Helicases can be divided into two classes on the basis of mechanism, those that ranslocate in a 5'-3' direction along single-stranded DNA and those that operate with the opposite polarity. For the 3'-5' helicases, there has been a considerable amount of recent structural and biochemical data to enlighten our understanding of the mechanism of these enzymes. The first crystal structure of a helicase was that of PcrA. The structure revealed that the enzyme comprised several domains, including two domains that are structurally similar to the ATPase domain of the recombination protein RecA. Subsequent structures of PcrA helicase in substrate and product complexes with a DNA substrate have provided details on the molecular basis of helicase mechanism.

For the 5'-3' helicases, structural information has come mainly from cryo-electron microscopy studies. These have demonstrated the conformational flexibility of the ring helicases in that species with both six-fold and three-fold symmetries, as well as many other forms, may be observed. The functional relevance of these is unclear.

Ring helicases frequently contain domains in addition to those required for helicase activity. For example, the T7 gene 4 protein comprises separate helicase and primase domains. The high resolution crystal structure of a part (4E) of this helicase domain has been determined by Ellenberger & co-workers. The structure confirmed predictions that this region of the protein would have structural homology with the ATPase domain of RecA and also retains the ability to bind and hydrolyse nucleoside triphosphates. However, this protein has lost the ability to hexamerise, instead forming helical filaments. By examining the gene 4E filament down the screw axis, a model was proposed for hexamer formation which involved the N-terminal region of the protein fragment.

Using limited proteolytic digestion, we have identified a fragment of the T7 gene 4 protein that comprises the helicase domain which we refer to as 4D (residues 241 to 566). This fragment forms a stable hexamer and has helicase activity. Using molecular replacement with the 4E structure combined with heavy-atom phasing we have determined the structure of the 4D hexamer.