Construction of DNA damage response gene pprI function-deficient and function-complementary mutants in Deinococcus radiodurans

PprI, a DNA damage response factor from the extraordinary radioresistant bacterium Deinococcus radi- odurans, plays a central regulatory role in multiple DNA damage repair. In this study, a fusion DNA fragment carry- ing kanamycin resistance gene with the D. radiodurans groEL promoter was cloned by PCR amplification and reversely inserted into the pprI locus in the genome of the wild-type strain R1. The resulting pprI-deficient strain, designated YR1, was very sensitive to ionizing radiation. Meanwhile, the re- combinant DNA fragment was cloned into the shuttle vector pRADZ3, and resulted in plasmid pRADK with kanamycin resistance in D. radiodurans. The fragments containing com- plete pprI gene and 3′-terminal deletion pprI△ were cloned into plasmid pRADK. The resulted plasmids designated pRADKpprI and pRADKpprI△ were then transformed to YR1. Results show that YR1 carrying pRADKpprI was able to fully restore the extreme radioresistance to the same level as the wild-type D. raiodurans R1, whereas YR1 pRADKpprI△ failed to do so. Construction of DNA repair switch PprI function-deficient and function-complementary mutants in D. radiodurans is not only useful to elucidating the relationship between domains and functions of PprI pro- tein, but also opens the door to the further studies of the bio- logical functions of PprI protein in vivo.

PprI, a DMA damage response factor from the extraordinary radloresistantbacterium Deinococcus radiodwans, plays a central regulatory role In multiple DNA damage repair. Inthis study, a fusion UNA fragment carrying Icanamycin resistance gene with the D. radiodurans groELpromoter was cloned by PCM amplification and reversely inserted into the pprl locus in the genome ofthe wild-type strain R1.The resultingpprl-deficient strain, designated YR1, was very sensitive toionizing radiation. Meanwhile, the recombinant DNA fragment was cloned into the shuttle vectorpRABZ3, and resulted in plasmid pRADK with kanamycin resistance in D. radiodurans. The fragmentscontaining complete pprl gene and 3'-terminal deletion pprl A were cloned into plasmid pRADK. Theresulted plasmids designated pRADKpprl and pRADKpprI were then transformed to YRI. Results show thatYR1 carrying pKADKpprl was able to fully restore the extreme radioresistance to the same level as(lie wild-type D. raiodurans Ml, whereas YRI pRADE CpprI failed to do so. Construction of DNA repairswitch Pprl function-deficient and function-complementary mutants in D, radiodurans is not onlyuseful to elucidating the relationship between domains and functions of Pprl protein, but also opensthe door to the further studies of the biological functions of Pprl protein in vivo.