The Deinococcus radiodurans uvrA gene: identification of mutation sites in two mitomycin-sensitive strains and the first discovery of insertion sequence element from deinobacteria

Abstract

Deinococcus radiodurans ( Dr) possesses a prominent ability to repair the DNA injury induced by various DNA-damaging agents including mitomycin C (MC), ultraviolet light (UV) and ionizing radiation. DNA damage resistance was restored in MC sensitive (MC S) mutants 2621 and 3021 by transforming with DNAs of four cosmid clones derived from the gene library of strain KD8301, which showed wild type (wt) phenotype to DNA-damaging agents. Gene affected by mutation ( mtcA or mtcB) in both mutants was cloned and its nucleotide (nt) sequence was determined. The deduced amino acid (aa) sequence of the gene product consists of 1016 aa and shares homology with many bacterial UvrA proteins. The mutation sites of both mutants were identified by analyzing the polymerase chain reaction (PCR) fragments derived from the genomic DNA of the mutants. A 144-base pair (bp) deletion including the start codon for the uvrA gene was observed in DNA of the mutant 3021, causing a defect in the gene. On the other hand, an insertion sequence (IS) element intervened in the uvrA gene of the mutant 2621, suggesting the insertional inactivation of the gene. The IS element comprises 1322-bp long, flanked by 19-bp inverted terminal repeats (ITR), and generated a 6-bp target duplication (TD). Two open reading frames (ORFs) were found in the IS element. The deduced aa sequences of large and small ORFs show homology to a putative transposase found in IS 4 of Escherichia coli ( Ec) and to a resolvase found in IS Xc5 of Xanthomonas campestris ( Xc), respectively. This is the first discovery of IS element in deinobacteria, and the IS element was designated IS 2621.