抗辐射菌DNA修复蛋白LexA的表达，纯化和鉴定
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摘要
抗辐射菌Deinoeoccus radiodurans(Dr)是一种对电离辐射，紫外线及其他一些DNA损伤剂具有显著抗性的微球菌。该菌能在辐照后几小时内准确无误地修复每条染色体上至少150多个DNA双链断裂(DSB)。已有的研究表明该独特抗性归功于其高效精确的DNA损伤修复能力。然而，对其修复路径及机制知之甚少。D.radiodurans中是否存在SOS修复路径，及在E.coli等菌中广泛存在的LexA修复蛋白在D.radiodurans 中的功能特性如何均是未解之迷。本研究利用已克隆构建的D.radiodurans lexA基因重组质粒pZA172，转化大肠杆菌JM109并在IPTG诱导下获得了高效表达，Dr LexA分子量约25.4KDa。实验表明IPTG浓度为0.1 mM，诱导时间为24 h时LexA蛋白表达效果最佳。大体积培养表达菌时，氨苄青霉素浓度为25 μg／ml及IPTG诱导浓度为0.1mM时，LexA表达量较高。
我们对表达产物进行了纯化并进一步研究了纯化LexA的功能特性。培养细胞经溶菌酶，反复冻融，超声等裂解破膜后，再通过高速离心，层析，超滤浓缩等技术使LexA逐步得到纯化。在层析分离阶段，Dr LexA表现出对生物肝素的高度亲和性，表明其为DNA结合蛋白。且与E.coli等菌LexA类似，Dr LexA亦与强阳离子交换柱相结合。大部分LexA蛋白经肝素柱及阳离子交换层析(SP Sepharose)后分离得到，洗脱峰分别在250 mM NaCl及400 mMNaCl左右。蛋白纯化程度达95％以上，产量为10L(约17.3 g)表达菌可获得约2 mg纯化蛋白。SDS-聚丙烯酰胺凝胶电泳结果显示纯化的蛋白为一分子质量25.4KDa的单一蛋白带。纯化蛋白用于制备抗体及进一步研究。
利用凝胶阻抑电泳实验分析了纯化的LexA蛋白的DNA结合特性。PCR扩增与DNA修复相关的D.radiodurans pprA,orf144c,recA基因上游序列并用地高辛标记作探针，与纯化Dr LexA反应后应用于非变性聚丙烯酰胺凝胶电泳。结果表明，与E.coli LexA不同，Dr LexA与D.radiodurans 修复基因pprA,orf144c,recA启动子序列无明显结合位点，而只与lexA基因有结合区域，说明其不参与RecA等蛋白的负反馈调节。然而类似E.coliLexA,Dr LexA的水解特性表现为在碱性条件下D.radiodurans LexA自动降解，以及生理pH
抗辐射菌DNA修复蛋白LexA的表达，纯化和鉴定 中文摘要
条件下可被激活的ReCA蛋白促进降解。单链DNA及Ah的存在则为ReCA
蛋白激活所必需。对x－射线损伤引起的不同D．radiodurans 菌株LexA蛋白
D 表达量变化的研究表明，RecA＋D．radiodurans 细胞受损后，细胞内 LexA蛋
白表达量明显下降，而recA基因缺陷的rec30照射后LexA蛋白减少不明显。
此结果与RecA蛋白促进的LexA降解反应相对应。研究同时发现在DNA损
伤敏感型D．radiodurans 细胞中，LexA蛋白的减少远远大于损伤抵抗型细胞，
原因尚不清楚。
我们在本研究中成功构建了D．radiodurans lexA基因缺损突变体XEI。
通过在 D．radiodurans lexA酶切位点 Ea 互插入一段长 0．9 Kb，含D catalase
启动子序列及CAT报道基因的外源DNA片段（KatCAT），进而使lexA基因
失活的重组方法，构建了突变基因重组质粒pXKE6。琼脂糖凝胶电泳证实其
DNA长度为 9石 Kb。利用Hind Ill酶切鉴定其插入KatCAT片段的方向。再
用含有正确插入方向的重组质粒进一步转化D．radiodurans KD8301细胞后，
经药物选择性筛选出同源重组突变体。突变体经PCR扩增及Southern杂交被
鉴定。Western杂交分析亦进一步证实了XE突变体中没有LexA蛋白的表达。
对 lexA基因的缺陷突变体 XEI 的研究表明，其辐射抗性与 lexA＋ D．
radiodurans KD8301相近，存活曲线上显示有一个宽大的肩区，证实 lexA基
因的缺陷对抗辐射菌的辐射抗性无明显影响，说明D．radioduans LexA蛋白
可能并不具有类似E。。h等LexA的负反馈调控相关DNA修复基因的机制。
同时，细胞受照后的蛋白印迹实验显示lexA基因的缺陷不干扰辐射诱导的D．
radiodurans RecA蛋白的表达，说明RecA的调控并不依赖于LexA蛋白，该
结论与LexA蛋白与recA上游基因序列无结合位点的证据相吻合。然而，与
lexA＋KD8301细胞受照后修复蛋白PprA的大量表达相反，受照后的XEI细
胞中没有检测到 PprA蛋白的表达，提示lexA基因的缺陷干扰了 D．radiodurans
pprA的转录。综合本文的研究结果，表明D．radiodurans LexA与其同源E。＊
LexA在功能及特性上同时存在相近及不同之处，提示了抗辐射菌的DNA损
、伤修复是一个多种基因参与的复杂的过程，有其独特的调节机制。
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 h 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?g/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 ? -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, lex A 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 Kat.CAT fragments is inserted into desired lexA region by PCR and southern blotti
我们对表达产物进行了纯化并进一步研究了纯化LexA的功能特性。培养细胞经溶菌酶，反复冻融，超声等裂解破膜后，再通过高速离心，层析，超滤浓缩等技术使LexA逐步得到纯化。在层析分离阶段，Dr LexA表现出对生物肝素的高度亲和性，表明其为DNA结合蛋白。且与E.coli等菌LexA类似，Dr LexA亦与强阳离子交换柱相结合。大部分LexA蛋白经肝素柱及阳离子交换层析(SP Sepharose)后分离得到，洗脱峰分别在250 mM NaCl及400 mMNaCl左右。蛋白纯化程度达95％以上，产量为10L(约17.3 g)表达菌可获得约2 mg纯化蛋白。SDS-聚丙烯酰胺凝胶电泳结果显示纯化的蛋白为一分子质量25.4KDa的单一蛋白带。纯化蛋白用于制备抗体及进一步研究。
利用凝胶阻抑电泳实验分析了纯化的LexA蛋白的DNA结合特性。PCR扩增与DNA修复相关的D.radiodurans pprA,orf144c,recA基因上游序列并用地高辛标记作探针，与纯化Dr LexA反应后应用于非变性聚丙烯酰胺凝胶电泳。结果表明，与E.coli LexA不同，Dr LexA与D.radiodurans 修复基因pprA,orf144c,recA启动子序列无明显结合位点，而只与lexA基因有结合区域，说明其不参与RecA等蛋白的负反馈调节。然而类似E.coliLexA,Dr LexA的水解特性表现为在碱性条件下D.radiodurans LexA自动降解，以及生理pH
抗辐射菌DNA修复蛋白LexA的表达，纯化和鉴定 中文摘要
条件下可被激活的ReCA蛋白促进降解。单链DNA及Ah的存在则为ReCA
蛋白激活所必需。对x－射线损伤引起的不同D．radiodurans 菌株LexA蛋白
D 表达量变化的研究表明，RecA＋D．radiodurans 细胞受损后，细胞内 LexA蛋
白表达量明显下降，而recA基因缺陷的rec30照射后LexA蛋白减少不明显。
此结果与RecA蛋白促进的LexA降解反应相对应。研究同时发现在DNA损
伤敏感型D．radiodurans 细胞中，LexA蛋白的减少远远大于损伤抵抗型细胞，
原因尚不清楚。
我们在本研究中成功构建了D．radiodurans lexA基因缺损突变体XEI。
通过在 D．radiodurans lexA酶切位点 Ea 互插入一段长 0．9 Kb，含D catalase
启动子序列及CAT报道基因的外源DNA片段（KatCAT），进而使lexA基因
失活的重组方法，构建了突变基因重组质粒pXKE6。琼脂糖凝胶电泳证实其
DNA长度为 9石 Kb。利用Hind Ill酶切鉴定其插入KatCAT片段的方向。再
用含有正确插入方向的重组质粒进一步转化D．radiodurans KD8301细胞后，
经药物选择性筛选出同源重组突变体。突变体经PCR扩增及Southern杂交被
鉴定。Western杂交分析亦进一步证实了XE突变体中没有LexA蛋白的表达。
对 lexA基因的缺陷突变体 XEI 的研究表明，其辐射抗性与 lexA＋ D．
radiodurans KD8301相近，存活曲线上显示有一个宽大的肩区，证实 lexA基
因的缺陷对抗辐射菌的辐射抗性无明显影响，说明D．radioduans LexA蛋白
可能并不具有类似E。。h等LexA的负反馈调控相关DNA修复基因的机制。
同时，细胞受照后的蛋白印迹实验显示lexA基因的缺陷不干扰辐射诱导的D．
radiodurans RecA蛋白的表达，说明RecA的调控并不依赖于LexA蛋白，该
结论与LexA蛋白与recA上游基因序列无结合位点的证据相吻合。然而，与
lexA＋KD8301细胞受照后修复蛋白PprA的大量表达相反，受照后的XEI细
胞中没有检测到 PprA蛋白的表达，提示lexA基因的缺陷干扰了 D．radiodurans
pprA的转录。综合本文的研究结果，表明D．radiodurans LexA与其同源E。＊
LexA在功能及特性上同时存在相近及不同之处，提示了抗辐射菌的DNA损
、伤修复是一个多种基因参与的复杂的过程，有其独特的调节机制。
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 h 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?g/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 ? -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, lex A 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 Kat.CAT fragments is inserted into desired lexA region by PCR and southern blotti
引文
1． Anderson, A., H.Nordan, R.Cain, G.Parrish, and D.Duggan.. Studies on a radioresistant micrococcus. I. Isolation, morphology, cultural charchteristoics, and resistance to gamma radiation. Food Technol. 1956;,10:575-578
2． Ferreira, A.C., M.F.Nobre, F.A.Rainey, M.T.Silva, F.Wait, J.Burghardt, A.P.Chung, and M.S.Da Costa. Deinococcus geothermalis sp. nov. and Deinococcus murrayi sp. nov, two extrimely radiationresistant and slightly thermophilic species from hot springs. Int. J. Syst. Bacteriol. 1997; 47:939-947
3． Minton, K.W. Repair of ionizing-radiation damage in the radiation resistant bacterium Deinococcus radiodurans. Mutant. Res. 1996; 363:1-7
4． Murray, R.G.E. The family Deinococcaceae, In A. Balows, H.G. Truper, M. Dworkin, W. Harder, and K.H. Schleifer(ed), The prokaryotes, vol. 4． Springer-verlag, New York, N.Y. 1992; 3732-3744．
5． Daly, M.J., Ouyang, P. Fuchs, and K.W.Minton. In vivo damage and recA-dependent repair of plasmid and chromosomal DNA in the radiation-resistant bacterium Deinococcus radiodurans. J. Bacteriol. 1994;176:3508-3517
6． Lange, C.C., L.P.Wackett, K.W.Minton, and M.J.Daly. Engineering a recombinant Deinococcus radiodurans for organopollutant degradation in radioactive mixed waste environments. Nat. Biotechnol. 1998; 16:929-933
7． Moseley, B.E., and D.M.Evans. Isolation and properties of strains of Micrococcus(Deinococcus) radiodurans unable to excise ultraviolet light-induced pyrimidine dimers from DNA: evidence for two excision pathways. J. Gen. Microbiol. 1983;12:2437-2445
8． Smith, K.C., and K.D.Martignoni. Protection of Escherichia coli cells against the lethal effects of ultraviolet and x irradiation by prior x irradiation: a genetic and physiological study. Photochem. Photobiol. 1976． 24:515-523
9． Hansen, M.T. Multiplicity of genome equivalents in the radiation-resistant bacterium Micrococcus radiodurans. J.Bacteriol. 1978; 134:71-75
10． Harsojo, S.Kitayama, and A. Matsuyama. Genome multiplicity and radiation resistance in Micrococcus radiodurans. J.Biochem. (Tokyo) 1990;877-880
11． Krasin, F., and F. Hutchinson. Repair of DNA double-strand breaks in Escherichia coli, which requires recA function and the presence of a duplicate genome. J.Mol.Biol 1997; 116:81-98
12． Majumdar, S., and A.K.Chandra. UV repair and mutagenesis in Azotobacter vinelandii. Zentbl. Mikrobiol. 1985;140:247-254
13． Sadoff, H.L., B.Shimel, and S.Ellis. Characterization of Azotobacter vinelandii deoxyribonucleic acid and folded chromosomes. J. Bacteriol. 1979; 138:871-877
14． Boiling, M.E., and J.K.Setlow. The resistance of Deinococcus radiodurans to ultraviolet radiation. III. A repair mechanism. Biochem. Biophys. Acta 1966;123:26-33
15． Daly, M.J., and K.W.Minton. Interchromosomal recombination in teh extremely radioresistant bacterium Deinococcus radiodurans. J.Bacteriol. 1995; 177:5495-5505
16． Battista, J.R.. Against all odds: the survival strategies of Deinococcus radiodurans. Annu.Rev.Microbiol. 1997; 51:203-224
17． Vukovic-Nagy, Bl,B.W.Fox, and M.Fox. The release of a deoxyribonucleic acid fragment after x-irradiation of Micrococcus radiodurans. Int. J.Radiat. Biol. Relat. Stud. Phys. Chem.Med. 1974; 25:329-337
18． Jordan, A., and P.Reichard. Ribonucleotide reductases. Annu. Rev. Biochem. 1998;67:71-98
19． Masters, C.I., B.E.Moseley, and K.W.Minton.. AP endonuclease and uracil DNA glycosylase activities in Deinococcus radiodurans. Mutat. Res. 1991; 254:263-272
20． Mun, C., J. Del Rowe, M. Sandigursky, K.W.Minton, and W.A.Franklin. DNA deoxyribophosphodiesterase and an activity that cleaves DNA containing thymine glycol adducts in Deinococcus radiodurans. Radiat. Res. 1994;
138:282-285
21． Moseley. B.E.B.,and H.J.R.Copland. Isolation and properties of a recombination deficient mutant of Micrococcus radiodurans. J.Bacteriol. 1975;121:42-48
22． Gutman, P.D., J.D.Carroll, C.I. Masters, and K.W.Minton. Sequencing, targeted mutagenesis of a DNA polymerase gene required for the extreme radioresistance of Deinococcus radiodurans. Gene 1994;141:31-37
23． Daly, M.J., O.Ling,and K.W.Minton. Interplasmidic recombination following irradiation of the radioresistant bacterium Deinococcus radiodurans. J.Bacteriol. 1994;176:7506-7615
24． Tan, ST., and R.B.Maxcy. Simple method to demonstrate radiation inducible radiation resistance in microbial cells. Appl. Environ. Microbiol. 1986;51:88-90
25． Wang.P., and H.E.Schellhorn. Induction of resistance to hydrogen peroxide and radiation in Deinococcus radiodurans. Can.J.Microbiol 1995; 41:170-176
26． Hansen, M.T. 1980． Four proteins synthesized in response to deoxyribonucleic acid damage in Micrococcus radiodurans. J.Bacteriol. 141:81-86
27． Tanaka, A., H. Hirano, M.Kikuchi, S.Kitayama, and H.Watanabe. Changes in cellular proteins of Deinococcus radiodurans following gamma-irradiation. Radiat. Environ. Biophs. 1996;35:95-99
28． Agostini, H.J., D.Carroll, and K.W.Minton. Identification and characterization of uvrA gene of Deinococcus radiodurans. J.Bacteriol. 1996; 178:6759-6765
29． Narumi, I1, K. Cherdchu, S.Kitayama, and H.Watanabe. The Deinococcus radiodurans uvrA gene: identification of mutation sites in two mitomycin-sensitive strains and the first discovery of insertion sequence lesment form deinobacteria. Gene 1997． ; 198:115-126
30． Gutman, P.D., P.Fuchs, L.Ouyang, and K.W.Minton. Identification, sequencing and targeted mytagenesis of a DNA polymerase gene required for the extreme radioresistance of Deinococcus radiodurans. J.Bacteriol. 1993;175:3581-3590
31． Carroll,J.D., M.J.Daly, and K.W.Minton. Expression of recA in Deinococcus radiodurans. J.Bacteriol. 1996;178:130-135
32． Narumi, I., K.Satoh, M. Kikuchi, T.Funayama, S.Kitayama, T.Yanagisawa, H.Watanabe, and K.Yamamoto. Molecular analysis of the Deinococcus radiodurans recA locus and identification of a mutation site in a DNA repair-deficient mutant, rec30． Mutat.Res. 1999;435:233-243
33． Daly, M.J., and K.W.Minton.. An alternative pathway of recombination of chromosomal fragments precedes recA-dependent recombination in the radioresiatant bacterium Deinococcus radiodurans. J. Bacteriol. 1996; 178:4461-4471
34． White O, Eisen JA, Heidelberg JF, et al. Genome sequence of the radioresistant bacterium Deinococcus radiodurans Rl. Science. 286(5444) : 1999;1571-1577
35． Mellon, I. and P.C.Hanawalt. Induction of the Escherichia coli lactose operon selectively increases repair of its transcribed DNA strand. Nature 1989;342:95-98
36． Narumi,I., Kong X.R., et al. Cloning, sequencing and expression of the lexA-like gene of D. radiodurans. 1997． Accession:AB003475 in Gnebank, DDBJ.
37． Ullmann A. and Perrin D. 1970． Complementation in B-galactosidase. In the lactose operon, pp. 143-172． Cold Sproing Harbor Laboratory, Cold Spring Harbor, New York.
38． Marianne Carlsson.1997． Partial purification of a DNA-binding protein using immobilized heparin. Science Tools from Pharmacia Biotech 2,1 8-9
39． Courcelle,J.A., Khodursky,A., Peter,B.,Brown,P.O.& Hanawalt,P.C. Comparative Gene Expression Profiles Following UV Exposure in Wild-Type and SOS-Deficient Escherichia coli. Genetics 2001． ; 158:41-64
40． Fernandez De Henestrosa, A.R., T. Ogi, S.Aoyagi, D.Chafin, and J.J.Hayes et al., Identification of additional genes belonging to the Lex A trgulon in Escherichia coli. Mol.Microbiol. 2000;35:1560-1572
41． Koch, W.H., and R.Woodgate. 1998． The SOS response, pp. 107-134 in DNA Damage and Repair:DNA Repair in Prokaryotes and Lower Eukaryotes, edited by
J.A.Nickolofff and M.F.Hoekstra. Humana Press, Totowa, NJ.
42． Narumi,I., Du,Z., Alatas,Z., Kitayama,S. and Watanabe,H. 1997． Isolation and characterization of pprA, a novel Deinococcus radiodurans gene involved in DNA repair. Accession:AB003475 in Gnebank, DDBJ.
43． Brandsma, J.A., D.Bosch, C.Backendorf, and P.Van de putte. A common regulatory region shared by divergently transcribed genes of the Escherichia coli SOS system. Natruel983． ;305:243-245
44． Friedberg, E.C., C. W. Graham and W.Siede. 1995． DNA repair and Mutagenesis. ASM Press, Washington, DC.
45． Little, J.W. Autodigestion of lexA and phage lambda repressers. Proc.Natl.Acad.Sci. USA 1984;81:1375-1379
46． Sassanfar,M. and J.W.Roberts.. 1990． Nature of the SOS-inducing signal in Escherichia coli. The involvement of DNA replication. J.Mol.Bi 1984;212:79-96
47． Slilaty,S.N.and J.W.Little. Lysine-156 and serine-119 are required for LexA Represser cleavage: a possible mechanism. Proc. Natl. Acad. Sci. USA 1987 ;84:3987-3991
48． Brent, R. and M.Ptashne.. Mechanism of action of the lexA gene product. Proc.Natl.Acad.Sci. USA 1981;78:4204-4208
49． Walker, G.C. Mutagenesis and inducible responses to deoxyribonucleic acid damage in Excherichia coli. Microbiol. Rev. 1984． ;48:60-93
50． Moseley, B.E.B. and Copeland. H.J.R. Isolation and properties of a recombination-deficient mutant of Micrococcus radiodurans. J.Bacteriol. 1975; 121:422-428
51． Mount, D.W. A mutant of Escherichia coli showing constiturive expression of the lysogenic induction and error-prone DNA repair pathways. Proc. Natl. Acad. Sci. USA 1977;74:300-304
52． Berta, C. Construction and characterization of two lexA mutants of Salmonella typhimurium with different UV sensitivities and UV mutabilities. J.Bacteriol. 1996;178:2890-2896
2． Ferreira, A.C., M.F.Nobre, F.A.Rainey, M.T.Silva, F.Wait, J.Burghardt, A.P.Chung, and M.S.Da Costa. Deinococcus geothermalis sp. nov. and Deinococcus murrayi sp. nov, two extrimely radiationresistant and slightly thermophilic species from hot springs. Int. J. Syst. Bacteriol. 1997; 47:939-947
3． Minton, K.W. Repair of ionizing-radiation damage in the radiation resistant bacterium Deinococcus radiodurans. Mutant. Res. 1996; 363:1-7
4． Murray, R.G.E. The family Deinococcaceae, In A. Balows, H.G. Truper, M. Dworkin, W. Harder, and K.H. Schleifer(ed), The prokaryotes, vol. 4． Springer-verlag, New York, N.Y. 1992; 3732-3744．
5． Daly, M.J., Ouyang, P. Fuchs, and K.W.Minton. In vivo damage and recA-dependent repair of plasmid and chromosomal DNA in the radiation-resistant bacterium Deinococcus radiodurans. J. Bacteriol. 1994;176:3508-3517
6． Lange, C.C., L.P.Wackett, K.W.Minton, and M.J.Daly. Engineering a recombinant Deinococcus radiodurans for organopollutant degradation in radioactive mixed waste environments. Nat. Biotechnol. 1998; 16:929-933
7． Moseley, B.E., and D.M.Evans. Isolation and properties of strains of Micrococcus(Deinococcus) radiodurans unable to excise ultraviolet light-induced pyrimidine dimers from DNA: evidence for two excision pathways. J. Gen. Microbiol. 1983;12:2437-2445
8． Smith, K.C., and K.D.Martignoni. Protection of Escherichia coli cells against the lethal effects of ultraviolet and x irradiation by prior x irradiation: a genetic and physiological study. Photochem. Photobiol. 1976． 24:515-523
9． Hansen, M.T. Multiplicity of genome equivalents in the radiation-resistant bacterium Micrococcus radiodurans. J.Bacteriol. 1978; 134:71-75
10． Harsojo, S.Kitayama, and A. Matsuyama. Genome multiplicity and radiation resistance in Micrococcus radiodurans. J.Biochem. (Tokyo) 1990;877-880
11． Krasin, F., and F. Hutchinson. Repair of DNA double-strand breaks in Escherichia coli, which requires recA function and the presence of a duplicate genome. J.Mol.Biol 1997; 116:81-98
12． Majumdar, S., and A.K.Chandra. UV repair and mutagenesis in Azotobacter vinelandii. Zentbl. Mikrobiol. 1985;140:247-254
13． Sadoff, H.L., B.Shimel, and S.Ellis. Characterization of Azotobacter vinelandii deoxyribonucleic acid and folded chromosomes. J. Bacteriol. 1979; 138:871-877
14． Boiling, M.E., and J.K.Setlow. The resistance of Deinococcus radiodurans to ultraviolet radiation. III. A repair mechanism. Biochem. Biophys. Acta 1966;123:26-33
15． Daly, M.J., and K.W.Minton. Interchromosomal recombination in teh extremely radioresistant bacterium Deinococcus radiodurans. J.Bacteriol. 1995; 177:5495-5505
16． Battista, J.R.. Against all odds: the survival strategies of Deinococcus radiodurans. Annu.Rev.Microbiol. 1997; 51:203-224
17． Vukovic-Nagy, Bl,B.W.Fox, and M.Fox. The release of a deoxyribonucleic acid fragment after x-irradiation of Micrococcus radiodurans. Int. J.Radiat. Biol. Relat. Stud. Phys. Chem.Med. 1974; 25:329-337
18． Jordan, A., and P.Reichard. Ribonucleotide reductases. Annu. Rev. Biochem. 1998;67:71-98
19． Masters, C.I., B.E.Moseley, and K.W.Minton.. AP endonuclease and uracil DNA glycosylase activities in Deinococcus radiodurans. Mutat. Res. 1991; 254:263-272
20． Mun, C., J. Del Rowe, M. Sandigursky, K.W.Minton, and W.A.Franklin. DNA deoxyribophosphodiesterase and an activity that cleaves DNA containing thymine glycol adducts in Deinococcus radiodurans. Radiat. Res. 1994;
138:282-285
21． Moseley. B.E.B.,and H.J.R.Copland. Isolation and properties of a recombination deficient mutant of Micrococcus radiodurans. J.Bacteriol. 1975;121:42-48
22． Gutman, P.D., J.D.Carroll, C.I. Masters, and K.W.Minton. Sequencing, targeted mutagenesis of a DNA polymerase gene required for the extreme radioresistance of Deinococcus radiodurans. Gene 1994;141:31-37
23． Daly, M.J., O.Ling,and K.W.Minton. Interplasmidic recombination following irradiation of the radioresistant bacterium Deinococcus radiodurans. J.Bacteriol. 1994;176:7506-7615
24． Tan, ST., and R.B.Maxcy. Simple method to demonstrate radiation inducible radiation resistance in microbial cells. Appl. Environ. Microbiol. 1986;51:88-90
25． Wang.P., and H.E.Schellhorn. Induction of resistance to hydrogen peroxide and radiation in Deinococcus radiodurans. Can.J.Microbiol 1995; 41:170-176
26． Hansen, M.T. 1980． Four proteins synthesized in response to deoxyribonucleic acid damage in Micrococcus radiodurans. J.Bacteriol. 141:81-86
27． Tanaka, A., H. Hirano, M.Kikuchi, S.Kitayama, and H.Watanabe. Changes in cellular proteins of Deinococcus radiodurans following gamma-irradiation. Radiat. Environ. Biophs. 1996;35:95-99
28． Agostini, H.J., D.Carroll, and K.W.Minton. Identification and characterization of uvrA gene of Deinococcus radiodurans. J.Bacteriol. 1996; 178:6759-6765
29． Narumi, I1, K. Cherdchu, S.Kitayama, and H.Watanabe. The Deinococcus radiodurans uvrA gene: identification of mutation sites in two mitomycin-sensitive strains and the first discovery of insertion sequence lesment form deinobacteria. Gene 1997． ; 198:115-126
30． Gutman, P.D., P.Fuchs, L.Ouyang, and K.W.Minton. Identification, sequencing and targeted mytagenesis of a DNA polymerase gene required for the extreme radioresistance of Deinococcus radiodurans. J.Bacteriol. 1993;175:3581-3590
31． Carroll,J.D., M.J.Daly, and K.W.Minton. Expression of recA in Deinococcus radiodurans. J.Bacteriol. 1996;178:130-135
32． Narumi, I., K.Satoh, M. Kikuchi, T.Funayama, S.Kitayama, T.Yanagisawa, H.Watanabe, and K.Yamamoto. Molecular analysis of the Deinococcus radiodurans recA locus and identification of a mutation site in a DNA repair-deficient mutant, rec30． Mutat.Res. 1999;435:233-243
33． Daly, M.J., and K.W.Minton.. An alternative pathway of recombination of chromosomal fragments precedes recA-dependent recombination in the radioresiatant bacterium Deinococcus radiodurans. J. Bacteriol. 1996; 178:4461-4471
34． White O, Eisen JA, Heidelberg JF, et al. Genome sequence of the radioresistant bacterium Deinococcus radiodurans Rl. Science. 286(5444) : 1999;1571-1577
35． Mellon, I. and P.C.Hanawalt. Induction of the Escherichia coli lactose operon selectively increases repair of its transcribed DNA strand. Nature 1989;342:95-98
36． Narumi,I., Kong X.R., et al. Cloning, sequencing and expression of the lexA-like gene of D. radiodurans. 1997． Accession:AB003475 in Gnebank, DDBJ.
37． Ullmann A. and Perrin D. 1970． Complementation in B-galactosidase. In the lactose operon, pp. 143-172． Cold Sproing Harbor Laboratory, Cold Spring Harbor, New York.
38． Marianne Carlsson.1997． Partial purification of a DNA-binding protein using immobilized heparin. Science Tools from Pharmacia Biotech 2,1 8-9
39． Courcelle,J.A., Khodursky,A., Peter,B.,Brown,P.O.& Hanawalt,P.C. Comparative Gene Expression Profiles Following UV Exposure in Wild-Type and SOS-Deficient Escherichia coli. Genetics 2001． ; 158:41-64
40． Fernandez De Henestrosa, A.R., T. Ogi, S.Aoyagi, D.Chafin, and J.J.Hayes et al., Identification of additional genes belonging to the Lex A trgulon in Escherichia coli. Mol.Microbiol. 2000;35:1560-1572
41． Koch, W.H., and R.Woodgate. 1998． The SOS response, pp. 107-134 in DNA Damage and Repair:DNA Repair in Prokaryotes and Lower Eukaryotes, edited by
J.A.Nickolofff and M.F.Hoekstra. Humana Press, Totowa, NJ.
42． Narumi,I., Du,Z., Alatas,Z., Kitayama,S. and Watanabe,H. 1997． Isolation and characterization of pprA, a novel Deinococcus radiodurans gene involved in DNA repair. Accession:AB003475 in Gnebank, DDBJ.
43． Brandsma, J.A., D.Bosch, C.Backendorf, and P.Van de putte. A common regulatory region shared by divergently transcribed genes of the Escherichia coli SOS system. Natruel983． ;305:243-245
44． Friedberg, E.C., C. W. Graham and W.Siede. 1995． DNA repair and Mutagenesis. ASM Press, Washington, DC.
45． Little, J.W. Autodigestion of lexA and phage lambda repressers. Proc.Natl.Acad.Sci. USA 1984;81:1375-1379
46． Sassanfar,M. and J.W.Roberts.. 1990． Nature of the SOS-inducing signal in Escherichia coli. The involvement of DNA replication. J.Mol.Bi 1984;212:79-96
47． Slilaty,S.N.and J.W.Little. Lysine-156 and serine-119 are required for LexA Represser cleavage: a possible mechanism. Proc. Natl. Acad. Sci. USA 1987 ;84:3987-3991
48． Brent, R. and M.Ptashne.. Mechanism of action of the lexA gene product. Proc.Natl.Acad.Sci. USA 1981;78:4204-4208
49． Walker, G.C. Mutagenesis and inducible responses to deoxyribonucleic acid damage in Excherichia coli. Microbiol. Rev. 1984． ;48:60-93
50． Moseley, B.E.B. and Copeland. H.J.R. Isolation and properties of a recombination-deficient mutant of Micrococcus radiodurans. J.Bacteriol. 1975; 121:422-428
51． Mount, D.W. A mutant of Escherichia coli showing constiturive expression of the lysogenic induction and error-prone DNA repair pathways. Proc. Natl. Acad. Sci. USA 1977;74:300-304
52． Berta, C. Construction and characterization of two lexA mutants of Salmonella typhimurium with different UV sensitivities and UV mutabilities. J.Bacteriol. 1996;178:2890-2896