Role of recombination-dependent DNA damage response mechanisms in the resolution of benzo[a]pyrene-induced DNA replication stress

重组依赖性 DNA 损伤反应机制在解决苯并[a]芘诱导的 DNA 复制应激中的作用

• 批准号: 288806128
• 负责人: Dr. Ann Liza Piberger
• 金额: --
• 依托单位: College of Medical and Dental Sciences
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2018-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/288806128?language=en
• 关键词: Role; recombination; dependent; DNA; damage

Occupational or smoking-related exposure towards the human carcinogen benzo[a]pyrene (B[a]P) is known to induce lung tumours. This carcinogenicity of B[a]P is significantly mediated by DNA adduct formation of its reactive metabolite anti-B[a]P-7,8-diol-9,10-epoxide (BPDE). Although these DNA adducts impair DNA replication fork progression if remaining unrepaired until S-phase, a process termed DNA replication stress, error-prone bypass by translesion synthesis (TLS) polymerases usually allows continued replication. TLS past BPDE adducts has been extensively studied and is implicated in B[a]P-caused mutagenicity. However, BPDE adducts also lead to homologous recombination (HR) and sister chromatid exchanges, suggesting that recombination factors act at forks that are substantially stalled or collapsed into DNA double-strand breaks (DSBs). The contributions of impaired replication fork progression and replication-dependent formation of DNA DSBs to B[a]P-induced toxicity as well as the role of recombination in resolution of this replication stress are very poorly understood. This project will apply experimental approaches typically used in fundamental research on DNA replication stress to illuminate whether BPDE adducts block or collapse the incoming replication apparatus and how recombination factors act at those forks in eukaryotes. Three interesting candidate proteins involved in remodelling and recombination at impaired forks, PARP1, RAD51, and ZRANB3, will be systematically investigated for their function and potential interplay at replication forks in response to BPDE treatment in mammalian cells. Their impact on slowing, stalling, as well as restart or collapse of individual forks and new origin firing will be examined. Analyses of protein dynamics at BPDE-stalled replication forks will additionally clarify which DNA damage response mechanisms and proteins are crucial for recovery. Finally, the role of PARP1, RAD51, and ZRANB3 in BPDE-induced recombination will be assessed to elucidate biochemical reasons for the engagement of HR in DNA damage response to BPDE adducts. As unfaithful recombination could give rise to gross chromosomal instability, a better understanding of recombination-dependent and potentially recombinogenic DNA damage response mechanisms involved in the resolution of BPDE-induced replication stress will ultimately provide novel insight into B[a]P-caused mutagenicity and toxicity. This might uncover innovative strategies for prevention or protection in future.

众所周知，职业或与吸烟有关的暴露于人类致癌物苯并[a]芘（B[a]P）会诱发肺肿瘤。B[a]P的这种致癌性通过其反应性代谢物抗B [a]P-7，8-二醇-9，10-环氧化物（BPDE）的DNA加合物形成而显著介导。尽管这些DNA加合物如果在S期之前保持未修复会损害DNA复制叉进展，但称为DNA复制应激的过程，通过跨损伤合成（TLS）聚合酶的易错旁路通常允许继续复制。已对TLS过去的BPDE加合物进行了广泛研究，并涉及B[a] P引起的致突变性。然而，BPDE加合物也会导致同源重组（HR）和姐妹染色单体交换，这表明重组因子作用于基本停滞或折叠成DNA双链断裂（DSB）的分叉。受损的复制叉进展和DNA DSB的复制依赖性形成对B[a] P诱导的毒性的贡献以及重组在解决这种复制应激中的作用知之甚少。该项目将应用通常用于DNA复制应激基础研究的实验方法，以阐明BPDE加合物是否会阻止或破坏传入的复制装置，以及重组因子如何在真核生物中的这些分叉处起作用。三个有趣的候选蛋白参与重建和重组受损叉，PARP 1，RAD 51和ZRANB 3，将系统地研究其功能和潜在的相互作用，在复制叉在哺乳动物细胞中响应BPDE治疗。他们的影响放缓，失速，以及重新启动或崩溃的个别叉和新的起源发射将被检查。BPDE停滞复制叉的蛋白质动力学分析将进一步阐明哪些DNA损伤反应机制和蛋白质对恢复至关重要。最后，PARP 1，RAD 51和ZRANB 3在BPDE诱导的重组中的作用将被评估，以阐明HR参与对BPDE加合物的DNA损伤反应的生化原因。由于不忠实的重组可能会引起总的染色体不稳定性，更好地了解重组依赖性和潜在的重组DNA损伤的反应机制，涉及的决议BPDE诱导的复制压力，最终将提供新的见解B[a] P引起的致突变性和毒性。这可能会揭示未来预防或保护的创新战略。