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

所有细胞都遭受外源性和内源性的复制压力，导致复制分叉和/或DNA合成的减慢或停止。真核生物已经进化出一种保守的监视系统——DNA复制检查点（DRC）来处理复制压力。我们目前对DRC的概念主要基于使用外源应激源的研究，如羟基脲（HU），一种RNR抑制剂，它可能具有脱靶效应，而不是dNTP耗竭。在我们的初步工作中，Lou实验室已经分离出几个低加工能力的酵母突变体pol2，编码前导链复制酶Pol ε的催化亚基。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背景下重新审视DRC的理想模型。基于Luke和Lou实验室之间的密切互动，以及自2015年以来两组之间未发表资源的共享，我们建议说明复制过程或速度如何影响分叉在不同基因组区域的进展，特别是难以复制或r环形成区域，包括端粒。我们计划(i)在出芽酵母中分离和表征慢复制突变体；（ii）阐明复制速度变化对全基因组r环形成、稳定性和端粒维持的影响；（iii）揭示DRC在内源性复制胁迫下的激活机制及其主要下游效应物；（四）将关键发现扩大到哺乳动物细胞系，并将这些发现应用于与基因组不稳定有关的人类疾病，包括癌症。这些研究将揭示DNA复制的一个被忽视的方面，即速度，对基因组稳定性维持的贡献，而大多数先前的研究都集中在DNA复制的保真度上。中国和德国团队的专业知识完全互补，专注于独特的慢复制模型，我们相信该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。

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 Based on the intensive interactions between the Luke and Lou labs, as well as the sharing of unpublished resources between two groups since 2015, we propose to illustrate how replication processivity or velocity affects fork progression through different genomic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.