Eichler D C, Lehman I R
J Biol Chem. 1977 Jan 25;252(2):499-503.
The deoxyribonuclease specified by the recB and recC genes of Escherichia coli (recBC DNase; exonuclease V) has been purified to near homogeneity by a new procedure. Although hydrolysis of even a single nucleotide from a duplex DNA molecule by the pure enzyme is absolutely dependent upon ATP, the extent of phosphodiester hydrolysis is strongly inhibited by ATP concentrations of 0.2 mm or greater, and the initial rate is unaffected. Under these conditions, the extent of DNA hydrolysis is proportional to enzyme concentration. In contrast, neither the rate nor the extent of hydrolysis of single-stranded DNA nor ATP is affected by high concentrations of ATP. The amount of large single-stranded polynucleotide generated by the action of the recBC DNase increases as the ATP concentration increases and, at 0.5 mM ATP, becomes equivalent to the amount of acid-soluble nucleotide formed. These findings suggest that high intracellular concentrations of ATP affect the mechanism of the recBC DNase so as to limit the extent of hydrolysis of duplex DNA, while at the same time favoring the formation of single-stranded regions within the duplex. Such regions may be essential intermediates in the recombination process.
通过一种新方法，已将大肠杆菌recB和recC基因所指定的脱氧核糖核酸酶（recBC DNase；外切核酸酶V）纯化至接近均一状态。尽管纯酶从双链DNA分子中水解哪怕单个核苷酸都绝对依赖于ATP，但磷酸二酯键的水解程度会受到0.2 mM或更高ATP浓度的强烈抑制，而初始速率不受影响。在这些条件下，DNA水解程度与酶浓度成正比。相比之下，高浓度ATP对单链DNA或ATP的水解速率和程度均无影响。随着ATP浓度增加，recBC DNase作用产生的大单链多核苷酸量也增加，在0.5 mM ATP时，其与形成的酸溶性核苷酸量相当。这些发现表明，细胞内高浓度的ATP会影响recBC DNase的作用机制，从而限制双链DNA的水解程度，同时有利于双链内单链区域的形成。这样的区域可能是重组过程中的关键中间体。
