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复制应激，这导致复制叉和/或DNA合成的减慢或停滞。真核生物已经进化出一个保守的监视系统，DNA复制检查点（DRC），以应对复制压力。我们目前对DRC的概念主要是基于使用外源性应激源如RNR抑制剂羟基脲（HU）的研究，其可能具有除dNTP耗尽之外的脱靶效应。在我们的初步工作中，Lou实验室已经分离出几种低合成能力的pol 2酵母突变体，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，而突变频率没有显着变化，因此代表了一个理想的模型，重新审视DRC的背景下，内源性复制应力。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，我们建议说明复制持续性或速度如何影响不同基因组区域的分叉进程，特别是难以复制或R环形成区域，包括端粒。我们计划（i）分离和表征芽殖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物响应内源性复制应激的机制;（iv）研究DRC的启动子和下游效应物。（iv）将主要研究结果扩展至哺乳动物细胞系，并将这些研究结果应用于与基因组不稳定性有关的人类疾病，包括癌症。这些研究将揭示DNA复制中一个被忽视的方面，即速度，基因组稳定性的维护，而大多数以前的研究都集中在DNA复制的保真度。凭借中德团队的完整互补专业知识，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性相关疾病的理解，并可能揭示复制速度作为未来治疗目标。

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 Based on the intensive interactions between the Luke and Lou labs, as well as the sharing of unpublished resources between two groups since 2015, we propose to illustrate how replication processivity or velocity affects fork progression through different genomic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