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.

DOI: https://doi.org/10.21954/ou.ro.00011f56

Abstract

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.