VISUALIZATION OF SSB-SSDNA COMPLEXES ACTIVE IN THE ASSEMBLY OF STABLE RECA-DNA FILAMENTS
VISUALIZATION OF SSB-SSDNA COMPLEXES ACTIVE IN THE ASSEMBLY OF STABLE RECA-DNA FILAMENTS
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DOI：
10.1101/sqb.1984.049.01.062
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发表时间：
1984-01-01
期刊：
影响因子：
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通讯作者：
REGISTER, J
中科院分区：
文献类型：
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作者：
GRIFFITH, JD;HARRIS, LD;REGISTER, J
在大肠杆菌中的同源重组依赖于两个DNA结合蛋白，单链结合蛋白(SSB)和recA产物(RecA)。来自体外研究的证据表明，这两种蛋白质以相互依赖的方式发挥作用(McEntee等人。1980年；考克斯等人。1983a，b)，而lexC突变，SSB基因的缺陷，在体内显示出典型的recA基因突变的特性(Johnson 1977；Glassberg等人)。1979年；Vales等人。1980)。同源重组的简单模型反应取决于这两种蛋白质的存在，据推测，SSB是启动链交换所必需的，但一旦异源双链连接被建立和交换已经开始，SSB就不那么重要了(West等人。1982年)。已经证明，在SSB的存在下，RecA显示出依赖于单链DNA(SsDNA)的ATPase活性大大降低(McEntee等人。1980年；Cohen等人。1983年)。此外，在SSB的存在下，RecA与ssDNA形成一种比在没有SSB的情况下更稳定的复合体(Cox和Lehman 1982)。到目前为止，还没有证据表明SSB和RecA在溶液中直接相互作用。由于每一个都是单链DNA结合蛋白，它们可能只有在其中一个或两个结合了DNA后才能相互作用。用电子显微镜(EM)研究了RecA与单链DNA的结合(Dicapua等人)。1982年；Dunn et al.1982；Flory和Radding1982)以及其他方法(Mc-Entee等人)。1981年；柴田等人。1981年；Cazenave等人。1983年)。在低盐和没有镁的情况下，RecA以合作的方式结合，包被单链DNA并将其粗略地延伸到DNA双链形式的长度60~(Dunn等人)。1982年；弗洛里和拉丁1982年)。这个络合物是相对稳定的。然而，当反应在Mg+§和ATP存在的情况下进行时，由于ADP的积累，结合不那么稳定，从而破坏了RecA-ssDNA复合体。在SSB和RecA蛋白存在下形成的与ssDNA的复合体显示出ATPase水平降低(Cox和Lehman 1982；Cox等人。1983b)，但尚不清楚其更高的稳定性是由于这一点本身，还是由于RecA与单链DNA结合的构象发生了变化。
Homologous recombination in Escherichia coli is dependent on two DNA-binding proteins, the singlestrand-binding protein (SSB) and the recA product (RecA). Evidence from in vitro studies suggests that these two proteins function in an interdependent way (McEntee et al. 1980; Cox et al. 1983a, b), and the lexC mutation, a defect in the gene for SSB, shows properties in vivo, typical of mutants in the recA gene (Johnson 1977; Glassberg et al. 1979; Vales et al. 1980). Simple model reactions of homologous recombination depend on the presence of both proteins, and it has been deduced that SSB is required for the initiation of strand exchange but is less important once the heteroduplex joint has been established and exchange has begun (West et al. 1982). It has been demonstrated that, in the presence of SSB, RecA shows a greatly diminished single-stranded DNA (ssDNA)-dependent ATPase activity (McEntee et al. 1980; Cohen et al. 1983). Also, in the presence of SSB, RecA associates with ssDNA to form a complex that is much more stable than any formed in the absence of SSB (Cox and Lehman 1982).To date, no evidence for a direct interaction of SSB and RecA in solution has been reported. Since each is a ssDNA-binding protein, they may interact only after one or both has bound DNA. The binding of RecA to ssDNA has been investigated by electron microscopy (EM)(DiCapua et al. 1982; Dunn et al. 1982; Flory and Radding 1982) as well as by other methods (Mc-Entee et al. 1981; Shibata et al. 1981; Cazenave et al. 1983). In low salt and in the absence of Mg § 247 RecA binds in a cooperative manner, coating ssDNA and extending it to rougly 60~ the length of the duplex form of the DNA (Dunn et al. 1982; Flory and Radding 1982). This complex is relatively stable. When reactions are carried out in the presence of Mg+ § and ATP, however, the binding is less stable due to the accumulation of ADP, which disrupts the RecA-ssDNA complex. The complex with ssDNA that forms in the presence of both SSB and RecA proteins exhibits a depressed ATPase level (Cox and Lehman 1982; Cox et al. 1983b), but it is not known if its greater stability is due to this alone or to an altered conformation of RecA bound to ssDNA.