Ganesan S, Smith G R
Fred Hutchinson Cancer Research Center, Seattle, WA 98104.
J Mol Biol. 1993 Jan 5;229(1):67-78. doi: 10.1006/jmbi.1993.1008.
RecBCD enzyme of Escherichia coli unwinds DNA from duplex DNA ends to produce single-stranded DNA, a central intermediate in the major (RecBCD) pathway of homologous recombination. To help elucidate the mechanism of unwinding we studied the complex of RecBCD enzyme bound to duplex DNA ends in the absence of ATP, a cofactor required for unwinding. In this complex the terminal 16 or 17 nucleotides of the 3' terminated strand and the terminal 20 or 21 nucleotides of the 5' terminated strand were protected from DNase I cleavage. u.v.-irradiation of the complex cross-linked the RecB subunit to the 3' terminated strand and the RecC and RecD subunits of the 5' terminated strand. Studies using pyridoxal 5-phosphate, an inhibitor of the RecBCD enzyme, corroborated the cross-linking studies and revealed a conformational change in the enzyme upon binding to DNA. Based on these results and proposals by others, we present a model for the mechanism of DNA unwinding by RecBCD enzyme.
大肠杆菌的RecBCD酶从双链DNA末端解开DNA以产生单链DNA，这是同源重组主要（RecBCD）途径中的核心中间体。为了帮助阐明解旋机制，我们研究了在没有ATP（解旋所需的辅助因子）的情况下与双链DNA末端结合的RecBCD酶复合物。在这个复合物中，3'端终止链的末端16或17个核苷酸以及5'端终止链的末端20或21个核苷酸免受DNase I切割。复合物的紫外线照射将RecB亚基与3'端终止链交联，以及5'端终止链的RecC和RecD亚基交联。使用磷酸吡哆醛（RecBCD酶的抑制剂）的研究证实了交联研究，并揭示了酶与DNA结合时的构象变化。基于这些结果和其他人的提议，我们提出了RecBCD酶解开DNA机制的模型。
