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

依赖于ATP的DNA酶广泛存在于各种微生物中，在添加ATP的情况下催化线形双链和单链DNA的外切降解，产生约5个核苷酸组成的寡核苷酸。同时，大肠杆菌recB和recC基因突变会导致表型突变，重组减少，对紫外光照射或丝裂霉素C治疗的敏感性增加。这两个基因也被发现控制着大肠杆菌细胞中依赖于ATP的DNA酶的产生。因此，本研究旨在大规模分离和纯化两种基因产物，并对其反应机理进行详细的分析。将大肠杆菌的ThyA、recC、recB和argA基因克隆到粘粒中，并分别亚克隆了recB和recC基因，证明这两个基因是由独立的顺反子组成的。对两个基因的碱基序列进行了分析，证实了REC-…其中B基因编码1180个氨基酸，recC基因编码1122个氨基酸，分别构建了recB和recC基因插入载体，并对其性质进行了研究。RecB突变体和recC突变体缺乏依赖于ATP的DNA酶，但分别导入携带recB和recC基因的质粒后，酶活性恢复较快。此外，还将载体上的recB和recC基因产物与相应的基因混合，在体外构建了依赖于ATP的DNA酶。用Maxicell方法对编码的蛋白质进行特异性标记，结果显示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依赖于ATP的脱氧核糖核酸酶在每一次磷酸二酯键断裂时消耗3个ATP，在每个6.3时消耗2.2个ATP，这表明这两种类型的反应都是催化的。较少

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