THE RECB PROTEIN OF ESCHERICHIA-COLI TRANSLOCATES ALONG SINGLE-STRANDED-DNA IN THE 3'-DIRECTION TO 5'-DIRECTION - A PROPOSED RATCHET MECHANISM

To investigate the role that the individual subunits play in the ATP-d ependent 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 ol igonucleotide 60 bases long. The results show that the only subunit wh ich can displace the 20-mers in the absence of the other subunits is t he RecB protein. Moreover, the 20-mer is displaced only if it is annea led to the 60-mer at the 5' end or the middle, suggesting that the Rec B protein translocates along the 60-mer in the 3' to 5' direction, dis placing annealed 20-mers as it proceeds. We have shown that reconstitu ted RecBC and RecBCD complexes displace the 20-mers but, unlike RecB, they do not require a 3'-ended single-stranded region for helicase act ion, but can displace the 20-mers from either end of the 60-mer. The l evel of helicase activity of the RecBC complex is considerably greater than that of RecB alone, and the activity of the RecBCD complex appea rs to be greater still. This hierarchy of activity is also shown by DN A binding studies, but is not reflected in the ATPase activities of th e enzymes. We have also shown that the ability of trypsin to cleave va rious 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, unidire ctional motion of the RecB translocase along single-stranded DNA. In t his model, the RecB molecule binds to single-stranded DNA and then tra nslocates 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 RecB C complex is proposed to act as a 'sliding clamp' which increases proc essivity by preventing dissociation.