Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Bethesda, MD, USA.
California NanoSystems Institute, University of California, Los Angeles, Los Angeles, CA, USA.
Nat Struct Mol Biol. 2020 Feb;27(2):119-126. doi: 10.1038/s41594-019-0363-2. Epub 2020 Feb 3.
A single enzyme active site that catalyzes multiple reactions is a well-established biochemical theme, but how one nuclease site cleaves both DNA strands of a double helix has not been well understood. In analyzing site-specific DNA cleavage by the mammalian RAG1-RAG2 recombinase, which initiates V(D)J recombination, we find that the active site is reconfigured for the two consecutive reactions and the DNA double helix adopts drastically different structures. For initial nicking of the DNA, a locally unwound and unpaired DNA duplex forms a zipper via alternating interstrand base stacking, rather than melting as generally thought. The second strand cleavage and formation of a hairpin-DNA product requires a global scissor-like movement of protein and DNA, delivering the scissile phosphate into the rearranged active site.
一个酶活性位点能够催化多种反应是一个被广泛认可的生化主题，但人们对于一个核酸酶位点如何切割双链 DNA 仍知之甚少。在分析起始 V(D)J 重组的哺乳动物 RAG1-RAG2 重组酶的位点特异性 DNA 切割时，我们发现活性位点为两个连续反应进行了重新配置，并且 DNA 双螺旋采用了截然不同的结构。对于 DNA 的初始切口，局部展开和未配对的 DNA 双链通过交替的链间碱基堆积形成一个拉链，而不是像通常认为的那样熔解。第二个链的切割和发夹 DNA 产物的形成需要蛋白质和 DNA 的全局剪刀样运动，将切割的磷酸基团输送到重新排列的活性位点。
