Cassuto E, West S C, Podell J, Howard-Flanders P
Nucleic Acids Res. 1981 Aug 25;9(16):4201-10. doi: 10.1093/nar/9.16.4201.
The recA protein of Escherichia coli promotes pairing in vitro between covalent circular duplex DNA and homologous circular duplex DNA containing a single stranded region. We have used a filter binding assay to investigate the frequency of homologous pairing between gapped and intact duplex DNA when unwinding of the free 3' and 5' ends of the gapped molecules was blocked. In order to obtain DNA without free ends, the gapped DNA was treated with trimethylpsoralen and 360 nm light so as to introduce about 6 crosslinks per DNA molecule and the double stranded regions on either side of the gaps were then digested up to the first crosslinks with exonuclease III and lambda exonuclease. This treatment did not diminish the frequency of homologous pairing, an observation which is difficult to reconcile with models for recombination requiring strand unwinding before pairing.
大肠杆菌的RecA蛋白在体外可促进共价闭合双链DNA与含有单链区域的同源环状双链DNA之间的配对。我们采用滤膜结合试验来研究当缺口分子的游离3'端和5'端的解旋被阻断时，缺口双链DNA与完整双链DNA之间同源配对的频率。为了获得无游离端的DNA，用三甲基补骨脂素和360nm光处理缺口DNA，以便每个DNA分子引入约6个交联，然后用核酸外切酶III和λ核酸外切酶将缺口两侧的双链区域消化至第一个交联处。这种处理并没有降低同源配对的频率，这一观察结果难以与那些认为在配对前需要链解旋的重组模型相协调。
