Collections > Electronic Theses and Dissertations > Biophysical and Biochemical Investigations of Proteins Involved in Genomic Stability
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The processes by which cells either make or avoid mutations in their DNA are varied and multifaceted. Often proteins involved in the repair of DNA damage are also found inducing mutations in the immune response. Despite the fact that these proteins and processes are found in all eukaryotes, the scientific community is just beginning to elucidate the mechanisms of these diverse pathways and the functions of the proteins involved in them. This dissertation looks at proteins in two seemingly different cellular processes: DNA mismatch repair and the immune response, as modulated by cytidine deaminases. The DNA mismatch repair protein MutLalpha has been long thought to function as a molecular matchmaker; facilitating interactions between proteins in the mismatch repair pathway. Since little was known about this protein, biophysical and biochemical assays were designed and carried out to elucidate what role binding of ATP and DNA may have on MutLalpha. Surprisingly, MutLalpha can exist in four different conformational states, and these conformational states are governed by adenine nucleotide binding (but not hydrolysis). It had been shown previously that MutLalpha binds to DNA in long, cooperative protein tracts. MutLalpha protein tracts do not appear to be effected by increasing salt concentration, but they are affected by the presence of ATP. Additionally, the ATP hydrolysis rate of full-length MutLalpha was measured, and found to be 2-6x larger than values previously reported for isolated N-terminal domains of MutLalpha, suggesting that conformational changes seen upon ATP binding, and the interactions that are likely made as a result of those conformational changes, may enhance the rate of ATP hydrolysis by MutLalpha. Two of the cytidine deaminases (AID and APOBEC3G) have been shown to be involved in the immune response. The third cytidine deaminase (APOBEC2) is only a cytidine deaminase by sequence homology; and as yet has unknown function. Since it is widely believed that all cytidine deaminases are dimeric or tetrameric, a study of the oligomeric states of these proteins (with and without substrate present) was undertaken. Despite all three proteins belonging to the same superfamily, they each appear to exist in a different oligomeric state.