Identification And Analysis Of Mismatch Repair Genes In The Extremely Radioresistant Bacterium Deinococcus Radiodurans

Deinococcus radiodurans R1 is one of the most radiation-resistant organisms discovered, which can reconstitute completely its genome from hundreds of DNA DSBs induced by y-radiation without mutagenesis or loss of viability. This bacterium has received considerable attention from the scientific community. Despite the release of its genome sequence and studies on any process associated with DNA repair, the molecular mechanisms underlying its resistance remain poorly understood. It was well known that DNA mismatch pathway plays an important role on preventing mutagensis, ensuring fidelity of replication and recombination in organisms. However, few reports were described on D. radiodurans mismatch repair. Here, identification and functional analysis of two putative mutS homologs (mutS1和 mutS2) in genome sequence were proformed by using sacB/Suc~S and rpoB/Rif~R mutational analysis.In this study, we have developed a mutation analysis system (sacB/Suc~S mutation system) by using Bacilllus subtilis sacB gene as a positive selection marker that confers a sucrose-sensitive phenotype for gram-negative or gram-positive bacteria. We have applied firstly this system for analyzing mutagenesis and the roles of the genes involved with mismatch repair in the extremely radioresistant bacterium, D. radiodurans. The frequencies of spontaneous mutation and radio-mutation and the mutation spectrum analysis of D. radiodurans wild-type, mutS1 and DR1975 gene deficient mutants constructed here were determined. It was showed that 4 kinds of insertion sequence (IS) elements (IS8031, IS2621, ISdrTCL2 and ISdrTCL1) with the transposition activity were trapped by sacB gene. The IS elements appeared more active while the mutS1, mutS2 and DR1975 genes inactivated, which mainly caused the spontaneous mutation and gamma radiation-induced mutation in D. radioduran. The results indicated that the inactivation of these genes resulted in 2~4-fold increase in the frequencies of spontaneous mutagenesis, and ten-fold increase in mutS2 gene default mutant for radiation-induced mutagenesis after the strain exposure to 8 kGy of y-radiation, comparing with D. radioduans wild-type strain. Our results strongly suggested that mutS1(DR1039), mutS2(DR1976) 及 DR1975 genes could efficiently restrain transposition activities of IS elements and played the important roles in preventing gene mutation and keeping the fidelity of genomic DNA in D. radiodurans.To further understand the functions of these genes, the rpoB/Rif~R mutation analysis system for analyzing base substitutions in Escherichia coli was used to D. radiodurans. Inactivation of mutSl and DR1975 gene led to 2 and 8 -fold increase in the frequencies of spontaneous Rif~R mutagenesis, while mutS2 gene disruption did not impact on the efficiency of Rif~R rpoB mutagenesis. Sequencing >100 mutations from wild-type, mutS1 and DR1975 gene deficient mutants of D. radiodurans, analysis of the mutation spectrum of Rif~R rpoB gene showed that inactivation of mutS1caused the mutational hotspot was changed from the position 1259(T→ G) to 1435(G→A), 1319(C → A)and 1441(A → C);for DR1975 gene deficient was increased another hotspots at the position 1441(A → C). The experiment of transformation of Rif~R rpoB gene with base substitute and 9 bp deletion mutation Rif~R rpoB fragments into D. radiodurans showed that the efficiency of transformation for all the gene deficient mutants were not different from wild-type, except for inactivationg of mutSl gene resulted in 3.5-fold increase in theefficiencies of transformation with base substitute t Rif* rpoB fragments. It was demonstrated the mutS2 gene was not involved in mismatch repair pathway, and MutSl encoded by DR1039 (mutSJ) was the crucial enzyme in mismatch repair pathway.