| DNA repair glycosylases locate and excise damaged bases from DNA during the first step of BER, playing central roles in the preservation of the genome and the prevention of disease. The Escherichia coli ( E. coli) glycosylases Fpg, MutY, and the human glycosylase hOGG1 are involved in the repair of the mutagenic oxidatively damaged guanine lesion, 8-oxo-7,8-dihydroguanine (8-OxoG or OG). To investigate the required features utilized for damage recognition and catalysis, a series of substituted indole nucleotides that mimic features of OG were tested as direct substrates for these glycosylases. Results demonstrate that the OG glycosylases hOGG1 and in particular, Fpg, can recognize and cleave non-polar OG mimics, despite their lack of hydrogen bonding groups and basic atoms. In contrast to Fpg and hOGG1, single turnover kinetic experiments conducted with MutY and the nonpolar OG analogs based paired to A resulted in no detectable A excision. The relative rates of base excision chemistry of the nonpolar analogs displayed by each glycosylase highlights key differences in the mechanisms of recognition and catalysis employed by Fpg, hOGG1, and MutY.;There are various diseases associated with defects in DNA repair. One such example is an inherited form of colon cancer, referred to as MUTYH-associated polyposis (MAP). MAP is caused by defects in the human homologue of MutY (MUTYH), which works to prevent G to T transversion mutations associated with the OG lesion. MutY is a unique glycosylase, which detects and removes the normal adenine when mispaired across from OG. To evaluate which interactions between MutY and the OG:A substrate are vital, a series of OG and adenine nucleotide analogs which vary from the natural substrate in the base pair stability, hydrogen bonding capability, and glycosidic bond stability were chosen. To determine which part of the MutY reaction pathway these substrate modifications affect, the binding affinity, the rate of catalysis, and the overall cellular repair were all measured. Results reported in this dissertation support previous observations demonstrating that any structural deviation from OG dramatically reduces binding, efficient catalysis in vitro, and successful repair in bacterial cells, demonstrating the critical importance of precise recognition of OG for efficient MutY activity. Interestingly, only modifications of adenine at the C-2 and N-1 positions where shown to negatively affect OG:A cellular repair. This suggests that efficient binding and modest catalytic activity are not the only requirements for MutY activity and underscores the importance of confirmation of adenine, in addition to the identification of OG in an OG:A mismatch.;Further oxidation of OG results in the production of the highly mutagenic hydantoin lesions, guanidinohydantion (Gh) and spiroiminodihydantoin (Sp). Interestingly, oxidation of OG in the presence of primary amines results in the formation of hydantoin amine adducts. The hydantoins, Gh and Sp, are known to be substrates for Fpg, Nei, and hNEIL1 BER glycosylases; however, bulky Sp-amine adducts may be more readily repaired by the nucleotide excision repair (NER) pathway. To delineate the contributions of NER and BER for hydantoin lesion repair, a series of Sp-amine adducts of varying size were prepared. In vitro experiments presented herein reveal that UvrABC excision of Gh and Sp is significantly greater than for OG, with rates of Sp removal similar to those observed for known NER substrates. The BER glycosylases Nei, Fpg, and hNEIL1 were also shown to mediate the removal Sp-amine bases from DNA. Detailed kinetic studies performed with hNEIL1 revealed that this glycosylase is relatively insensitive to the size of the Sp-amine adducts and exhibited robust base removal activity under single-turnover conditions for the entire series. Studies suggest that hydantoin lesions may be efficiently repaired in cells by both NER and BER pathways.;Taken together, the work in this dissertation provides unique insight into the chemical features utilized by each of the BER and NER DNA repair enzymes to recognize and mediate repair of oxidatively damaged DNA bases. Illustrating that fine-tuning of overall similar catalytic strategies allows for broad versus specific substrate processing and the exquisite control needed to preserve the genome. |
