Construction of DNA damage response genepprI function-deficient and function-complementary mutants inDeinococcus radiodurans
- 51 Downloads
PprI, a DNA damage response factor from the extraordinary radioresistant bacteriumDeinococcus radi-odurans, plays a central regulatory role in multiple DNA damage repair. In this study, a fusion DNA fragment carrying kanamycin resistance gene with theD. radiodurans groEL promoter was cloned by PCR amplification and reversely inserted into thepprI locus in the genome of the wild-type strain R1. The resultingpprI-deficient strain, designated YR1, was very sensitive to ionizing radiation. Meanwhile, the recombinant DNA fragment was cloned into the shuttle vector pRADZ3, and resulted in plasmid pRADK with kanamycin resistance inD. radiodurans. The fragments containing completepprI gene and 3′-terminal deletionpprI † 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-typeD. raiodurans Rl, whereas YR1 pRADKpprI† failed to do so. Construction of DNA repair switch PprI function-deficient and function-complementary mutants inD. radiodurans is not only useful to elucidating the relationship between domains and functions of Pprl protein, but also opens the door to the further studies of the biological functions of PprI proteinin vivo.
KeywordsDeinococcus radiodurans Rl pprI mutants complementary
Unable to display preview. Download preview PDF.
- 3.Anderson, A. W., Nordan, H. C., Cain, R. F. et al., Studies on the radio-resistant micrococcus. I. The isolation, morphology, cultural characteristics and resistance to g radiation, Food Technol., 1956, 10: 575–578.Google Scholar
- 15.Hua, Y. J., Gao, G. J., Comparative genomics of genes contributing to DNA repair the in radiation-resistantDeinococcus radiodurans, Acta Microbiol. Sinica, 2003, 43: 120–126.Google Scholar
- 16.Maniatis, T., Fritsch, E. F., Sambrook, J., Molecular Cloning, A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 1982.Google Scholar