Regulation of metazoan replisome disassembly at stalled replication forks and during mitosis

Regulation of metazoan replisome disassembly at stalled replication forks and during mitosis Background: The CUL2LRR1 ubiquitin ligase drives ubiquitylation and disassembly of the CMG helicase during DNA replication termination in animal cells.

Metazoa also have a second pathway of CMG helicase disassembly that acts during mitosis and requires the TRAIP ubiquitin ligase.

We showed that TRAIP is required during mitosis to process sites of incomplete DNA replication that would otherwise impede chromosome segregation during anaphase.

These findings suggest that TRAIP facilitates chromosome segregation and preserves genome integrity, whenever cells enter mitosis with incomplete DNA replication, by inducing the ubiquitylation and disassembly of the CMG helicase.

We have also found that the deubiquitylase USP37, which is an interesting target for new anti-cancer therapies, associates with the mammalian replisome and is thus a strong candidate for a factor that counteracts CMG ubiquitylation and thereby allows cells to survive DNA replication stress.

Aims: Test the following hypotheses: - that the USP37 deubiquitylase protects the mammalian replisome from premature disassembly before DNA replication termination. - that metazoa regulate the TRAIP ubiquitin ligase during mitosis by post-translational modifications and association with partner proteins - that the mitotic TRAIP pathway is one of multiple mechanisms in metazoa that preserve genome integrity during mitosis, by processing DNA replication forks at sites of incomplete DNA replication.

Methods: We propose to dissect the molecular basis for the mitotic regulation of TRAIP activity, identifying the factors and post-translational modifications that are required for TRAIP to support mitotic CMG ubiquitylation and disassembly.

We will also explore the physiological significance of the TRAIP pathway and its relation to other genome stability pathways that act during mitosis to preserve genome integrity.

We will map and mutate the interaction of USP37 with the replisome, to study the importance of this interaction for replisome stability.

To investigate the mechanism, we will also study the regulation of CMG ubiquitylation by USP37 in vitro using reconstituted replication / ubiquitylation reactions based on purified proteins. How the results of the research will be used: Our findings will be published and materials made freely available.

These findings will relevant to the therapeutic exploitation of inherent DNA replication defects in cancer cells.