Taylor A F, Smith G R
Fred Hutchinson Cancer Research Center, Seattle, Washington 98104, USA.
J Biol Chem. 1995 Oct 13;270(41):24451-8. doi: 10.1074/jbc.270.41.24451.
RecBCD enzyme is a multifunctional nuclease that is essential for the major pathway of homologous genetic recombination in Escherichia coli. It has a potent helicase activity that uses ATP hydrolysis to unwind very long stretches of DNA. The functional form of RecBCD enzyme has been unclear, since M(r) of 250,000-655,000 have been previously reported. We have isolated two oligomeric forms of the enzyme, one (monomeric) containing a single copy of the RecB, RecC, and RecD polypeptides, and the other (dimeric) containing two copies of each polypeptide. We show here that the monomeric form of the enzyme (M(r) approximately 330,000) can form a stable initiation complex on the end of ds DNA. Depending on the nature of the ds end, KD estimates ranged from approximately 0.1 nM to approximately 0.7 nM in the presence of Mg2+ ions, which enhanced but was not required for binding. We further showed that the complex of monomeric RecBCD enzyme and a ds DNA end was competent to unwind DNA. A general model for the action of helicases has been proposed that uses repeated conformational changes between two states of a complex between DNA and a dimeric form of the enzyme. Our results make such a model unlikely for RecBCD enzyme.
RecBCD酶是一种多功能核酸酶，对大肠杆菌中同源基因重组的主要途径至关重要。它具有强大的解旋酶活性，利用ATP水解来解开很长的DNA片段。RecBCD酶的功能形式一直不清楚，因为之前报道的分子量在250,000 - 655,000之间。我们分离出了该酶的两种寡聚形式，一种（单体）包含RecB、RecC和RecD多肽的单拷贝，另一种（二聚体）包含每种多肽的两个拷贝。我们在此表明，该酶的单体形式（分子量约330,000）可在双链DNA末端形成稳定的起始复合物。根据双链末端的性质，在存在Mg2+离子的情况下，解离常数估计值在约0.1 nM至约0.7 nM之间，Mg2+离子增强了结合但不是结合所必需的。我们进一步表明，单体RecBCD酶与双链DNA末端的复合物能够解开DNA。已经提出了一种解旋酶作用的通用模型，该模型利用DNA与酶的二聚体形式之间复合物的两种状态之间的重复构象变化。我们的结果使得这种模型不太可能适用于RecBCD酶。
