Mechanisms Of Genome Stability In The Hyperthermophilic Archaeon Sulfolobus acidocaldarius
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University of Cincinnati / OhioLINK
2011
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ndltd-OhioLink-oai-etd.ohiolink.edu-ucin13213701822021-08-03T06:15:05Z Mechanisms Of Genome Stability In The Hyperthermophilic Archaeon Sulfolobus acidocaldarius Sakofsky, Cynthia J. Biology archaea Sulfolobus DNA repair genome stability Hyperthermophilic archaea (HA) have evolved cellular mechanisms for adapting to extreme environmental conditions. Little, however, is understood about how these organisms repair or tolerate DNA damage in order to maintain genome stability. Thus, the aim of this dissertation was to elucidate strategies employed by these organisms to maintain genomic integrity by utilizing HA Sulfolobus acidocaldarius as a model species. Five studies were conducted which include (i) investigating cellular responses to chemically-induced damage by cisplatin, (ii) determining the function of genes implicated in UV photoproduct repair, (iii) verifying the functions of an S. acidocaldarius translation synthesis polymerase (TLS) Dbh, (iv) examining the role of TLS bypass in tolerating abasic lesions, and (v) investigating the effect of promoter strength on mutagenesis in reporter gene lacS. The chemical cisplatin was utilized to evaluate cellular sensitivity and genetic effects to bulky-adducts. This project involved experiments that were technically challenging and therefore the results were very preliminary. However, the data suggested that S. acidocaldarius has the ability to repair or tolerate large numbers of cisplatin damage. Additionally, there was no evidence that cisplatin-damage was mutagenic, and the process of homologous recombination seemed to be unaffected by cisplatin-induced damage. Studies regarding UV photoproduct repair analyzed disruptant mutants, Phr and Uvde, and showed that the photolyase protein homologue Phr is required for the repair of UV damage in the presence of light, while the UV-endonuclease UVDE homologue did not seem to play a critical role in dark-repair of UV damage. TLS Dbh polymerase disruption mutants were used to determine the role of this polymerase in DNA damage sensitivity and in replication accuracy of spontaneous mutations in a selectable gene pyrE. The absence of Dbh did not seem to have an affect on the sensitivity of the cells to chemicals, however the mutation spectrum sampled from Dbh- mutants versus Dbh+ strains did show significant differences in the types and positions of mutations occurring in these strains. The role of TLS bypass in tolerating abasic lesions was determined by introducing a site-specific abasic lesion into the genome and selecting recombinants that successfully tolerated the damage. Genotyping techniques were then used to identify which base was present at the site of the damage. The results suggest that Dbh does not play a prominent role in bypassing abasic lesions. Finally, to examine how the rate of transcription may affect mutagenesis, mutation rates and the specific activity for reporter gene lacS, found in different genetic contexts and under the control of different promoters, was measured. The results suggested that stronger promoters regulating lacS in S. acidocaldarius yielded higher mutation rates. The results are consistent with the phenomenon of transcription-associated mutagenesis (TAM), where highly transcribed genes have been observed to yield higher mutation rates. In conclusion, the data obtained from these experiments have brought insight into various DNA damage repair and tolerance mechanisms, while setting the foundation for future experiments. 2011 English text University of Cincinnati / OhioLINK http://rave.ohiolink.edu/etdc/view?acc_num=ucin1321370182 http://rave.ohiolink.edu/etdc/view?acc_num=ucin1321370182 unrestricted This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws. |
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NDLTD |
| language |
English |
| sources |
NDLTD |
| topic |
Biology archaea Sulfolobus DNA repair genome stability |
| spellingShingle |
Biology archaea Sulfolobus DNA repair genome stability Sakofsky, Cynthia J. Mechanisms Of Genome Stability In The Hyperthermophilic Archaeon Sulfolobus acidocaldarius |
| author |
Sakofsky, Cynthia J. |
| author_facet |
Sakofsky, Cynthia J. |
| author_sort |
Sakofsky, Cynthia J. |
| title |
Mechanisms Of Genome Stability In The Hyperthermophilic Archaeon Sulfolobus acidocaldarius |
| title_short |
Mechanisms Of Genome Stability In The Hyperthermophilic Archaeon Sulfolobus acidocaldarius |
| title_full |
Mechanisms Of Genome Stability In The Hyperthermophilic Archaeon Sulfolobus acidocaldarius |
| title_fullStr |
Mechanisms Of Genome Stability In The Hyperthermophilic Archaeon Sulfolobus acidocaldarius |
| title_full_unstemmed |
Mechanisms Of Genome Stability In The Hyperthermophilic Archaeon Sulfolobus acidocaldarius |
| title_sort |
mechanisms of genome stability in the hyperthermophilic archaeon sulfolobus acidocaldarius |
| publisher |
University of Cincinnati / OhioLINK |
| publishDate |
2011 |
| url |
http://rave.ohiolink.edu/etdc/view?acc_num=ucin1321370182 |
| work_keys_str_mv |
AT sakofskycynthiaj mechanismsofgenomestabilityinthehyperthermophilicarchaeonsulfolobusacidocaldarius |
| _version_ |
1719433490706989056 |