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- ...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.展开更多
基金This work was supported by the National Basic Research Program of China(Grant No.2004CB 19604)the Distin-guished Young Scientists of China(Grant No.30425038)the Na-tional Natural Science Foundation of China(Grant No.30330020).
文摘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.
