Muench K A, Bryant F R
Department of Biochemistry, Johns Hopkins University, School of Hygiene and Public Health, Baltimore, Maryland 21205.
J Biol Chem. 1990 Jul 15;265(20):11560-6.
We recently described a mutant recA protein in which glycine 160 of the recA polypeptide was replaced by an asparagine residue (Bryant, F. R. (1988) J. Biol. Chem. 263, 8716-8723). Although the [Asn-160]recA protein has a ssDNA-dependent ATPase activity that is similar to that of the wild-type recA protein, the mutant protein is unable to promote the ATP-dependent three-strand exchange reaction under standard reaction conditions (pH 7.5, 1 mM ATP). We have found that the [Asn-160]recA protein is able to carry out the three-strand exchange reaction at pH 6.0 to 6.7, but that the strand exchange activity is abolished at higher pH. The induction of strand exchange activity at low pH correlates directly with a pH-mediated activation of an ATP-dependent isomerization of the [Asn-160]recA protein. This ATP-dependent isomerization is characterized by the conversion of the [Asn-160]recA protein to a form that is not displaced from ssDNA by the Escherichia coli SSB protein. In contrast to the pronounced pH sensitivity of the [Asn-160]recA protein, the wild-type recA protein undergoes ATP-dependent isomerization, and is able to carry out the three-strand exchange reaction, over the range of pH 6.0 to 8.4. These results show that the [Asn-160] mutation disrupts the ATP-dependent isomerization of the recA protein and suggest that protonation of the [Asn-160]recA protein (or the [Asn-160]recA-ssDNA complex) relieves this mechanistic defect. Furthermore, the direct correlation between ATP-dependent isomerization and the strand exchange activity of the [Asn-160]recA protein strongly suggests that the ATP-dependent isomerization is an obligatory step in the recA protein-promoted strand exchange mechanism.
我们最近描述了一种突变型RecA蛋白，其中RecA多肽的第160位甘氨酸被天冬酰胺残基取代（布莱恩特，F.R.（1988年）《生物化学杂志》263卷，8716 - 8723页）。尽管[天冬酰胺-160]RecA蛋白具有与野生型RecA蛋白相似的单链DNA依赖性ATP酶活性，但在标准反应条件（pH 7.5，1 mM ATP）下，该突变蛋白无法促进ATP依赖性三链交换反应。我们发现，[天冬酰胺-160]RecA蛋白能够在pH 6.0至6.7下进行三链交换反应，但在较高pH值下链交换活性被消除。低pH值下链交换活性的诱导与pH介导的[天冬酰胺-160]RecA蛋白的ATP依赖性异构化直接相关。这种ATP依赖性异构化的特征是[天冬酰胺-160]RecA蛋白转变为一种不会被大肠杆菌单链结合蛋白从单链DNA上取代的形式。与[天冬酰胺-160]RecA蛋白明显的pH敏感性相反，野生型RecA蛋白在pH 6.0至8.4范围内会发生ATP依赖性异构化，并且能够进行三链交换反应。这些结果表明，[天冬酰胺-160]突变破坏了RecA蛋白的ATP依赖性异构化，并表明[天冬酰胺-160]RecA蛋白（或[天冬酰胺-160]RecA - 单链DNA复合物）的质子化缓解了这种机制缺陷。此外，[天冬酰胺-160]RecA蛋白的ATP依赖性异构化与链交换活性之间的直接相关性强烈表明，ATP依赖性异构化是RecA蛋白促进的链交换机制中的一个必要步骤。
