Phillips R J, Hickleton D C, Boehmer P E, Emmerson P T
Department of Biochemistry and Genetics, Medical School, University of Newcastle upon Tyne, UK.
Mol Gen Genet. 1997 Apr 16;254(3):319-29. doi: 10.1007/pl00008605.
To investigate the role that the individual subunits play in the ATP-dependent helicase activity of the RecBCD protein we have investigated the ability of the RecB, RecC and RecD proteins to displace various 20-mer oligonucleotides annealed to either end or to the centre of an oligonucleotide 60 bases long. The results show that the only subunit which can displace the 20-mers in the absence of the other subunits is the RecB protein. Moreover, the 20-mer is displaced only if it is annealed to the 60-mer at the 5' end or the middle, suggesting that the RecB protein translocates along the 60-mer in the 3' to 5' direction, displacing annealed 20-mers as it proceeds. We have shown that reconstituted RecBC and RecBCD complexes displace the 20-mers but, unlike RecB, they do not require a 3'-ended single-stranded region for helicase action, but can displace the 20-mers from either end of the 60-mer. The level of helicase activity of the RecBC complex is considerably greater than that of RecB alone, and the activity of the RecBCD complex appears to be greater still. This hierarchy of activity is also shown by DNA binding studies, but is not reflected in the ATPase activities of the enzymes. We have also shown that the ability of trypsin to cleave various sites on the RecB molecule is modified by the presence of ATP or ATP-gamma-S, suggesting that conformational changes may be induced in RecB upon ATP binding. We discuss a model for the ATP-driven, unidirectional motion of the RecB translocase along single-stranded DNA. In this model, the RecB molecule binds to single-stranded DNA and then translocates along it, one base at a time, in the 3' to 5' direction, by a 'ratchet' mechanism in which repeated stretching and contraction of the protein is coupled to ATP hydrolysis. The RecC protein in the RecBC complex is proposed to act as a 'sliding clamp' which increases processivity by preventing dissociation.
为了研究各个亚基在RecBCD蛋白的ATP依赖性解旋酶活性中所起的作用，我们研究了RecB、RecC和RecD蛋白取代与一条60个碱基长的寡核苷酸的一端、中间或两端退火的各种20聚体寡核苷酸的能力。结果表明，在没有其他亚基的情况下，唯一能够取代20聚体的亚基是RecB蛋白。此外，只有当20聚体在5'端或中间与60聚体退火时，它才会被取代，这表明RecB蛋白沿60聚体从3'到5'方向移位，在移位过程中取代退火的20聚体。我们已经表明，重组的RecBC和RecBCD复合物能够取代20聚体，但与RecB不同的是，它们的解旋酶作用不需要3'端单链区域，而是可以从60聚体的任一端取代20聚体。RecBC复合物的解旋酶活性水平远高于单独的RecB，而RecBCD复合物的活性似乎更高。这种活性层次在DNA结合研究中也有体现，但在这些酶的ATP酶活性中并未反映出来。我们还表明，胰蛋白酶切割RecB分子上各个位点的能力会因ATP或ATP-γ-S的存在而改变，这表明ATP结合可能会诱导RecB发生构象变化。我们讨论了一个关于RecB转位酶沿单链DNA进行ATP驱动的单向运动的模型。在这个模型中，RecB分子与单链DNA结合，然后通过一种“棘轮”机制沿其从3'到5'方向一次移动一个碱基，在这种机制中，蛋白质的反复拉伸和收缩与ATP水解相偶联。RecBC复合物中的RecC蛋白被认为起到“滑动夹”的作用，通过防止解离来增加持续性。
