NITROSATIVE STRESS AND DNA REPAIR IN MICROORGANISMS

DNA can be damaged by reactive nitrogen intermediates (RNI) and reactive oxygen intermediates (ROI) from both endogenous and exogenous sources. Under nitrosative stress conditions, DNA may be modified through base deamination or crosslinking. Uracil (U), xanthine (X) and oxanine (O), hypoxanthine (I), and thymine (T) are the corresponding deamination products derived from cytosine (C), guanine (G), adenine (A), and 5-methylcytosine, respectively. To remove DNA damage, cells are equipped with a variety of DNA repair enzymes which participate in different repair pathways. My dissertation work involves studies of genes and enzymes in nucleotide excision repair (NER) and base excision repair (BER) pathways. Chapter 1 is an overview of DNA damage and repair pathways. Chapter 2 presents a genetic investigation on screening of genes involved in resistance to nitrosative stress in intracellular bacterial pathogen Coxiella burnetii and characterization of DNA repair genes uvrB, uvrA and uvrC. This work shows the relationship between NER and RNI (Reactive nitrogen intermediates) resistance in C. burnetii. Chapter 3 shows a biochemical study on deaminated repair activities in DNA repair enzymes in E. coli. Finally, chapter 4 discusses the deaminated base repair properties of a TDG/MUG family enzyme in the UDG superfamily from fungal pathogen Penicillium marneffei (Pma). This study shows Pma MUG can excise all four deaminated bases from DNA with strongest activity on hypoxanthine. Mutational analysis coupled with molecular modeling was used to pinpoint amino acids in the active site that are important for its catalytic function. Overall, these studies provide new insights on the role of NER and BER genes in resistance to nitrosative stress and repair of deaminated base damage in bacteria and fungi