Functional Assessment of Mismatch Repair and Nucleotide Excision Repair in a Sulfolobus species

University Of Cincinnati Main Campus, Cincinnati OH

Investigators

Abstract

The goal of this project is to evaluate two important DNA repair capabilities in one of the hyperthermophilic archaea (prokaryotes that live in geothermal environments and are unrelated to bacteria). Specifically, the project will compare Sulfolobus acidocaldarius to Escherichia coli with respect to the efficiency of removing certain DNA lesions, and thereby provide experimental evidence either for or against the operation of some functional equivalent of DNA mismatch repair (MMR) and nucleotide excision repair (NER) in Sulfolobus spp. This information is significant, because complete genome sequences of hyperthermophilic archaea show no sign of encoding the proteins that initiate mismatch repair (MMR) or nucleotide excision repair (NER) in nearly all other organisms. The absence of such genes seems illogical, as the harsh growth conditions of these archaea should make highly accurate and efficient DNA repair essential for cellular viability and evolutionary success. The project will also construct and test new genetic elements for gene disruption in hyperthermophilic archaea. One element (a transposon) will be designed to insert randomly into archaeal genomes, whereas the other (a cassette) will recombine into any site of interest, defined by short DNA sequences attached to its ends. The former will allow genes important for DNA repair and other non-essential processes to be identified without knowing their location or nucleotide sequence, whereas the latter will allow the role of candidate genes identified by sequence analysis to be confirmed experimentally. Broader impacts of this work include i) the training of graduate and undergraduate students in techniques of microbiology and molecular genetics at an urban, comprehensive research university, ii) incorporation of the knowledge gained from this research into teaching and scientific discourse, iii) new microbial strains and genetic constructs made available to other researchers, and iv) insight into novel molecular aspects of DNA repair that may lead to new diagnostic technologies with corresponding benefits for society.