Replication fork remodeling and genomic stability

 描述（由申请方提供）：基因组DNA不断受到细胞代谢过程中自发诱导或外源性DNA损伤剂产生的DNA损伤的挑战。在DNA复制过程中，DNA损伤会导致复制叉的停滞或崩溃。在复制叉损伤之后，DNA损伤信号转导网络的激活促进复制叉的重新启动，从而保持基因组的完整性。我们最近确定ZRANB 3作为一种新的因子，在复制叉损伤后保持基因组的完整性。特别是，我们表明，ZRANB 3，一种非典型的DNA损伤酶，表现出DNA移位酶和核酸内切酶的活动，促进重新启动的复制叉逮捕的DNA损伤和保护细胞从代理，造成复制叉损伤。尽管有这些重要的观察结果，ZRANB 3在复制叉处保持基因组完整性的确切作用机制目前还缺乏完整的理解。在复制叉损伤后未能保持基因组完整性导致突变和基因组重排的积累，并易于发生癌症。与ZRANB 3作为假定的肿瘤抑制因子的作用一致，最近的癌症基因组研究报道ZRANB 3在子宫内膜癌中显著突变。此外，在黑色素瘤和其他肿瘤类型中也发现了预测影响ZRANB 3功能的突变。该提案的目标是定义ZRANB 3为抑制基因组不稳定性而显示的精确生物化学和细胞活性，并研究这些活性如何受到肿瘤中鉴定的ZRANB 3突变的影响。我们特别建议：1）确定ZRANB 3在复制叉处保持基因组完整性所表现出的活性; 2）阐明ZRANB 3与复制叉损伤后保持基因组完整性的其他DNA损伤因子之间的相互作用; 3）评估ZRANB 3癌症相关突变对基因组不稳定性的贡献。我们的方法将利用新的体外酶测定、创新的蛋白质组学方法、基因组DNA纤维上的复制叉重启测定、染色体畸变测试和下一代基因组测序分析。我们预计，我们的研究将确定ZRANB 3抑制基因组不稳定性的独特机制，并将为ZRANB 3癌症相关突变对肿瘤发展的潜在贡献提供见解。

 DESCRIPTION (provided by applicant): 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. Following replication fork damage, activation of the DNA damage signal transduction network promotes the restart of replication forks thereby preserving genomic integrity. We recently identified ZRANB3 as a novel factor that maintains genomic integrity after replication fork damage. In particular, we showed that ZRANB3, an atypical DNA damage enzyme that exhibits both DNA translocase and endonuclease activities, facilitates the restart of replication forks arrested by DNA lesions and protects cells from agents that cause replication fork damage. Despite these important observations, a complete understanding of the precise mechanisms of action exhibited by ZRANB3 to maintain genomic integrity at replication forks is currently lacking. Failure to maintain genomic integrity after replication fork damage results in the accumulation of mutations and genomic rearrangements and predisposes to cancer development. In agreement with a role of ZRANB3 as a putative tumor suppressor, recent cancer genomic studies have reported that ZRANB3 is significantly mutated in endometrial cancer. In addition, mutations predicted to affect the function of ZRANB3 are also found in melanoma and other tumor types. The goal of this proposal is to define the precise biochemical and cellular activities displayed by ZRANB3 for suppressing genomic instability and examine how these activities are affected by ZRANB3 mutations identified in tumors. In particular, we propose: 1) to define the activities exhibited by ZRANB3 for preserving genomic integrity at replication forks; 2) to elucidate the interplay between ZRANB3 and other DNA damage factors that maintain genomic integrity after replication fork damage; 3) to evaluate the contribution of ZRANB3 cancer-associated mutations to genomic instability. Our approach will utilize new in vitro enzymatic assays, innovative proteomic methods, replication fork restart assays on genomic DNA fibers, chromosomal aberration tests and next-generation genome sequencing analyses. We anticipate that our studies will define the unique mechanisms by which ZRANB3 suppresses genomic instability and will provide insights into the potential contribution of ZRANB3 cancer-associated mutations to tumor development.