Chen J, Kanaar R, Cozzarelli N R
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Genes Dev. 1994 Jun 1;8(11):1356-66. doi: 10.1101/gad.8.11.1356.
Strand exchange protein 1 (Sep1) from the yeast Saccharomyces cerevisiae promotes the transfer of one strand of a linear duplex DNA to a homologous single-stranded DNA circle. Using a nitrocellulose filter binding assay and electron microscopy, we find that Sep1 promotes the pairing of homologous DNA molecules via a paranemic joint. In this joint there is no net intertwining of the parental DNA molecules, as in the standard plectonemic double helix. The paranemic joints form with as little as 41 bp of homology between the parental DNA molecules. The substrates used were a circular molecule (either single-stranded DNA or duplex supercoiled DNA) and a linear duplex with heterologous regions at both ends to bar duplex plectonemic intertwining. We excluded the possibility that the exonuclease activity of Sep1 exposes complementary single-stranded regions that constitute the joint. The paranemic joint is the key intermediate in the search for homologous DNA by the RecA protein of Escherichia coli. Our results imply that the search process in a eukaryote such as yeast can be mechanistically similar.
来自酿酒酵母的链交换蛋白1(Sep1)可促进线性双链DNA的一条链转移至同源单链DNA环上。通过硝酸纤维素滤膜结合试验和电子显微镜观察,我们发现Sep1通过平行配对接头促进同源DNA分子的配对。在这种接头中,亲本DNA分子不像标准的双螺旋那样有净缠绕。亲本DNA分子之间同源性低至41bp时就可形成平行配对接头。所用的底物是一个环状分子(单链DNA或双链超螺旋DNA)和一个两端带有异源区域以阻止双链螺旋缠绕的线性双链。我们排除了Sep1的核酸外切酶活性暴露构成接头的互补单链区域的可能性。平行配对接头是大肠杆菌RecA蛋白寻找同源DNA的关键中间体。我们的结果表明,在诸如酵母这样的真核生物中,搜索过程在机制上可能是相似的。