Reaction Mechanism of ATP-dependent DNase and its Role in the genetic Recombination Process in the Cell.

ATP依赖性DNA酶的反应机制及其在细胞遗传重组过程中的作用。

• 批准号: 60440105
• 负责人: TAKAGI Yasuyuki
• 金额: $ 20.29万
• 依托单位: Fujita-Gakuen Health University, Medical School
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (A)
• 财政年份: 1985
• 资助国家: 日本
• 起止时间: 1985 至 1988
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-60440105/
• 关键词: ATP-dependent DNase; recBC Enzyme; Genetic Recombination; 細胞内遺伝子組換え; ATP依存性DNase

ATP-dependent DNase is widely distributed among various microorganisms, and catalyzes the exonucleolytic degradation of lineal double and single stranded DNA in the presence of added ATP, producing the oligonucleotides consisted of about 5 nucleotides. Meanwhile mutations in the Escherichia coli recB and recC genes are known to lead to a mutant phenotype, reduced recombination and increased sensitivity to ultraviolet-light irradiation or mitomycin C treat-ment. Both genes are also found to control the production of the ATP-dependent dnase in E. coli cells. Therefore in this research project, it was aimed to isolate both ganes and purify the gene products in large scale, then to analyze the reaction mechanism in detail.The E. coli thyA, recC, recB and argA gene region was cloned in a cosmid, and the recB and recC genes were separately subcloned, proving that these two genes consist of independent cistrons. The base sequences of both genes were analyzed, and it was confirmed that the rec … More B gene encodes a protein of 1180 amino acids, and the recC gene 1122 amino acids.Then the properties of the recB and recC gene products were studied using recB and recC gene-inserted plasmids. recB mutants and recC mutants lacked ATP-dependent DNase, but showed apar-ent recovery of enzyme activity on introduction of plasmids carrying the recB and recC gene, respectively. The ATP-dependent DNase was also constructed in vitro by mixing the recB and recC gene products by the plasmids with the corresponding gene. Specific labeling of plasmid-encoded proteins by the maxicell method showed that the recB and recC gene products were 135,000 and 125,000 dalton protein, respectively. However a possibility that the addition of another gene product is requested to construct the whole enzyme was suggested.Finally it was found that ATP-dependent DNase of Bacillus laterosporus consumes 3 ATPs at pH 8.3, and 2.2 at 6.3 per every one phosphodiester bond-cleavage, suggesting to catalyze the reaction of two types. Less

依赖ATP的DNA酶广泛存在于各种微生物中，在ATP存在下催化线性双链和单链DNA的外切降解，产生约5个核苷酸的寡核苷酸。同时，大肠杆菌recB和recC基因的突变已知导致突变表型、重组减少和对紫外线照射或丝裂霉素C处理的敏感性增加。在大肠杆菌中，这两个基因也被发现控制ATP依赖性DNA酶的产生。coli细胞。因此本研究的目的是大规模分离纯化这两种加内斯基因产物，并对其反应机理进行详细分析。colithyA、recC、recB和argA基因片段克隆到粘粒中，recB和recC基因分别亚克隆，证明这两个基因由独立的顺反子组成。对两个基因的碱基序列进行分析，证实了rec ...更多信息 B基因编码1180个氨基酸的蛋白质，recC基因编码1122个氨基酸的蛋白质。recB突变体和recC突变体缺乏ATP依赖性DNA酶，但分别导入携带recB和recC基因的质粒后，酶活性有部分恢复。通过将重组质粒的recB和recC基因产物与相应的基因混合，在体外构建ATP依赖性DNA酶。通过maxicell方法对质粒编码蛋白的特异性标记显示，recB和recC基因产物分别为135，000和125，000道尔顿蛋白。最后发现侧孢芽孢杆菌的ATP依赖性DNA酶在pH8.3时每裂解一个磷酸二酯键消耗3个ATP，在pH6.3时每裂解一个磷酸二酯键消耗2.2个ATP，表明它催化两种类型的反应。少

项目成果

Sasaki, M.: "Nucleotide Sequence Analysis of Escherichia coli recC Gene" J. Biochem.

Sasaki, M.：“大肠杆菌recC 基因的核苷酸序列分析”J. Biochem。

Sasaki, M.: "Nucleotide Sequence Analysis of Escherichia coli recB Gene" J. Biochem.

Sasaki, M.：“大肠杆菌recB 基因的核苷酸序列分析”J. Biochem。

Sasaki,M.: J.Biochem.

佐佐木，M.：J.Biochem。