Displacement of a DNA binding protein by Dda helicase.
Displacement of a DNA binding protein by Dda helicase.
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DOI：
10.1093/nar/gkl369
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发表时间：
2006
影响因子：
14.9
通讯作者：
Raney KD
中科院分区：
文献类型：
--
作者：
Byrd AK;Raney KD
噬菌体T4Dda解旋酶最近被证明是一种活性单体，用于解离短双链寡核苷酸和取代3‘-生物素化寡核苷酸中的链霉亲和素。然而，它对链霉亲和素的置换和DNA解离的活性已经被证明随着与底物分子结合的dda分子的数量的增加而增加。设计了一种底物来解决DDA取代DNA结合蛋白的能力。将大肠杆菌Trp抑制子的DNA结合位点引入含有单链悬垂的DDA解旋酶的寡核苷酸底物中。在这里，我们证明了在单一周转条件下，尽管底物被展开，当单链悬垂足够长以容纳两个dda分子时，阻遏物发生位移，但dda单体不足以将E.coliTrp阻遏物从dsDNA中置换出来。当底物能够容纳两个以上的DDA分子时，形成的产物的数量增加。这些结果表明，多个DDA分子以一种与DNA解离活性和链霉亲和素置换活性相关的方式作用于DNA结合蛋白。我们提出了一个描述DDA解旋酶活性的合作尺寸虫模型。
Bacteriophage T4 Dda helicase has recently been shown to be active as a monomer for unwinding of short duplex oligonucleotides and for displacing streptavidin from 3′-biotinylated oligonucleotides. However, its activity for streptavidin displacement and DNA unwinding has been shown to increase as the number of Dda molecules bound to the substrate molecule increases. A substrate was designed to address the ability of Dda to displace DNA binding proteins. A DNA binding site for the Escherichia coli trp repressor was introduced into an oligonucleotide substrate for Dda helicase containing single-stranded overhang. Here we show that a Dda monomer is insufficient to displace the E.coli trp repressor from dsDNA under single turnover conditions, although the substrate is unwound and the repressor displaced when the single-stranded overhang is long enough to accommodate two Dda molecules. The quantity of product formed increases when the substrate is able to accommodate more than two Dda molecules. These results indicate that multiple Dda molecules act to displace DNA binding proteins in a manner that correlates with the DNA unwinding activity and streptavidin displacement activity. We suggest a cooperative inchworm model to describe the activities of Dda helicase.
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