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

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 Based on the intensive interactions between the Luke and Lou labs, as well as the sharing of unpublished resources between two groups since 2015, we propose to illustrate how replication processivity or velocity affects fork progression through different genomic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