TelA: an agrobacterial telomere resolvase
| dc.contributor.committeeMember | Wilson, Joyce | |
| dc.contributor.committeeMember | White, Aaron | |
| dc.contributor.committeeMember | Chelico, Linda | |
| dc.contributor.committeeMember | Koester, Wolfgang | |
| dc.creator | McGrath, Siobhan L | |
| dc.date.accessioned | 2020-05-12T20:27:40Z | |
| dc.date.available | 2020-05-12T20:27:40Z | |
| dc.date.created | 2020-05 | |
| dc.date.issued | 2020-05-12 | |
| dc.date.submitted | May 2020 | |
| dc.date.updated | 2020-05-12T20:27:40Z | |
| dc.description.abstract | In contrast to the majority of prokaryotes, both the Agrobacterium and Borrelia genera have linear replicons, including both chromosomes and plasmids, which are terminated by covalently closed hairpin loops commonly referred to as hairpin telomeres. These hairpin telomeres are resolved from a dimeric replication intermediate through a process called telomere resolution. A small, diverse class of enzyme, referred to as telomere resolvases are responsible for this activity. To date, three telomere resolvases have been well-characterized including TelK from Klebsiella phage KO2, TelA from the Agrobacterium genus, and ResT from Borrelia species. Of these ResT is the best biochemically characterized telomere resolvase. Previous studies have revealed that aside from the expected property of telomere resolution, ResT can anneal ssDNA and also possesses an ATP-dependent, 3’-5’ unwinding activity. These results suggest ResT is a multifunctional enzyme. However, at present the in vivo function of these annealing and unwinding activities is unknown. In the work of Bandy et al. conditional expression of ResT in vivo showed ResT depleted cells unexpectedly ceased DNA replication and did not filament as expected, suggesting ResT could be directly or indirectly involved in DNA replication. We sought to analyze a second member of this diverse enzyme family to determine whether these unexpected activities were unique to ResT or represented a property of the enzyme family; and if these activities were identified, to create separation-of-function mutants to eventually assess their function and importance in an in vivo context. We selected the telomere resolvase, TelA from Agrobacterium tumefaciens as it is the closest to ResT in both size and sequence homology and has available partial structural data. We have demonstrated that TelA also has the ability to promote the annealing of complementary ssDNA, and removal of the N-terminal domain of TelA produces a mutant deficient for annealing activity while still maintaining its telomere resolution capabilities. Our data suggests TelA binds ATP; however, it does not possess the ssDNA-dependent ATPase and ATP-dependent unwinding activities observed in ResT, suggesting these activities are not conserved among the telomere resolvase enzyme family. Under certain conditions, ATP can interfere with TelA’s ability to perform telomere resolution and appears to reduce TelA’s affinity for its replicated telomere substrate. Mutation of the active-site nucleophile of TelA (TelA Y405F), while expectedly eliminating telomere resolution, still maintains its annealing activity. Collectively, we have identified the N-terminal domain to be required for TelA’s annealing activity while the catalytic domain alone is sufficient to perform telomere resolution, representing domain separation-of-function in TelA. | |
| dc.format.mimetype | application/pdf | |
| dc.identifier.uri | http://hdl.handle.net/10388/12833 | |
| dc.subject | telomere resolvase | |
| dc.subject | DNA enzymes | |
| dc.subject | agrobacterium tumefaciens | |
| dc.subject | hairpin telomeres | |
| dc.subject | DNA-protein interactions | |
| dc.title | TelA: an agrobacterial telomere resolvase | |
| dc.type | Thesis | |
| dc.type.material | text | |
| thesis.degree.department | Microbiology and Immunology | |
| thesis.degree.discipline | Microbiology and Immunology | |
| thesis.degree.grantor | University of Saskatchewan | |
| thesis.degree.level | Masters | |
| thesis.degree.name | Master of Science (M.Sc.) |