Cai L, Marquardt U, Zhang Z, Taisey M J, Chen J
Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, USA.
Nucleic Acids Res. 2001 Mar 15;29(6):1389-98. doi: 10.1093/nar/29.6.1389.
To initiate homologous recombination, sequence similarity between two DNA molecules must be searched for and homology recognized. How the search for and recognition of homology occurs remains unproven. We have examined the influences of DNA topology and the polarity of RecA-single-stranded (ss)DNA filaments on the formation of synaptic complexes promoted by RecA. Using two complementary methods and various ssDNA and duplex DNA molecules as substrates, we demonstrate that topological constraints on a small circular RecA-ssDNA filament prevent it from interwinding with its duplex DNA target at the homologous region. We were unable to detect homologous pairing between a circular RecA-ssDNA filament and its relaxed or supercoiled circular duplex DNA targets. However, the formation of synaptic complexes between an invading linear RecA-ssDNA filament and covalently closed circular duplex DNAs is promoted by supercoiling of the duplex DNA. The results imply that a triplex structure formed by non-Watson-Crick hydrogen bonding is unlikely to be an intermediate in homology searching promoted by RecA. Rather, a model in which RecA-mediated homology searching requires unwinding of the duplex DNA coupled with local strand exchange is the likely mechanism. Furthermore, we show that polarity of the invading RecA-ssDNA does not affect its ability to pair and interwind with its circular target duplex DNA.
为启动同源重组，必须搜索两个DNA分子之间的序列相似性并识别同源性。同源性的搜索和识别是如何发生的仍未得到证实。我们研究了DNA拓扑结构和RecA单链（ss）DNA细丝的极性对RecA促进的突触复合体形成的影响。使用两种互补方法以及各种ssDNA和双链DNA分子作为底物，我们证明了对小的环状RecA-ssDNA细丝的拓扑限制会阻止其在同源区域与其双链DNA靶标相互缠绕。我们无法检测到环状RecA-ssDNA细丝与其松弛或超螺旋环状双链DNA靶标之间的同源配对。然而，双链DNA的超螺旋促进了入侵的线性RecA-ssDNA细丝与共价闭合环状双链DNA之间突触复合体的形成。结果表明，由非沃森-克里克氢键形成的三链结构不太可能是RecA促进的同源性搜索中的中间体。相反，RecA介导的同源性搜索需要双链DNA解旋并伴有局部链交换的模型可能是其机制。此外，我们表明入侵的RecA-ssDNA的极性不会影响其与环状靶标双链DNA配对和相互缠绕的能力。
