Dissertation
Recombinase filament dynamics and paralog involvement during homologous recombination in the hyperthermophilic crenarchaeon Sulfolobus solfataricus.
Doctor of Philosophy (PhD), Washington State University
01/2014
Handle:
https://hdl.handle.net/2376/117109
Abstract
Homologous recombination (HR) is a high-fidelity mechanism used by cells to repair DNA double stranded breaks and collapsed replication forks. Recombinases perform the major steps in HR and are highly conserved. In addition to recombinases, all organisms encode other ancillary and mediator proteins that promote efficient and effective HR. The Haseltine laboratory uses a hyperthermophilic archaeal model organism named Sulfolobus solfataricus to probe the functions of HR proteins. We are especially interested in the mechanism of action of the S. solfataricus recombinase, SsoRadA, and the three SsoRadA paralog proteins: SsoRal1, SsoRal2, and SsoRal3.
This dissertation describes original research undertaken to determine the previously undescribed mechanism of the recombinase paralog proteins encoded by S. solfataricus and expands on the mechanism of ATP and metal cofactor utilization by the SsoRadA recombinase. The information herein is organized into five chapters. Chapter one gives a detailed overview of current knowledge related to function of recombinase paralog proteins and mechanism of presynaptic filament formation. Chapter two describes the function of the SsoRal3 protein, its biochemical characteristics, and its mechanistic relationship to SsoRadA. We found that SsoRal3 acts as a stabilization factor for the presynaptic filament and has unusual DNA binding characteristics. The presence of ATP regulates SsoRal3 preference for single-stranded DNA versus double stranded DNA. In chapter three, we describe the study undertaken to characterize the SsoRal1 protein and its interaction with SsoRadA. Similar to SsoRal3, SsoRal1 works as a presynaptic filament stabilization factor but can only bind ssDNA. Chapter four describes the SsoRadA recombinase and elaborates on two major methods of recombinase functional control. We found that certain SsoRadA functions are vastly changed in the presence of specific divalent metal cations and that regulation of both ATP binding and hydrolysis are critical to full SsoRadA function. The last chapter of this document includes conclusions about how this work has advanced the field and also discusses potential future directions for this project.
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Details
- Title
- Recombinase filament dynamics and paralog involvement during homologous recombination in the hyperthermophilic crenarchaeon Sulfolobus solfataricus.
- Creators
- William J. Graham
- Contributors
- Cynthia A Haseltine (Advisor)Chengtao Her (Committee Member)William B Davis (Committee Member)Michael Smerdon (Committee Member)
- Awarding Institution
- Washington State University
- Academic Unit
- Molecular Biosciences, School of
- Theses and Dissertations
- Doctor of Philosophy (PhD), Washington State University
- Number of pages
- 194
- Identifiers
- 99900581535401842
- Language
- English
- Resource Type
- Dissertation