Functional dissection of the S. cerevisiae helicase activating factor Cdc45
Thesis (S.M.)--Massachusetts Institute of Technology, Department of Biology, 2013.
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| Format: | Thesis |
| Language: | eng |
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Massachusetts Institute of Technology
2014
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| Online Access: | http://hdl.handle.net/1721.1/84876 |
| _version_ | 1811082929222189056 |
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| author | Chan, Sze Ham (Sze Ham Bena) |
| author2 | Stephen P. Bell. |
| author_facet | Stephen P. Bell. Chan, Sze Ham (Sze Ham Bena) |
| author_sort | Chan, Sze Ham (Sze Ham Bena) |
| collection | MIT |
| description | Thesis (S.M.)--Massachusetts Institute of Technology, Department of Biology, 2013. |
| first_indexed | 2024-09-23T12:13:58Z |
| format | Thesis |
| id | mit-1721.1/84876 |
| institution | Massachusetts Institute of Technology |
| language | eng |
| last_indexed | 2024-09-23T12:13:58Z |
| publishDate | 2014 |
| publisher | Massachusetts Institute of Technology |
| record_format | dspace |
| spelling | mit-1721.1/848762019-04-11T03:13:12Z Functional dissection of the S. cerevisiae helicase activating factor Cdc45 Chan, Sze Ham (Sze Ham Bena) Stephen P. Bell. Massachusetts Institute of Technology. Department of Biology. Massachusetts Institute of Technology. Department of Biology. Biology. Thesis (S.M.)--Massachusetts Institute of Technology, Department of Biology, 2013. Cataloged from PDF version of thesis. Includes bibliographical references. Eukaryotic DNA replication initiation requires two essential steps: helicase loading and activation. During the G1 to S transition, loaded Mcm2-7 helicases are activated by the S-CDK (S phase cyclin-dependent kinase)- and DDK (Dbf4-dependent kinase)- dependent recruitment of the helicase activating proteins Cdc4S and GINS. The resulting Cdc45/Mcm2-7/GINS (CMG) complex unwinds double-stranded DNA at the replication fork. Although it is clear that CMG complex possesses robust helicase activity in vitro that is dependent on GINS and Cdc45, it is unclear how the CMG is assembled in vivo. Moreover, the mechanism of Cdc45 and GINS stimulation of Mcm2-7 helicase activity is unclear. In budding yeast, recruitment of Cdc45 and GINS occurs sequentially with Cdc45 associating with Mcm2-7 in a DDK- and Sd3- dependent manner. Subsequently, S-CDK stimulates the association of GINS in a Sd2-, DNA Pol epsilon- and Dpbll-mediated event. To better understand the mechanism of helicase activation, I have dissected the function of Cdc45 using a combination of in vivo and in vitro approaches. I generated a series of deletion and site-specific mutations of Cdc45 based on homology to RecJ, structural prediction algorithms and sequence conservation. Functional domain mapping of Cdc45 revealed that both N- and C-terminal segments are essential in vivo. Analysis of site-specific mutations identified five lethal and three temperature-sensitive site-specific mutations. Three of these mutations are within a putative DHH phosphoesterase domain related to bacterial RecJ, suggesting a critical role for the RecJ-homology region for Cdc45 function. Two lethal site-specific mutations were found within a predicted intrinsically disordered region (IDR). Oddly, a complete deletion of the IDR is dispensable for Cdc45 function in vivo. Mutants that failed to fully complement a CDC45 deletion in vivo were purified and tested for their ability to complement Cdc45 function biochemically. Purified Cdc45 mutants were found to be associated with Hsp70 chaperones, and subsequently failed to fully restore in vitro replication in a Cdc45-dependent biochemical assay. Co-immunoprecipitation studies revealed an interaction of Cdc45 with Sd3 and S1d7 and mapped a Cdc45 region that interferes with the Sld7 interaction. Together, these preliminary data provide a starting point for future mechanistic studies to understand role of Cdc45 in helicase activation. by Sze Ham (Bena) Chan. S.M. 2014-02-10T16:57:24Z 2014-02-10T16:57:24Z 2013 Thesis http://hdl.handle.net/1721.1/84876 868669217 eng M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission. http://dspace.mit.edu/handle/1721.1/7582 102 pages application/pdf Massachusetts Institute of Technology |
| spellingShingle | Biology. Chan, Sze Ham (Sze Ham Bena) Functional dissection of the S. cerevisiae helicase activating factor Cdc45 |
| title | Functional dissection of the S. cerevisiae helicase activating factor Cdc45 |
| title_full | Functional dissection of the S. cerevisiae helicase activating factor Cdc45 |
| title_fullStr | Functional dissection of the S. cerevisiae helicase activating factor Cdc45 |
| title_full_unstemmed | Functional dissection of the S. cerevisiae helicase activating factor Cdc45 |
| title_short | Functional dissection of the S. cerevisiae helicase activating factor Cdc45 |
| title_sort | functional dissection of the s cerevisiae helicase activating factor cdc45 |
| topic | Biology. |
| url | http://hdl.handle.net/1721.1/84876 |
| work_keys_str_mv | AT chanszehamszehambena functionaldissectionofthescerevisiaehelicaseactivatingfactorcdc45 |