STABLE DNA HETERODUPLEX FORMATION CATALYZED BY THE ESCHERICHIA-COLI RECA PROTEIN IN THE ABSENCE OF ATP HYDROLYSIS
STABLE DNA HETERODUPLEX FORMATION CATALYZED BY THE ESCHERICHIA-COLI RECA PROTEIN IN THE ABSENCE OF ATP HYDROLYSIS
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DOI：
10.1073/pnas.87.1.21
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发表时间：
1990-01-01
影响因子：
11.1
通讯作者：
KOWALCZYKOWSKI, SC
中科院分区：
文献类型：
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作者：
MENETSKI, JP;BEAR, DG;KOWALCZYKOWSKI, SC
关于RecA蛋白的功能还有一个有待回答的问题，那就是ATP在DNA链交换反应中的作用。在这篇文章中，我们描述了在没有ATP水解的情况下，以腺苷5‘-[γ-硫代]三磷酸(ATP[γS])作为核苷酸辅因子，形成联合分子。当加入双链DNA后，ATP[Gamma.S]-RecA蛋白-单链DNA突触前复合体可以形成同源配对的分子，并且脱蛋白后是稳定的。这些联合分子的形成既需要同源，也需要自由的同源末端，这表明它们本质上是血小板增多的。该反应对镁离子浓度非常敏感，在4-5 mM的醋酸镁时反应速度和幅度最大。在此条件下，联结分子中异源双链DNA的平均长度为2.4-3.4千碱基对。因此，RecA蛋白可以在ATP[γS]的存在下形成广泛的异源双链DNA区域，这表明在没有ATP水解的情况下，DNA分子的同源配对和交换可以发生。提出了一个包含这些结果的RecA蛋白催化的DNA链交换反应的模型，并将其与真核重组酶的机制相关联。
A question remaining to be answered about RecA protein function concerns the role of ATP hydrolysis during the DNA-strand-exchange reaction. In this paper we describe the formation of joint molecules in the absence of ATP hydrolysis, using adenosine 5''-[.gamma.-thio]triphosphate (ATP[.gamma.S]) as nucleotide cofactor. Upon the addition of double-stranded DNA, the ATP[.gamma.S]-RecA protein-single-stranded DNA presynaptic complexes can form homologously paired molecules that are stable after deproteinization. Formation of these joint molecules requires both homology and a free homologous end, suggesting that they are plectonemic in nature. This reaction is very sensitive to magnesium ion concentration, with a maximum rate and extent observed at 4-5 mM magnesium acetate. Under these conditions, the average length of heteroduplex DNA within the joint molecules is 2.4-3.4 kilobase pairs. Thus, RecA protein can form extensive regions of heteroduplex DNA in the presence of ATP[.gamma.S], suggesting that homologous pairing and the exchange of the DNA molecules can occur without ATP hydrolysis. A model for the RecA protein-catalyzed DNA-strand-exchange reaction that incorporates these results and its relevance to the mechanisms of eukaryotic recombinases are presented.