The Role of Replication Protein A in the Radiation-Resistance of Halobacterium salinarum ssp. NRC-1

Ionizing radiation is used for a number of applications involving biological systems, including sterilization of items such as medical equipment and mail. The effectiveness of these applications depends on the formation of DNA double-strand breaks in the genomes of the irradiated microbes. To understand the processes by which organisms withstand irradiation, we have used the model haloarchaeon Halobacterium salinarum ssp. NRC-1. H. salinarum is among the most resistant organisms documented, and as an archaeon, discoveries made here have applications in bacteria and eukaryotes. In addition to its extreme radiation-resistance, H. salinarum is a well-developed model, with a sequenced genome and post-genomic tools such as whole-genome DNA microarrays and a targeted gene deletion system.;Previous work described the characterization of mutants of H. salinarum with increased resistance to ionizing radiation. These mutants were created through repeated exposure to high doses of electron-beam radiation. Microarray analysis of their transcriptional profiles revealed a single common change: up-regulation of the rfa3 operon, encoding proteins homologous to the large and medium subunits of eukaryotic Replication Protein A (RPA). RPA is a single-stranded DNA binding protein with central roles in DNA replication and repair, and we have examined its role in the radiation-resistance of H. salinarum. Exposure of the aforementioned mutants to ultraviolet radiation unexpectedly revealed that increased RPA expression does not lead to increased UV-tolerance. Microarray analysis of H. salinarum and the mutants after irradiation identified a potential pathway for DNA repair reminiscent of Extended Synthesis-Dependent Strand Annealing in Deinococcus radiodurans. In addition, we have deleted the genes for several RPAs in H. salinarum and determined their essentiality in radiation-resistance as well as for normal growth. Using a newly-developed system for alteration of chromosomal loci, we have extended findings in the microarray analyses to the protein level and determined the levels of the Rfa3 protein in H. salinarum. Finally, we have further enhanced the radiation-resistant mutants and determined the levels of radiation-reistance in other halophiles. This study has shed more light on the role of RPA in radiation-resistance, and lays the framework for further studies to determine the exact role of RPA in H. salinarum. .