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Dissertation
The branch migration of Holliday junctions by the homologous recombination proteins RecA, Rad51, and Rad54
Doctor of Philosophy (Ph.D.), Drexel University
Dec 2010
DOI:
https://doi.org/10.17918/00009372
Abstract
Homologous recombination is the process responsible for repairing double-strand DNA breaks, exchanging genetic material, and ensuring faithful segregation of homologous chromosomes. Individuals with deficiencies in homologous recombination have dramatically increased incidents of cancer, premature aging diseases, and genome instability. During homologous recombination, two homologous DNA duplexes are paired by the bacterial recombinase protein RecA or its eukaryotic homologue Rad51 to form a cross-stranded structure termed a Holliday junction. Several helicase-like proteins, including Rad54, are known to bind Holliday junctions and translocate this structure along DNA in a process known as branch migration. Branch migration is a highly regulated process with multiple possible outcomes. We believe that in order to understand how homologous recombination functions and is controlled on a cellular level, we must first understand how the branch migration of Holliday junctions occurs on a molecular level. Therefore, we used a variety of in vitro assays that utilize intricate DNA substrates to study the molecular mechanisms of the branch migration reaction as promoted by the homologous recombination proteins RecA, Rad51, and Rad54. RecA and Rad51 promote the branch migration of Holliday junctions despite the fact they do not bind Holliday junctions preferentially and do not translocate along DNA. The mechanism of branch migration promoted by RecA/RAD51 is unknown. Here, we demonstrate that cycles of RecA/Rad51 polymerization and dissociation coupled with ATP hydrolysis drives the branch migration of Holliday junctions. Rad54 is a more prototypical branch migration protein that binds specifically to Holliday junctions and translocates on DNA. Rad54 performs its functions during homologous recombination in an association with Rad51. We studied the stimulatory effect that RAD51 has on the branch migration activity of RA054 and found this stimulation to be driven by specific protein-protein interactions between these proteins. We also performed a detailed investigation of Rad54's branch migration activity and found that Rad54 possesses a non-canonical helicase activity that allows it to bypass significant regions of heterology during branch migration; and we have identified a motif of Rad54 that supports its oligomerization and is critical to its branch migration activity.
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Details
- Title
- The branch migration of Holliday junctions by the homologous recombination proteins RecA, Rad51, and Rad54
- Creators
- Matthew J. Rossi
- Contributors
- Alexander V. Mazin (Advisor) - Drexel University, Drexel University (1970-)
- Awarding Institution
- Drexel University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- xvii, 255 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- College of Medicine; Drexel University
- Other Identifier
- 991021888947204721