REPSUBREP · Regulatory mechanisms coordinating replication integrity and intra-S repair

The RecQ family of helicases, including BLM in human and its Saccharomyces cerevisiae homolog Sgs1, have important roles in maintaining genome stability. Sgs1 plays a specific role in response to intra-S DNA damage by resolving the pseudo double HJs resulting from replication-related sister chromatid junctions (SCJs). These catenated DNA structures are thought to represent template switch (TS) intermediates. Although the molecular mechanism is largely unknown, TS is thought to represent a major mechanism to bypass damage during replication. We recently showed that two genetic TS pathways, both of which involve homologous recombination (HR) factors, can lead to bypass of damage by means of SCJs and that their choice is regulated by sumoylation of a key replication factor, PCNA. We also found that the main TS pathway operating in cells involves the coordinated action of HR with the post-replication repair proteins Rad18, Rad5 and Mms2-Ubc13, which exert their role in promoting SCJ formation by means of ubiquitilating PCNA. Previously we showed that the resolution activity of Sgs1 and Top3 is regulated by Ubc9- and Mms21-dependent sumoylation, but the SUMO targets implicated with Sgs1 in this process remained unknown. In this project we plan to characterize the pathways that lead to TS and to understand the factors that promote different steps of this process and their regulation, using in vivo and in vitro approaches. We will attempt to understand how sumoylation is activated by intra-S damage and to address how the formation and subsequent resolution of these X-structures is coordinated with chromatin remodeling functions and topological transitions. We will characterize the X-shaped molecules formed during replication and we will attempt their visualization.