Construction of DNA damage response

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

PprI, a DNA damage response factor from the extraordinary radioresistant bacterium Deinococcus radiodurans, plays a central regulatory role in multiple DNA damage repair. In this study, a fusion DNA fragment carrying 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 complete 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 protein, but also opens the door to the further studies of the biological functions of PprI protein in vivo.

作 者： GAO Guanjun LU Huiming Huang Lifen HUA Yuejin   作者单位： Institute of Nuclear-Agricultural Science, Zhejiang University, Hangzhou 310029, China  刊 名： 科学通报（英文版）  SCI 英文刊名： CHINESE SCIENCE BULLETIN  年，卷(期)： 2005 50(4)  分类号： Q5  关键词： Deinococcus radiodurans R1   pprI   mutants   complementary  

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