Understanding DNA Repair and Damage-Tolerance Mechanisms in the Hyperthermophilic Crenarchaeote Sulfolobus acidocaldarius
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University of Cincinnati / OhioLINK
2019
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ndltd-OhioLink-oai-etd.ohiolink.edu-ucin15759674800865572021-12-17T05:24:09Z Understanding DNA Repair and Damage-Tolerance Mechanisms in the Hyperthermophilic Crenarchaeote Sulfolobus acidocaldarius Jain, Rupal Microbiology Hyperthermophilic Archaea DNA repair Damage tolerance Oxidative damage Accessory polymerases AP endonuclease Hyperthermophilic Archaea have adapted to unusual and extreme habitats and the mechanisms they employ to maintain their genomic integrity have been elusive for quite some time now. The hyperthermophilic archaeon Sulfolobus acidocaldarius, despite its extreme optimal growth conditions of about 80° C and pH 3, is capable of preserving its genomic integrity. Hence, it will be used as the model organism to understand the DNA damage coping mechanisms representing hyperthermophilic Archaea. The three specific goals of this thesis are as follows: 1) Investigate genes implicated to deal with oxidative stress, 2) Identify AP endonuclease genes and investigate their biological roles and, 3) Examine the association of accessory polymerases with DNA repair and damage-tolerance pathways. The approach involved the construction of mutant strain(s) of the genes implicated to be involved in DNA repair or damage-tolerance and, investigation of its effects using a combination of in vitro and in vivo assays. Reactive oxygen species predominantly lead to the oxidation of deoxyguanosine in DNA to 8-oxo-7,8-dihydro-2'-deoxyguanine (8-oxoG). If left unrepaired this often leaves a genetic mark in the form of G:C to T:A transversion mutation. The deletion of a putative OxoG glycosylase and Dbh TLS polymerase individually led to a significant increase in the mutagenic signature left behind by 8-oxoG. However, the deletion of both the genes led to a five-fold increase in the mutation rate and a 47-fold increase in the G:C to T:A mutagenic signature. To analyze the bypass of 8-oxoG specifically, all four strains were transformed with a synthetic oligo carrying an 8-oxoG lesion within it, and in each resulting transformant, the base inserted opposite the lesion was identified. The results showed that the double mutant exclusively inserts a dAMP across the 8-oxoG lesion whereas the wild type strain inserts dCMP. Apurinic/apyrimidinic sites are one of the most common DNA lesions which can either arise spontaneously or as regular intermediates of the base excision repair pathway. The deletion of S. acidocaldarius’ putative AP endonuclease gene resulted in a ~98% reduction in the AP endonuclease specific enzymatic activity when compared to the wild type strain, indicating it be the major AP endonuclease. However, the deletion of the major endonuclease gene had little if any effect on the efficiency to transform with an abasic oligo. Accessory polymerases have been found to be important in the DNA repair and damage-tolerance pathways in all domains of life. However, S. acidocaldarius can tolerate the deletion of all its accessory polymerase without any obvious phenotypic deficiencies. The deletion of all accessory polymerases had no effect on the mutation rate however, its mutation spectrum revealed a decrease in the occurrence of G:C to A:T transition mutation when compared to the wild type and dbh single mutant strain. The 8-oxoG bypass specificity of the triple polymerase mutant strain was similar to the dbh single mutant strain. To my knowledge, this is the first study to construct the mutant strains of the DNA repair and damage-tolerance genes under investigation and, carry out in-depth experiments to understand their functions. 2019 English text University of Cincinnati / OhioLINK http://rave.ohiolink.edu/etdc/view?acc_num=ucin1575967480086557 http://rave.ohiolink.edu/etdc/view?acc_num=ucin1575967480086557 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 |
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English |
| sources |
NDLTD |
| topic |
Microbiology Hyperthermophilic Archaea DNA repair Damage tolerance Oxidative damage Accessory polymerases AP endonuclease |
| spellingShingle |
Microbiology Hyperthermophilic Archaea DNA repair Damage tolerance Oxidative damage Accessory polymerases AP endonuclease Jain, Rupal Understanding DNA Repair and Damage-Tolerance Mechanisms in the Hyperthermophilic Crenarchaeote Sulfolobus acidocaldarius |
| author |
Jain, Rupal |
| author_facet |
Jain, Rupal |
| author_sort |
Jain, Rupal |
| title |
Understanding DNA Repair and Damage-Tolerance Mechanisms in the Hyperthermophilic Crenarchaeote Sulfolobus acidocaldarius |
| title_short |
Understanding DNA Repair and Damage-Tolerance Mechanisms in the Hyperthermophilic Crenarchaeote Sulfolobus acidocaldarius |
| title_full |
Understanding DNA Repair and Damage-Tolerance Mechanisms in the Hyperthermophilic Crenarchaeote Sulfolobus acidocaldarius |
| title_fullStr |
Understanding DNA Repair and Damage-Tolerance Mechanisms in the Hyperthermophilic Crenarchaeote Sulfolobus acidocaldarius |
| title_full_unstemmed |
Understanding DNA Repair and Damage-Tolerance Mechanisms in the Hyperthermophilic Crenarchaeote Sulfolobus acidocaldarius |
| title_sort |
understanding dna repair and damage-tolerance mechanisms in the hyperthermophilic crenarchaeote sulfolobus acidocaldarius |
| publisher |
University of Cincinnati / OhioLINK |
| publishDate |
2019 |
| url |
http://rave.ohiolink.edu/etdc/view?acc_num=ucin1575967480086557 |
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AT jainrupal understandingdnarepairanddamagetolerancemechanismsinthehyperthermophiliccrenarchaeotesulfolobusacidocaldarius |
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