Functions Analysis DNA Damage Repair Key Genes DdrB And RecO In Deinococcus Radiodurans

Deinococcus radiodurans is best known for its astonishing ability to resistant to lethal effects of ionizing radiation,as well as many other DNA-damaging agents,including UV,hydrogen peroxide and desiccation.The radiation resistance phenotype of Deinococcus radiodurans is largely contributed by its extraordinary capacity for double-stranded DNA breaks repair.Exposure 6 500Gy ionizing radiation to cells leads to shatter chromosome into hundreds of shattered DNA fragments.It could reconstruction a functional chromosome within hours.It is believed that Cells recovering from the effect of high doses of ionizing radiations show biphasic kinetics of DSB repairs:the phase I,which is RecA-independent,precedes subsequent to RecA-dependent mechanisms of DSB repair.It was proposed that the earliest phase of DNA repair is dominated by a recA-independent single-strand annealing(SSA)or extended synthesis dependent strand annealing(ESDSA).Transcriptome analyses have identified some highly induced genes during post-radiation by several groups.Genetic studies revealed that at least exist ddrA and ddrB two RecA-independent DNA repair pathway in this organism.DdrA binds specifically the 3'-ends of single strand DNA in vitro and could protect it from nuclease degradation.However,the function of DdrB is unknown.Genome sequencing and comparative genomics have revealed that some of repair genes,such as,recB,recC,reeE,recT,are absent from D.radiodurans genome.In contrast,the genes of ReeF pathway,such as recA,reeF,recJ,recN,recO,recQ,recR,ssb,ruvA,ruvB,and ruvC,are present in D.radiodurans genome.However,little in vivo evidence has been presented to date for the roles of the unique genes(recF,recO and recR)of RecF pathway in D.radiodurans' radioresistance.The roles of RecF pathway in D.radiodurans' radioresistance still need to be addressed experimentally.In this study,the roles of ddrB and the key gene recO of RecFOR pathway in D.radiodurans was analysis and the relationship between ddrB and recO in DNA repair of D.radiodurans were further studied.The main work and its results are appended following:1.A fusion DNA fragment carrying kanamycin resistance gene with the D.radiodurans groEL promoter was cloned by PCR amplification and inserted into the ddrB(Dr0070)locus in the genome of the wild-type strain R1.The resulting ddrB null mutant strain was constructed. Results show that and ddrB null mutant strain was very sensitive to ionizing radiation,but not to H₂O₂.Therefore,it was presumed that ddrB was the necessary gene in maintaining the extreme resistance to ionizing radiation.2.A recA/ddrB double mutant strain was constructed.The kinetics of reassembly of broken chromosomes after radiation of recA and recA/ddrB were compared by PFGE.The resulting showed,compared with recA,the first phase repair process of recA/ddrB double mutant was compromised.This result indicates ddrB involve in the first phase repair process3.The DdrB protein was expressed by E.coli expression system and then purified.And the biochemical properties were further characterized.DdrB interacted with DNA in nonspecific manner and preferred interaction with ssDNA.The protein could protect dsDNA from degenerating by nuclease.DdrB promoted single strand annealing reaction and SSB stimulated the reaction.SSB and DdrB could physical interaction in vitro.Furthermore,two proteins could corporate binding with ssDNA.It was presumed that DdrB-mediated repair largely via SSA.4.In order to study the function of recO in D.radiodurans's radiotelorence,a null mutant of recO was constructed.The mutant demonstrated growth defect and was very sensitive to irradiation with gamma rays and UV light.This result suggested DNA repair in this organism was mainly via recF pathway.5.A recO/ddrB double mutant strain was constructed.The kinetics of reassembly of broken chromosomes after radiation of recO and recO/ddrB were compared by PFGE.The repair kinetics of recO was comparable with recA and recO/ddrB was comparable with recA/ddrB.These indicate that recO involve in RecA-dependent phase and ddrB involve in RecA-independent phase.