Response of DNA repair and replication systems to exocyclic nucleic acid base damage
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2012.
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| Format: | Thesis |
| Language: | eng |
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Massachusetts Institute of Technology
2012
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| Online Access: | http://hdl.handle.net/1721.1/71468 |
| _version_ | 1826201232855793664 |
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| author | Śrīvāstava, Nidhi |
| author2 | John M. Essigmann. |
| author_facet | John M. Essigmann. Śrīvāstava, Nidhi |
| author_sort | Śrīvāstava, Nidhi |
| collection | MIT |
| description | Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2012. |
| first_indexed | 2024-09-23T11:48:41Z |
| format | Thesis |
| id | mit-1721.1/71468 |
| institution | Massachusetts Institute of Technology |
| language | eng |
| last_indexed | 2024-09-23T11:48:41Z |
| publishDate | 2012 |
| publisher | Massachusetts Institute of Technology |
| record_format | dspace |
| spelling | mit-1721.1/714682019-04-12T20:17:26Z Response of DNA repair and replication systems to exocyclic nucleic acid base damage Response of deoxyribonucleic acid repair and replication systems to exocyclic nucleic acid base damage Śrīvāstava, Nidhi John M. Essigmann. Massachusetts Institute of Technology. Dept. of Biological Engineering. Massachusetts Institute of Technology. Dept. of Biological Engineering. Biological Engineering. Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2012. Cataloged from PDF version of thesis. Includes bibliographical references. Genomes experience an often hostile environment that creates a vast array of damages that can give rise to myriad biological outcomes. Fortunately, cells are equipped with networks such as direct reversal, base excision repair, nucleotide excision repair, homologous recombination, and translesion synthesis that help preserve informational integrity. The first part of this dissertation focuses on whether or not bulky alkyl lesions at the N2 atom of guanine are addressed in vivo by the DinB bypass polymerase. In the work described herein, a collection of N2-guanine lesions was inserted in single-stranded M13 genomes and evaluated in strains possessing or lacking DinB via the competitive replication and adduct bypass (CRAB) and restriction endonuclease and postlabeling (REAP) assays. It was found that DinB could in fact bypass the N2-furfuryl-guanine lesion and its saturated homolog in vivo. The second part of this work describes how we systematically investigated the role that the distance from an origin of replication may have in the mutagenesis of an adduct. Our hypothesis was that a lesion farther from the origin of replication would be less mutagenic since it would be afforded more time for detection and removal before the replicative polymerase traversed it, fixing the mutation. We inserted 0-methylguanine in single-stranded M13 genomes at different distances from the origin of replication and analyzed progeny phage by the REAP assay. Our findings were in contrast with the hypothesis; a higher mutation frequency was obtained at the site distal from the origin of replication. Alternative hypotheses and future experiments are discussed as part of this work. The third part of this dissertation seeks to expand the spectrum of known substrates for the enzyme AIkB, which mediates direct reversal of DNA damage. AlkB is an iron- and CCketoglutarate- dependent dioxygenase that is part of the adaptive response in E. coli, and has homologs in many species. On basis of in vitro data we created the hypothesis that the N 2 guanine lesions as well as 6-methyladenine would be substrates for the enzyme AlkB in vivo. We found, however, in this case the in vitro results did not predict the biology observed in cells. by Nidhi Shrivastav. Ph.D. 2012-07-02T15:43:38Z 2012-07-02T15:43:38Z 2012 2012 Thesis http://hdl.handle.net/1721.1/71468 795194172 eng MIT theses are protected by copyright. They may be viewed, downloaded, or printed from this source but further reproduction or distribution in any format is prohibited without written permission. http://dspace.mit.edu/handle/1721.1/7582 201, 4524-4527, 8 p. application/pdf Massachusetts Institute of Technology |
| spellingShingle | Biological Engineering. Śrīvāstava, Nidhi Response of DNA repair and replication systems to exocyclic nucleic acid base damage |
| title | Response of DNA repair and replication systems to exocyclic nucleic acid base damage |
| title_full | Response of DNA repair and replication systems to exocyclic nucleic acid base damage |
| title_fullStr | Response of DNA repair and replication systems to exocyclic nucleic acid base damage |
| title_full_unstemmed | Response of DNA repair and replication systems to exocyclic nucleic acid base damage |
| title_short | Response of DNA repair and replication systems to exocyclic nucleic acid base damage |
| title_sort | response of dna repair and replication systems to exocyclic nucleic acid base damage |
| topic | Biological Engineering. |
| url | http://hdl.handle.net/1721.1/71468 |
| work_keys_str_mv | AT srivastavanidhi responseofdnarepairandreplicationsystemstoexocyclicnucleicacidbasedamage AT srivastavanidhi responseofdeoxyribonucleicacidrepairandreplicationsystemstoexocyclicnucleicacidbasedamage |