Relationship of the physical and enzymatic properties of Escherichia coli recA protein to its strand exchange activity.
Relationship of the physical and enzymatic properties of Escherichia coli recA protein to its strand exchange activity.
复制标题
大肠杆菌recA 蛋白的物理和酶性质与其链交换活性的关系。
DOI：
10.1021/bi00371a020
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发表时间：
1986
期刊：
影响因子：
2.9
通讯作者：
Kowalczykowski,SC
中科院分区：
文献类型：
--
作者：
Roman,LJ;Kowalczykowski,SC
西北大学医学院分子生物学系，芝加哥，伊利诺伊州，60611收到1986年3月21日；修订稿件收到1986年7月21日摘要：我们已经证明在乙酸酯阴离子存在下进行recA蛋白催化的链交换反应，而不是通常使用的氯离子，极大地提高了反应速度。在以醋酸盐为基础的缓冲液中，有大肠杆菌单链DNA结合蛋白(SSB蛋白)存在时，反应的初始速度大约是在氯化物中的3-4倍，在没有SSB蛋白的情况下，反应的初始速度是在氯化物中的2倍。为了确定醋酸盐缓冲液的这种刺激作用的酶基础，我们研究了recA蛋白的一些物理和酶性质与链交换反应的关系。我们发现，尽管醋酸盐阴离子对recA蛋白的聚集特性和单链DNA依赖的ATPase活性有一定的影响，但这些影响不能解释在醋酸盐缓冲液中链交换活性的增强。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相关。第一个是recA蛋白从单链DNA中取代SSB蛋白的能力，这一事件对突触前复合体的形成至关重要。当醋酸盐中的镁浓度低于氯缓冲液中的镁浓度时，RecA蛋白能够抵抗SSB蛋白的置换。链交换的镁离子浓度依赖关系恰好符合这一行为。与链交换相关的第二个活性是recA蛋白的双链DNA依赖的ATPase活性。我们发现，在各种各样的氯化钠和醋酸钠浓度下，这种依赖于DNA的双链ATPase活性与链交换反应中形成的产物的数量呈线性关系。我们推测，在链交换的分支迁移步骤中，这种依赖于DNA的双链ATPase活性在双链DNA的变性过程中是重要的，并确定了这个反应是非常有效的，每个交换的碱基对的ATP分子水解数为0.75±0.25。此外，RecA蛋白催化的环状单链和线性双链DNA分子之间的链交换是不可逆的，并提出了这种不可逆性的可能解释。^]R.ECA蛋白是一种37.8千道尔顿(KDa)1蛋白，已被证明在遗传重组中发挥重要作用[有关综述，请参阅McEntee&Weinstock(1981)，Radding1982和Dressier&Potter(1982)]。RecA突变体在重组方面表现出高达6个数量级的缺陷，并对紫外线辐射表现出更高的敏感性。这种敏感性部分是由于recA蛋白在SOS诱导中所起的作用，在ATP和单链DNA存在的情况下，它催化LexA蛋白的蛋白水解性切割[有关综述，请参阅Little&mount(1982)]。
Department of Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611 Received March 21, 1986; Revised Manuscript Received July 21, 1986 abstract: 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 cannotexplain the enhanced strand exchange activity in an acetate-based buffer. We do find, however, that two aspects of recAprotein activity closely parallel the ability of this protein to catalyze strand exchange. The first is the ability of recAprotein 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 strandexchange reaction. We postulate that this duplex DNA-dependent ATPase activity is important inthe 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.^] R. ecA protein is a 37.8-kilodalton (kDa) 1 protein that has been shown to play a vital role in genetic recombination [for reviews, see McEntee & Weinstock (1981), Radding (1982), and Dressier & Potter (1982)]. RecA mutants are pheno-typically deficient in recombination by as much as 6 orders of magnitude and show an increased sensitivity to UV irra-diation. This sensitivity is due partly to the rolethat recA protein plays in SOS induction where it catalyzes the proteolytic cleavage of lexA protein in the presence of ATP and single-stranded DNA [for a review, see Little & Mount (1982)].