Binding specificity determines polarity of DNA unwinding by the Sgs1 protein of S-cerevisiae
Binding specificity determines polarity of DNA unwinding by the Sgs1 protein of S-cerevisiae
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DOI：
10.1006/jmbi.1999.2739
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发表时间：
1999-06-04
影响因子：
5.6
通讯作者：
Wang, JC
中科院分区：
文献类型：
--
作者：
Bennett, RJ;Keck, JL;Wang, JC
酿酒酵母Sgs 1蛋白是RecQ DNA解旋酶家族的成员，该家族还包括人类Bloom综合征和Werner综合征基因的产物。我们已经研究了重组SGS 1解旋酶（SGS 1蛋白的氨基酸残基400-1268）的底物特异性。Sgs 1显示出对结合分支DNA底物的强烈偏好，包括具有3'单链突出端的双链体结构和具有多个分支的DNA连接。具有5'而不是3'单链尾部的双链DNA不被Sgs 1识别或解旋。DNA酶I和Sgs 1-DNA复合物的羟基自由基足迹显示，该蛋白质特异性结合双链DNA的连接处及其3'突出端。如果3 '突出端的主链极性在接合区域中反转，则具有3'突出端的双链体DNA的结合和解旋大大减少，但如果在远离接合点的四个核苷酸处发生极性反转，则不受影响。这些结果表明，由重组Sgs 1蛋白解旋的3'至5'极性是解旋酶与单链/双链DNA接合处的结合及其对接合处单链DNA极性的识别的直接结果。重组Sgs 1也解开四路路口（合成霍利迪路口），结果可能是显着的抑制体内DNA重组的作用。(C)北京：科学出版社.
Saccharomyces cerevisiae Sgs1 protein is a member of the RecQ DNA helicase family which also includes the products of the human Bloom's syndrome and Werner's syndrome genes. We have studied the substrate specificity of a recombinant Sgs1 helicase (amino acid residues 400-1268 of the Sgs1 protein). Sgs1 shows a strong preference for binding branched DNA substrates, including duplex structures with a 3' single-stranded overhang and DNA junctions with multiple branches. Duplex DNA with a 5' rather than a 3' single-stranded tail is not recognized or unwound by Sgs1. DNase I and hydroxyl radical footprinting of the Sgs1-DNA complex shows that the protein binds specifically to the junction of a double-stranded DNA and its 3' overhang. Binding and unwinding of duplex DNA with a 3' overhang are much reduced if the backbone polarity of the 3' overhang is reversed in the junction region, but are unaffected if polarity reversal occurs four nucleotides away from the junction. These results indicate that the 3' to 5' polarity of unwinding by the recombinant Sgs1 protein is a direct consequence of the binding of the helicase to the single-stranded/double-stranded DNA junction and its recognition of the polarity of the single-stranded DNA at the junction. The recombinant Sgs1 also unwinds four-way junctions (synthetic Holliday junctions), a result that may be significant in terms of its role in suppressing DNA recombination in vivo. (C) 1999 Academic Press.