%0 Journal Article
%T Sulfolobus Mutants, Generated via PCR Products, Which Lack Putative Enzymes of UV Photoproduct Repair
%A Cynthia J. Sakofsky
%A Laura A. Runck
%A Dennis W. Grogan
%J Archaea
%D 2011
%I Hindawi Publishing Corporation
%R 10.1155/2011/864015
%X In order to determine the biological relevance of two S. acidocaldarius proteins to the repair of UV photoproducts, the corresponding genes (Saci_1227 and Saci_1096) were disrupted, and the phenotypes of the resulting mutants were examined by various genetic assays. The disruption used integration by homologous recombination of a functional but heterologous pyrE gene, promoted by short sequences attached to both ends via PCR. The phenotypic analyses of the disruptants confirmed that ORF Saci_1227 encodes a DNA photolyase which functions in vivo, but they could not implicate ORF Saci_1096 in repair of UV- or other externally induced DNA damage despite its similarity to genes encoding UV damage endonucleases. The success of the gene-disruption strategy, which used 5กไ extensions of PCR primers to target cassette integration, suggests potential advantages for routine construction of Sulfolobus strains. 1. Introduction Gene inventories of hyperthermophilic archaea (HA) suggest that this deeply branching clade of prokaryotes may employ unusual molecular strategies during DNA replication and repair. For example, HA have certain DNA enzymes and enzymatic properties not found in mesophilic archaea or bacteria. These include reverse DNA gyrase, a type I topoisomerase that introduces positive superhelical turns into DNA [1], and family-B DNA polymerases which stall specifically ahead of dU residues in the template strand [2, 3]. Conversely, HA lack certain DNA-repair proteins that are widely conserved in other organisms; they encode no homologues of DNA mismatch repair proteins MutS and MutL [4], and they also lack the UvrABC homologues that mediate nucleotide excision repair (NER) in bacteria and mesophilic archaea [5]. Whereas HA do encode homologues of the eukaryotic helicases and structure-specific nucleases that complete the process of NER in eukaryotes, they do not have the corresponding proteins that initiate NER. The fact that no HA encode homologues of known NER-specific damage-recognition proteins seems significant, since these proteins are required for NER function in other organisms, and are the only proteins specific to NER in eukaryotes. Experimental data, nevertheless, suggest that HA can excise UV photoproducts from DNA although the mechanism and proteins responsible have not been identified. Biochemical assays indicate time-dependent loss of cyclobutane pyrimidine dimers from genomic DNA of intact Sulfolobus solfataricus cells at physiological temperature [6, 7]. In Sulfolobus acidocaldarius conjugation assays, UV enhances recombinant formation,
%U http://www.hindawi.com/journals/archaea/2011/864015/