Single-Molecule Studies of Replication Kinetics in Response to DNA Damage
Authors
Iyer, Divya RamalingamFaculty Advisor
Nick Rhind, PhDAcademic Program
Interdisciplinary Graduate ProgramUMass Chan Affiliations
Department of Biochemistry and Molecular PharmacologyDocument Type
Doctoral DissertationPublication Date
2017-05-24Keywords
single-molecule replication kineticsDNA combing
fork rate
fork stalling
origin firing rate
DNA damage
intra-S checkpoint
fission yeast
Biochemistry
Cell Biology
Genetics
Molecular Biology
Metadata
Show full item recordAbstract
In response to DNA damage during S phase, cells slow DNA replication. This slowing is orchestrated by the intra-S checkpoint and involves inhibition of origin firing and reduction of replication fork speed. Slowing of replication allows for tolerance of DNA damage and suppresses genomic instability. Although the mechanisms of origin inhibition by the intra-S checkpoint are understood, major questions remain about how the checkpoint regulates replication forks: Does the checkpoint regulate the rate of fork progression? Does the checkpoint affect all forks, or only those encountering damage? Does the checkpoint facilitate the replication of polymerase-blocking lesions? To address these questions, we have analyzed the checkpoint in the fission yeast Schizosaccharomyces pombe using a single-molecule DNA combing assay, which allows us to unambiguously separate the contribution of origin and fork regulation towards replication slowing, and allows us to investigate the behavior of individual forks. Moreover, we have interrogated the role of forks interacting with individual sites of damage by using three damaging agents—MMS, 4NQO and bleomycin—that cause similar levels of replication slowing with very different frequency of DNA lesions. We find that the checkpoint slows replication by inhibiting origin firing, but not by decreasing fork rates. However, the checkpoint appears to facilitate replication of damaged templates, allowing forks to more quickly pass lesions. Finally, using a novel analytic approach, we rigorously identify fork stalling events in our combing data and show that they play a previously unappreciated role in shaping replication kinetics in response to DNA damage.DOI
10.13028/M27M28Permanent Link to this Item
http://hdl.handle.net/20.500.14038/32286Rights
Copyright is held by the author, with all rights reserved.ae974a485f413a2113503eed53cd6c53
10.13028/M27M28