Regulation of DNA synthesis in response to DNA damage

项目摘要 基因组DNA不断受到DNA损伤的挑战，DNA损伤或在细胞增殖过程中自发诱导， 代谢或由外源性DNA损伤剂产生。在DNA复制过程中，DNA损伤可以 导致复制分叉停止或崩溃。分叉塌陷导致DNA双链的形成 中断（DSB）。MCM 8和MCM 9（MCM 8 -9）形成解旋酶复合物，其通过以下方式促进DSB的修复： 同源重组我们最近发现MCM 8IP是MCM 8 -9的一种新型相互作用物， 复制应激后的基因组完整性特别是，我们发现MCM 8IP通过以下方式促进DSB修复： 同源重组，促进被DNA损伤阻止的复制叉的重新启动，并保护细胞 复制应激诱导剂造成的DNA损伤尽管这些重要的初步 研究结果表明，在DNA降解过程中，MCM 8IP，MCM 8和MCM 9表现出的确切作用机制 重组和复制仍有待阐明。据报道，MCM 8或MCM 9可作为肿瘤细胞， 抑制因子和最近的癌症基因组分析已经证明了在肿瘤中MCM 8、MCM 9和MCM 8IP的突变， 多种癌症类型。该提案的目标是定义精确的生物化学和细胞活动 MCM 8 IP-MCM 8 -9复合物显示的抑制基因组不稳定性的功能，并研究这些功能是如何发挥作用的。 活性受到肿瘤中鉴定的MCM 8IP、MCM 8和MCM 9中的突变的影响。我们尤其 建议1）定义MCM 8IP-MCM 8 -9表现出的生物化学活性和物理相互作用 2）阐明MCM 8 IP-MCM 8 -9的作用机制， 促进DSB修复并确保复制叉进展以响应DNA损伤; 3）评估 MCM 8IP、MCM 8和MCM 9癌症相关突变对基因组不稳定性的贡献。我们的方法 将利用创新的蛋白质组学方法，最先进的基因组编辑技术，单分子 复制动力学分析、电子显微镜和超分辨率成像。我们预计， 研究将确定MCM 8 IP-MCM 8 -9复合物抑制基因组表达的独特机制。 不稳定性，并将提供对MCM 8IP，MCM 8和MCM 9癌症的潜在贡献的见解- 与癌症病因相关的突变。

PROJECT SUMMARY Genomic DNA is constantly challenged by DNA damage either spontaneously induced during cellular metabolism or generated by exogenous DNA damaging agents. During DNA replication, DNA lesions can cause the stalling or collapse of replication forks. Fork collapse results in the formation of DNA double-strand breaks (DSBs). MCM8 and MCM9 (MCM8-9) form a helicase complex that promotes the repair of DSBs by homologous recombination. We recently identified MCM8IP as a novel interactor of MCM8-9 that maintains genomic integrity after replication stress. In particular, we showed that MCM8IP promotes DSB repair by homologous recombination, facilitates the restart of replication forks arrested by DNA lesions and protects cells from DNA damage generated by replication stress-inducing agents. Despite these important preliminary findings, the precise mechanisms of action exhibited by MCM8IP, MCM8 and MCM9 during DNA recombination and replication remain to be elucidated. MCM8 or MCM9 have been reported to act as tumor suppressors and recent cancer genomic analyses have evidenced mutations in MCM8, MCM9 and MCM8IP in multiple cancer types. The goals of this proposal are to define the precise biochemical and cellular activities displayed by the MCM8IP-MCM8-9 complex for suppressing genomic instability and to examine how these activities are affected by mutations in MCM8IP, MCM8 and MCM9 identified in tumors. In particular, we propose 1) to define the biochemical activities and physical interactions exhibited by the MCM8IP-MCM8-9 complex for preserving genomic integrity; 2) to elucidate the mechanisms by which MCM8IP-MCM8-9 promotes DSB repair and ensures replication fork progression in response to DNA damage; 3) to evaluate the contribution of MCM8IP, MCM8 and MCM9 cancer-associated mutations to genomic instability. Our approach will utilize innovative proteomic methods, state-of-the-art genome editing technologies, single-molecule analyses of replication dynamics, electron microscopy and super-resolution imaging. We anticipate that our studies will define the unique mechanisms employed by the MCM8IP-MCM8-9 complex to suppress genomic instability and will provide insights into the potential contribution of MCM8IP, MCM8 and MCM9 cancer- associated mutations to cancer etiology.