免受核酸酶攻击的可能性。我们表明，在没有金属酶的情况下，停滞的复制叉能正常切割，这是一个重要且新颖的结果，因为先前的一项研究表明金属酶核酸酶对及时的复制叉重新启动很重要。双敲除对依托泊苷的敏感性与单敲除一样，这表明金属酶和EEPD1之间存在一定程度的上位性。我们提出，EEPD1通过切割停滞的复制叉启动复制叉重新启动，而金属酶可能通过处理同源重组中间体和/或诱导H3K36me2招募DDR因子来促进复制叉重新启动。通过加速复制叉重新启动，金属酶和EEPD1减少了停滞的复制叉采用有毒或基因组不稳定结构的可能性，防止基因组不稳定和癌症。金属酶和EEPD1在某些癌症中过表达，因此也可能促进对基因毒性疗法的抗性。
