极端耐辐射奇异球菌Deinococcus sp.BR501切除修复系统突变株的构建及功能的鉴定

极端辐射异常球菌Deinococcussp.BR501的核苷酸切除修复系统,可能包含两条途径:依赖于UV损伤内切酶β(UvsE)的修复途径和依赖于UV损伤内切酶α(UvrABC)的修复途径,其关键酶分别为UvsE(dr1819编码)和UvrABC(A亚基dr1771编码)。根据Deinococcus radiodurans的序列,采用同源克隆的方法,设计PCR引物、克隆基因和体外阻断目的基因,再通过PCR产物线性转化的方法,筛选到同源双交换的重组阻断突变株△dr1771、△dr1819和△dr1771与△dr1819的双突变株。对此突变株和野生型均进行UV辐射抗性和化学损伤试剂MMC与H2O2的损伤修复能力的分析。结果表明了BR501中两条核苷酸切除修复途径的存在,并且只有两条途径同时缺失时,才能使菌体对UV和DNA交联剂MMC敏感。

DISRUPTION AND CHARACTERIZATION OF THE EXCISION REPAIR PATHWAY IN THE EXTREMELY RADIORESISTANT BACTERIUM Deinococcus SP.BR501

Deinococcus sp.BR501,an extremely radioresistant bacterium may contain two nucleotide excision repair pathways: the UV damage endonuclease β(UvsE)-dependent excision repair pathway and the UvrABC-dependent pathway.And the UvsE(coded by dr1819) and UvrABC(Unit A coded by dr1771) are their key enzymes respectively.PCR primers were designed and homologous genes were cloned and disrupted in vitro according to the completely nucleotide sequence of Deinococcus radiodurans R1 genome.Then PCR production was transformed to BR501,and the disrupted mutants(△dr1771,△dr1819 and △dr1771dr1819) were checked and confirmed by homologous recombination.These mutants and the wild type were irradiated by UV light and exposed to the DNA-damaging agents MMC and H_2O_2.The results showed that these pathways were existed in BR501 and only the two pathway losses could result in increased sensitivity to UV and MMC.