Muench K A, Bryant F R
Department of Biochemistry, Johns Hopkins University School of Hygiene and Public Health, Baltimore, Maryland 21205.
J Biol Chem. 1991 Jan 15;266(2):844-50.
We have used site-directed mutagenesis to replace histidine 163 of the recA polypeptide with an alanine residue. The new [Ala-163]recA protein catalyzes single-stranded (ss) DNA-dependent ATP hydrolysis with a turnover number that is similar to that of the wild-type recA protein. Despite being proficient in ssDNA-dependent ATP hydrolysis, the [Ala-163]recA protein is unable to promote the ATP-dependent three-strand exchange reaction under standard reaction conditions, pH 7.5. The [Ala-163]recA protein does exhibit three-strand exchange activity at pH 6.0-7.0, however, and the induction of strand exchange activity at low pH correlates directly with the activation of an ATP-dependent isomerization of the mutant protein. Thus, the [Ala-163]recA protein is functionally similar to our previously described mutant [Asn-160]recA protein (Bryant, F.R. (1988) J. Biol. Chem. 263, 8716-8723; Muench, K.A., and Bryant, F. R. (1990) J. Biol. Chem. 265, 11560-11566). Trypsin proteolysis studies indicate that the [Ala-163]recA and [Asn-160]recA proteins, like the wild-type recA protein, are organized into carboxyl-terminal and amino-terminal domains of nearly equal size. According to this structural model, the [Ala-163]recA and [Asn-160]recA mutations may lie in a linker region joining these two domains. We speculate that the [Ala-163]recA and [Asn-160]recA mutations interfere with an ATP-dependent conformational change of the recA protein that perhaps involves a change in the relative orientation of the carboxyl-terminal and amino-terminal domains.
我们利用定点诱变技术，将recA多肽的组氨酸163替换为丙氨酸残基。新的[Ala-163]recA蛋白催化单链（ss）DNA依赖性ATP水解，其周转数与野生型recA蛋白相似。尽管[Ala-163]recA蛋白在ssDNA依赖性ATP水解方面表现出色，但在标准反应条件（pH 7.5）下，它无法促进ATP依赖性三链交换反应。然而，[Ala-163]recA蛋白在pH 6.0 - 7.0时确实表现出三链交换活性，并且低pH下链交换活性的诱导与突变蛋白的ATP依赖性异构化的激活直接相关。因此，[Ala-163]recA蛋白在功能上与我们之前描述的突变体[Asn-160]recA蛋白相似（布莱恩特，F.R.（1988年）《生物化学杂志》263卷，8716 - 8723页；门奇，K.A.，和布莱恩特，F.R.（1990年）《生物化学杂志》265卷，11560 - 11566页）。胰蛋白酶蛋白水解研究表明，[Ala-163]recA和[Asn-160]recA蛋白与野生型recA蛋白一样，被组织成大小几乎相等的羧基末端和氨基末端结构域。根据这个结构模型，[Ala-163]recA和[Asn-160]recA突变可能位于连接这两个结构域的连接区域。我们推测，[Ala-163]recA和[Asn-160]recA突变干扰了recA蛋白的ATP依赖性构象变化，这可能涉及羧基末端和氨基末端结构域相对取向的改变。
