A single-molecule approach to understanding sister chromatid cohesion establishment
File(s)
Author(s)
Glaser, Samson
Type
Thesis
Abstract
Sister chromatid cohesion is established by the cohesin complex during DNA replication in S phase. This process requires cohesin to transition from binding to one DNA strand to co-entrapping the two newly synthesised sister chromatids. Passage of the eukaryotic replisome through the ring-shaped cohesin complex has been proposed as a fail-safe mechanism to ensure that cohesin entraps the two sister chromatids, enabling their faithful segregation during cell division. However, whether replisomes can indeed pass through cohesin remains unknown, and the details of replisome-cohesin encounters are poorly understood.
Here, I use single molecule fluorescence microscopy to directly visualise encounters between biochemically reconstituted DNA replication forks and cohesin complexes. On a linear forked DNA, I find that the translocating replicative CMG helicase acts as an obstacle to cohesin. The obstacle is overcome by cohesin at low frequencies, which increase in presence of added replisome components with known sister chromatid cohesion establishment functions. When lateral cohesin diffusion is prevented, CMG passage, during which cohesin retains topological DNA entrapment, becomes the predominant outcome. Finally, I find that the likelihood of passage also increases when an active replisome encounters cohesin, resulting in successful cohesion establishment between the two replication products. Interestingly, when cohesin’s ability to diffuse on DNA is blocked, replication fork passage is efficiently achieved independently of cohesion establishment factors.
My findings resolve a long-held puzzle by visualising live sister chromatid cohesion establishment as the replisome passes the cohesin ring, providing new insights into the molecular mechanisms of sister chromatid cohesion establishment.
Here, I use single molecule fluorescence microscopy to directly visualise encounters between biochemically reconstituted DNA replication forks and cohesin complexes. On a linear forked DNA, I find that the translocating replicative CMG helicase acts as an obstacle to cohesin. The obstacle is overcome by cohesin at low frequencies, which increase in presence of added replisome components with known sister chromatid cohesion establishment functions. When lateral cohesin diffusion is prevented, CMG passage, during which cohesin retains topological DNA entrapment, becomes the predominant outcome. Finally, I find that the likelihood of passage also increases when an active replisome encounters cohesin, resulting in successful cohesion establishment between the two replication products. Interestingly, when cohesin’s ability to diffuse on DNA is blocked, replication fork passage is efficiently achieved independently of cohesion establishment factors.
My findings resolve a long-held puzzle by visualising live sister chromatid cohesion establishment as the replisome passes the cohesin ring, providing new insights into the molecular mechanisms of sister chromatid cohesion establishment.
Version
Open Access
Date Issued
2024-09-29
Date Awarded
01/06/2025
License URL
Advisor
Uhlmann, Frank
Diffley, John F. X.
Rueda, David
Publisher Department
Department of Infectious Disease
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)