Roman L J, Kowalczykowski S C
Biochemistry. 1986 Nov 18;25(23):7375-85. doi: 10.1021/bi00371a020.
We have shown that performing the recA protein catalyzed strand exchange reaction in the presence of acetate anions, rather than chloride which is commonly used, greatly increases the rate of the reaction. The initial rate of the reaction in an acetate-based buffer is approximately 3-4 times higher in the presence of Escherichia coli single-stranded DNA binding protein (SSB protein) and 2 times higher in its absence than the initial rate in chloride. To determine the enzymatic basis for this stimulatory effect of acetate buffer, we investigated the relationship between a number of physical and enzymatic properties of recA protein and the strand exchange reaction. We have found that although the acetate anion has some effect on the aggregation properties and the single-stranded DNA-dependent ATPase activity of recA protein, these effects cannot explain the enhanced strand exchange activity in an acetate-based buffer. We do find, however, that two aspects of recA protein activity closely parallel the ability of this protein to catalyze strand exchange. The first is the ability of recA protein to displace SSB protein from single-stranded DNA, an event critical to presynaptic complex formation. RecA protein is able to resist displacement by SSB protein at a lower magnesium concentration in acetate than in chloride buffer. The magnesium ion concentration dependence of strand exchange coincides exactly with this behavior. The second activity correlated to strand exchange is the duplex DNA-dependent ATPase activity of recA protein. We find that over a wide variety of sodium chloride and sodium acetate concentrations, this duplex DNA-dependent ATPase activity is linearly related to the amount of product formed in the strand exchange reaction. We postulate that this duplex DNA-dependent ATPase activity is important in the denaturation of the duplex DNA during the branch migration step of strand exchange and have also determined that this reaction is quite efficient, with the number of ATP molecules hydrolyzed per base pair exchanged being 0.75 +/- 0.25. In addition, recA protein catalyzed strand exchange between circular single-strand and linear duplex DNA molecules is shown to be irreversible, and a possible explanation for this irreversibility is presented.
我们已经表明，在醋酸根阴离子存在的情况下进行RecA蛋白催化的链交换反应，而不是常用的氯离子存在的情况下，会大大提高反应速率。在基于醋酸盐的缓冲液中，反应的初始速率在存在大肠杆菌单链DNA结合蛋白（SSB蛋白）时比在氯离子存在时高约3 - 4倍，在不存在SSB蛋白时比在氯离子存在时高2倍。为了确定醋酸盐缓冲液这种刺激作用的酶学基础，我们研究了RecA蛋白的一些物理和酶学性质与链交换反应之间的关系。我们发现，尽管醋酸根阴离子对RecA蛋白的聚集性质和单链DNA依赖性ATP酶活性有一些影响，但这些影响无法解释在基于醋酸盐的缓冲液中增强的链交换活性。然而，我们确实发现，RecA蛋白活性的两个方面与该蛋白催化链交换的能力密切平行。第一个方面是RecA蛋白从单链DNA上置换SSB蛋白的能力，这一事件对突触前复合物的形成至关重要。在醋酸盐缓冲液中，RecA蛋白能够在比氯离子缓冲液更低的镁离子浓度下抵抗被SSB蛋白置换。链交换对镁离子浓度的依赖性与这种行为完全一致。与链交换相关的第二个活性是RecA蛋白的双链DNA依赖性ATP酶活性。我们发现，在各种氯化钠和醋酸钠浓度范围内，这种双链DNA依赖性ATP酶活性与链交换反应中形成的产物量呈线性关系。我们推测这种双链DNA依赖性ATP酶活性在链交换的分支迁移步骤中双链DNA的变性过程中很重要，并且还确定该反应相当高效，每交换一个碱基对水解的ATP分子数为0.75±0.25。此外，RecA蛋白催化的环状单链和线性双链DNA分子之间的链交换被证明是不可逆的，并给出了这种不可逆性的一种可能解释。
