Replisome components in sister chromatid cohesion establishment
File(s)
Author(s)
Shrestha, Sudikchya
Type
Thesis
Abstract
The chromosomal cohesin complex establishes sister chromatid cohesion during S phase, which forms the basis for the recognition of DNA replication products for their faithful segregation in mitosis. Here, I investigate the non-essential S. cerevisiae replication fork components Tof1, Csm3 and Mrc1, whose absence leads to defective sister chromatid cohesion. How these three proteins contribute to cohesion establishment is not yet known.
Tof1, Csm3 and Mrc1 serve known roles during DNA replication, including mediating replication checkpoint signalling, accelerating replication fork progression as well as the recruitment of topoisomerase I and FACT to the replisome. By modulating each of these replication functions independently of Tof1, Csm3 and Mrc1 and using separation of function mutants, I rule out a possible contribution of these functions to the establishment of sister chromatid cohesion. Instead, using purified recombinant components, I reveal direct protein interactions between Tof1/Csm3 and Mrc1 and the cohesin complex. Preliminary results from crosslinking mass spectrometry to identify interaction surfaces narrows down a small cluster of Tof1 and Mrc1 interaction sites in Smc1 and Smc3 subunits of cohesin. Tof1-Csm3 also directly interacts with the Chl1 helicase, another replisome component that contacts cohesin and contributes to cohesion establishment. My findings open the possibility that a series of direct physical interactions between replication fork components and the cohesin complex facilitate successful establishment of sister chromatid cohesion during DNA replication.
Tof1, Csm3 and Mrc1 serve known roles during DNA replication, including mediating replication checkpoint signalling, accelerating replication fork progression as well as the recruitment of topoisomerase I and FACT to the replisome. By modulating each of these replication functions independently of Tof1, Csm3 and Mrc1 and using separation of function mutants, I rule out a possible contribution of these functions to the establishment of sister chromatid cohesion. Instead, using purified recombinant components, I reveal direct protein interactions between Tof1/Csm3 and Mrc1 and the cohesin complex. Preliminary results from crosslinking mass spectrometry to identify interaction surfaces narrows down a small cluster of Tof1 and Mrc1 interaction sites in Smc1 and Smc3 subunits of cohesin. Tof1-Csm3 also directly interacts with the Chl1 helicase, another replisome component that contacts cohesin and contributes to cohesion establishment. My findings open the possibility that a series of direct physical interactions between replication fork components and the cohesin complex facilitate successful establishment of sister chromatid cohesion during DNA replication.
Version
Open Access
Date Issued
2022-11
Date Awarded
2023-08
Copyright Statement
Creative Commons Attribution NonCommercial Licence
License URL
Advisor
Uhlmann, Frank
Sponsor
Francis Crick Institute
Boehringer Ingelheim Pharmaceuticals
Publisher Department
Institute of Clinical Sciences
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)