DNA修复基因pprA和lexA-like的表达及功能研究

Deinococcus radiodurans (Dr) is remarkable for its extraordinary resistance to ionizing, UV irradiation and many other agents that damage DNA. This organism can mend at least 150 double-strand breaks (DSBs) per chromosome induced by ionizing radiation without lethality or mutagenesis within several hours. This resistance is known to be due to D. radiodurans' extremely proficient DNA repair processes. However, very little is known about the repair pathways employed by this organism. Also, the existence of SOS response and characterization of Dr LexA remains unclear. The gene coding for the Deinococcus radiodurans homologue of LexA, which represses a number of genes involved in the response to DNA damage, has been cloned. In this study, the D. radiodurans LexA protein is over expressed in E. coli JM109 and induced by IPTG, the molecular weight is 25.4 KDa. The most suitable induction is obtained from the 24 hr culture in which 0.1 mM IPTG is added. In large scale culture, the relatively high lexA expression is achieved in the culture containing 25 μg/ml of Ap and 0.1 mM IPTG..Dr LexA is further purified and its functional activities is investigated. The cultured cell is lysed followed by freeze thrawing and sonication. After ultracentrifugation, column chromatography and ultrafiltration, LexA protein has been purified. During the chromatography process, it is found that, Dr LexA binds tightly to heparin-agarose, showing that it is a DNA binding protein. Furthermore, Dr LexA bound to cationic ion exchange column with properties similar to those reported for other LexA. The majority of LexA is eluted from the heparin column at approximately 250 mM NaCl, while eluted about at 400 mM NaCl from SP sephrose column. By this protocol, Dr LexA has been purified to greater than 95% purity , with a yield of approximately 2 mg of pure protein from 10 liters of induced culture. SDS-PAGE analysis of the purified protein showed only one band with molecular mass of 25.4KDa. Purified LexA is used for preparation of antibody and further study..Electrophoretic mobility shift assay is performed to determine DNA binding affinity of LexA. The PCR products for DNA repair gene pprA, orf144c, recA upsteam sequence are amplified and then labeled by DIG. The reaction mixture with Dr LexA are loaded onto the native polyacrylamide gel. We demonstrated that, unlike E. coli LexA, the purified Dr LexA can only bind to the upstream regions of its own gene, but have no apparent affinity to DNA rapair-related genes pprA, orf144c, recA upsteam regions, suggesting that Dr LexA is not involved in the negative control of RecA. However, like E. coli LexA, D. radiodurans LexA undergoes an autocatalytic reactions at alkaline pH. The cleavage reaction can also be mediated in vitro under more physiological conditions by the RecA protein. Activation of RecA requires single-stranded DNA and nucleoside triphosphate. Corresponding to its cleavage by activated RecA following DNA damage, the level of LexA is significantly reduced in recA+ D.radiodurans cells following exposure to γ-rays. However, in the recA-defective strain, rec30, LexA disappearance is not obvious.Meanwhile, LexA disappearance in DNA damage sensitive cells appeared to be greater than that in their parental DNA damage resistant cells. The reason for this difference is unknown at present. .In this work, lexA mutant of D. radiodurans XE1 has been successfully constructed. By inserting a 0.9 kb Hinc II fragment carrying the promoter region of Dr catalase gene and reporter gene coding sequence, into the Eag I site of Dr lexA, Dr lexA is inactivated. pXKE6 is confirmed to be recombinant plasmid, having a size of 9.6 kb fragments by 0.7 % agarose gel. The orientation of the inserts is confirmed by Hind III restriction fragment analysis. The yielding plasmid is selected to transform KD8301cells by the procedure of natural transformation of D. radiodurans and transformants are selected. It is confirmed that 0.9 kb KatCAT fragments is inserted into desired lexA region by PCR and southern blotting. Moreov

在最新找到并克隆了抗辐射菌DNA修复基因pprA和lexA-like的研究基础上，构建其高效分泌型表达质粒，纯化蛋白产物。进一步研究pprA和LexA蛋白在DNA修复机制中的作用以及其对溉槔喽锵赴涿舾行缘挠跋欤云诓骺狗渚咝NA修复能力的奥秘且为其今后在生物医学上的应用打下基础，并使我国在DNA损伤和修复领域的研究跻身世界领先。