Mechanisms and Factors Promoting Faithful Replication of Problematic Genomic Regions

Agashe, Sumedha (2020). Mechanisms and Factors Promoting Faithful Replication of Problematic Genomic Regions. PhD thesis The Open University.



Natural Pausing Sites (NPSs) are complex genomic regions predisposed to fragility. Our lab previously uncovered that the Smc5/6 complex is critical for replication through NPSs. We hypothesized that Smc5/6 maintains NPS integrity by coupling replication fork pausing at different repeat elements with recombination and recombination intermediate resolution. The Sgs1-Top3-Rmi1 (STR) complex and Smc5/6 co-localize genome-wide in G2/M, and prevent accumulation of recombination structures at damaged forks. Here we use several genome-wide and locus-specific methods to investigate the mechanisms and factors involved in NPS metabolism. We find evidence that Smc5/6 collaborates tightly with STR and coordinates various resolvases at NPSs to support replication completion.

Smc5/6 chromatin clusters overlap with the ones of Top3 and Rmi1 and are enriched at NPSs, where Smc5/6 facilitates Top3 retention. Further, we observe that Smc6 mutants that accumulate recombination intermediates at replication termination regions encode variants that bind less efficiently to NPSs. Both Smc5/6 dysfunction and STR depletion cause accumulation of recombination intermediates at stalled NPSs. A newly discovered intragenic mutation of smc6-56 restores Top3 binding but causes additivity with various sgs1 mutants, suggesting defects in other resolvases, possibly Mus81-Mms4. We further observe a role for Smc5/6, STR and DNA damage tolerance (DDT) pathways mediated by the polymerase clamp PCNA at topologically constrained regions along with Top2. We observe aggravated top2-4 temperature sensitivity for mutants of the above-mentioned factors, which is independent of Rad51 dependent recombination.

Taken together, our results indicate a role for the STR complex in collaboration with Smc5/6 in NPS maintenance by resolving recombination intermediates to allow faithful segregation of these complex genomic regions. We further observe a role for STR, DDT and Smc5/6 along Top2 in facilitating resolution of topological stress before and during mitosis.

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