Dixon D A, Kowalczykowski S C
Department of Cell, Molecular, and Structural Biology, Northwestern University Medical School, Chicago, Illinois 60611.
Cell. 1991 Jul 26;66(2):361-71. doi: 10.1016/0092-8674(91)90625-9.
Genetic recombination in Escherichia coli is stimulated at DNA sequences known as Chi sites, 5'-GCT-GGTGG-3'. We describe the in vitro formation of homologously paired joint molecules that is dependent upon this recombination hotspot. Chi-dependent joint molecule formation requires RecA, RecBCD, and SSB proteins and a Chi site in the donor linear dsDNA. The donor dsDNA is unwound by RecBCD enzyme, and the invasive strand is generated by nicking at Chi. This Chi-dependent invading strand must contain homology to the recipient supercoiled DNA substrate at its newly formed 3' end for efficient joint molecule formation. Action at Chi generates invasive ssDNA from the 5' but not the 3' side of Chi, suggesting that the nuclease activity of RecBCD enzyme is attenuated upon encountering a Chi site. These results support the view that RecBCD enzyme action can precede RecA protein action and reconcile the seemingly opposing degradative and recombination functions of RecBCD enzyme.
在大肠杆菌中，遗传重组在被称为Chi位点（5'-GCT-GGTGG-3'）的DNA序列处受到刺激。我们描述了同源配对连接分子的体外形成，这种形成依赖于这个重组热点。依赖Chi的连接分子形成需要RecA、RecBCD和SSB蛋白以及供体线性双链DNA中的一个Chi位点。供体双链DNA被RecBCD酶解开，侵入链在Chi处通过切口产生。这种依赖Chi的侵入链在其新形成的3'端必须与受体超螺旋DNA底物具有同源性，才能有效地形成连接分子。在Chi处的作用从Chi的5'端而不是3'端产生侵入性单链DNA，这表明RecBCD酶的核酸酶活性在遇到Chi位点时会减弱。这些结果支持了RecBCD酶的作用可以先于RecA蛋白的作用这一观点，并调和了RecBCD酶看似相反的降解和重组功能。
