Dna Mismatch Repair and Response to Oxidative Stress in the Extremely Halophilic Archaeon Halobacterium Sp. Strain Nrc-1

Title of Document: DNA MISMATCH REPAIR AND RESPONSE TO OXIDATIVE STRESS IN THE EXTREMELY HALOPHILIC ARCHAEON HALOBACTERIUM SP. STRAIN NRC-1 Courtney Rae Busch, Doctor of Philosophy, 2008 Directed By: Assistant Professor Dr. Jocelyne DiRuggiero, Department of Cell Biology and Molecular Genetics Halobacterium is an extremely halophilic archaeon that has homologs of the key proteins, MutS and MutL used in DNA mismatch repair in both Bacteria and Eukarya. To determine whether Halobacterium has a functional mismatch repair system, we calculated the spontaneous mutation rate and determined the spectrum of mutation in Halobacterium using fluctuation tests targeting genes of the UMP biosynthesis pathway and we performed a sequence analysis of the mutated genes. We found that Halobacterium has a low incidence of mutation indicating that some form of DNA repair is taking place, however the mutational spectrum in the Archaea is different from that seen in Bacteria and Eukarya suggesting differences between the archaeal, bacterial, and eukaryal repair systems. To test if the MutS and MutL homologs in Halobacterium are essential for the low incidence of mutation, we used in-frame targeted gene deletion and characterized the mutant phenotypes. We found no phenotypic differences between the mutant strains and the background strain indicating that the MutS and MutL protein homologs found in Halobacterium are not essential for maintaining the low incidence of mutation. Since much of the replication and repair processes in Halobacterium are similar to that of Eukarya, deciphering how MMR occurs in the Archaea could lead to a new understanding of pathway interactions based on the recruitment of repair enzymes from both bacterial and eukaryal counterparts. In addition, we elucidated the oxidative stress response in Halobacterium to hydrogen peroxide and paraquat using a whole genome transcriptional array, in-frame targeted gene deletion, and survival analysis of mutant phenotypes. We showed an overall effort of the cells to scavenge reactive oxygen species and repair damages to the DNA, which has also been seen in response to gamma irradiation. From the mutant analyses, we were able to deduce that Sod1 and PerA proteins played an essential role in removing oxidative stress in Halobacterium. Deciphering the stress response to hydrogen peroxide and paraquat in an extreme halophile that lives in an environment subject to long periods of desiccation can further our understanding of the DNA repair and protection systems to oxidative stress in general. DNA MISMATCH REPAIR AND RESPONSE TO OXIDATIVE STRESS IN THE EXTREMELY HALOPHILIC ARCHAEON HALOBACTERIUM SP. STRAIN NRC-1