The interplay between DNA replication speed and R-loop stability regulation and its consequences on genome/telomere integrity

DNA复制速度和R环稳定性调节之间的相互作用及其对基因组/端粒完整性的影响

• 批准号: 468884468
• 负责人: Professor Dr. Brian Luke, Ph.D.
• 金额: --
• 依托单位: National Natural Science Foundation of China
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/468884468?language=en
• 关键词: interplay; between; DNA; replication; speed

All cells suffer exogenous and endogenous replication stress that causes the slowing or stalling of replication forks and/or DNA synthesis. Eukaryotes have evolved a conserved surveillance system, the DNA replication checkpoint (DRC), to deal with replication stress. Our current concepts of the DRC are mostly based on studies using exogenous stressors like hydroxyurea (HU), an RNR inhibitor, which might have off-target effects other than dNTP depletion. In our preliminary work, the Lou lab has isolated several low-processivity yeast mutants of pol2, encoding the catalytic subunit of the leading strand replicase Pol ε. These mutants exhibit slow DNA synthesis without a significant change in mutation frequency, thus representing an ideal model to revisit DRC in the context of endogenous replication stress. Based on the intensive interactions between the Luke and Lou labs, as well as the sharing of unpublished resources between two groups since 2015, we propose to illustrate how replication processivity or velocity affects fork progression through different genomic regions, particularly difficult-to-replicate or R-loop forming areas including telomeres. We plan to (i) isolate and characterize the slow-replication mutants in budding yeast; (ii) elucidate the effects of replication velocity changes on genome-wide R-loop formation and stability, and telomere maintenance; (iii) reveal the mechanisms of DRC activation and its major downstream effectors in response to endogenous replication stress; (iv) expand the key findings to mammalian cell lines and implement these findings to genome instability-related human diseases including cancer.These studies will reveal the contribution of an overlooked aspect of DNA replication, the velocity, to genome stability maintenance, whereas most previous studies have focused on the fidelity of DNA replication. With the complete complementary expertise of Chinese and German teams to focus on a distinctive slow-replication model, we believe this project will expand our understanding of aging and genome instability-related diseases and may reveal replication velocity as a future therapeutic target.

所有细胞都承受外源性和内源性复制压力，导致复制分叉和/或DNA合成减慢或停滞。真核生物已经进化出一种保守的监视系统，即DNA复制检查点(DRC)，以应对复制压力。我们目前对DRC的概念大多基于使用外源性应激源的研究，如RNR抑制剂羟基脲(HU)，它可能具有dNTP耗竭以外的非靶向影响。在我们的初步工作中，楼的实验室已经分离了几个POL2的低加工酵母突变体，编码领先链复制酶POLε的催化亚单位。这些突变体表现出缓慢的DNA合成，而突变频率没有显著变化，因此代表了在内源性复制应激的背景下重新研究DRC的理想模型。基于Luke和Lou实验室之间的密集相互作用，以及自2015年以来两组之间共享未发表的资源，我们建议说明复制处理速度或速度如何影响不同基因组区域的分叉进展，特别是包括端粒在内的难以复制或R环形成区域。我们计划(I)分离和鉴定发芽酵母中的慢复制突变体；(Ii)阐明复制速度变化对全基因组R环的形成和稳定性以及端粒维持的影响；(Iii)揭示DRC激活及其主要下游效应因子响应内源复制压力的机制；(Iv)将关键发现扩展到哺乳动物细胞系，并将这些发现应用于包括癌症在内的与基因组不稳定相关的人类疾病。这些研究将揭示DNA复制的一个被忽视的方面，即速度，对维持基因组稳定性的贡献，而以前的大多数研究都集中在DNA复制的保真度上。随着中国和德国团队完全互补的专业知识专注于一种独特的慢复制模式，我们相信这个项目将扩大我们对衰老和基因组不稳定相关疾病的理解，并可能揭示复制速度作为未来的治疗目标。