In the known monoclinic crystals the 3-dimensionalstructure of the hexameric, replicative helicaseRepA encoded by plasmid RSF1010 shows 6-foldrotational symmetry. In contrast, in the cubic crystalform at 2.55 A˚ resolution described here RepA has3-fold symmetry and consists of a trimer of dimers.To study structure–function relationships, a seriesof repA deletion mutants and mutations yieldingsingle amino acid exchanges were constructed andthe respective gene products were analyzed in vivoand in vitro. Hexamerization of RepA occurs via theN-terminus and is required for NTP hydrolysis. TheC-terminus is essential both for the interaction withthe replication machinery and for the helicaseactivity. Functional analyses of RepA variants withsingle amino acid exchanges confirmed most of thepredictions that were based on the published 3-dimensional structure. Of the five motifs conservedin family 4 helicases, all residues conserved inRepA and T7 gp4 helicases participate in DNAunwinding. Residues K42, E76, D77, D139 and H178,proposed to play key roles in catalyzing the hydroly-sis of NTPs, are essential for RepA activity. ResidueH178 of motif H3 couples nucleotide consumptionto DNA strand separation.INTRODUCTIONDNA helicases are ubiquitous motor proteins that utilize theenergy obtained by the hydrolysis of nucleoside triphosphates(NTPs) to unwind double-stranded nucleic acids. The proteinsplay key roles in a variety of biological processes like DNAreplication, recombination, repair and transcription. Theenzymes have received increasing attention since it becameknown that at least six hereditary diseases, like xerodermapigmentosum and Cockayne syndrome, are caused by variantsof DNA helicases or of putative helicases (1). There are twogroups of structurally known DNA helicases, one forminghexameric rings that operate at the DNA replication fork toseparate both strands of the duplex DNA ahead of the DNApolymerase complexes, whereas the other group includesmonomeric or dimeric enzymes.Plasmid RSF1010 encodes its own replication initiationsystem, making its replication independent of the hostinitiation machinery. Proteins RepC and RepB are requiredfor origin recognition and primer synthesis, respectively,whereas RepA is the hexameric replicative helicase essentialfor RSF1010 replication (2,3). RepA has 5¢fi3¢ polarity andrequires a forked DNA substrate for optimal activity. Amongribonucleoside triphosphates ATP is the preferred lowmolecular weight substrate. The pH optimum of the helicaseactivity is at pH 5.5–6, which corresponds with optimalbinding to single-stranded (ss)DNA (3). In contrast to otherhexameric helicases RepA assembles into stable hexamers inthe absence of any nucleotide or metal cofactor, as demon-strated by chemical cross-linking, gel filtration and electronmicroscopy (3).In monoclinic RepA crystals grown at pH 6.0, dimers ofhexamers in head-to-head orientation are observed (5). Bothimage reconstruction of electron microscopy data and the highresolution 3-dimensional crystal structures (2.4 and 1.95 A˚)revealed a 6-fold rotational symmetry (3,5,6). The fivehelicase motifs H1, H1a and H2–H4 conserved in DnaB-likeenzymes (7) are present in RepA and spatially clusteredaround the NTP binding pocket. The proposed catalytic sitefor ATP hydrolysis is located at the interface of neighboringmonomers, with the adenine base being sandwiched betweenR85 of the NTP binding monomer and Y242 of the adjacentsubunit.Besides RepA, the 3-dimensional structures of several otherhelicases have been determined, e.g. the monomeric homologsBacillus stearothermophilus PcrA and Escherichia coli Rephelicases, (8,9), the T7 helicase domain [amino acids 272–566(10) and 241–566 (11)] and the 130 amino acid RNA bindingdomain of the E.coli Rho RNA helicase (12). The latter twoenzymes are hexamers. A low resolution structure for thehexameric prototype E.coli DnaB has been determined byelectron microscopy and 3-dimensional image reconstruction(13), but high resolution data of hexameric replicative DnaB-type helicases are still not available except for a short stretchof an N-terminal domain (14).For translocation of helicases along DNA a variety ofmodels have been proposed (15; reviewed in 16). For themonomeric or dimeric helicases the ‘inchworm’ model, the