Hel308 is a superfamily 2 helicase conserved in eukaryotes and archaea. It is thought to function in the early stages of recombination following replication fork arrest and has a specificity for removal of the lagging strand in model replication forks. A homologous helicase constitutes the N-terminal domain of human DNA polymerase Q. The Drosophila homologue mus301 is implicated in double strand break repair and meiotic recombination. We have solved the high resolution crystal structure of Hel308 from the crenarchaeon Sulfolobus solfataricus, revealing a five-domain structure with a central pore lined with essential DNA binding residues. The fifth domain is shown to act as an autoinhibitory domain or molecular brake, clamping the single-stranded DNA extruded through the central pore of the helicase structure to limit the helicase activity of the enzyme. This provides an elegant mechanism to tune the processivity of the enzyme to its functional role. Hel308 can displace streptavidin from a biotinylated DNA molecule, and this activity is only partially inhibited when the DNA is pre-bound with abundant DNA-binding proteins RPA or Alba1, whereas prebinding with the recombinase RadA has no effect on activity. These data suggest that one function of the enzyme may be in the removal of bound proteins at stalled replication forks and recombination intermediates.DNA helicases unwind duplex DNA and are essential components of the DNA replication, recombination, and repair machinery in all cellular organisms and many viruses. DNA helicases utilize the energy released by ATP hydrolysis to undergo conformational cycling and translocate along singlestranded DNA (ssDNA), 4 displacing a duplex DNA strand in the process. Many helicases belong to one of three superfamilies (SF1, 2, and 3), classified according to the conservation of specific sequence motifs (1). SF1 and SF2 helicases possess two motor domains with RecA-like folds that couple ATP hydrolysis to DNA translocation (2). SF2 DNA helicases include RecG in bacteria, hepatitis C virus NS3, and the RecQ family helicases, which all translocate along ssDNA with a 3Ј to 5Ј polarity (3). The RecQ helicases play a key role in maintaining genomic integrity by stabilizing stalled replication forks and removing intermediates of DNA recombination (4). Previous studies have shown that RecQ proteins target specialized DNA structures, specifically branched substrates that mimic replication forks and Holliday junctions. In humans, RecQ family helicases include the BLM and WRN proteins, mutated in certain rare inherited diseases in humans (5). The Hel308 family SF2 helicases, like RecQ, are implicated in DNA repair, recombination, and genome stability. The founding member, Mus308 from Drosophila melanogaster, was identified in a screen for mutations conferring hypersensitivity to DNA cross-linking reagents (6). Mus308 consists of an N-terminal SF2 helicase fused to a C-terminal DNA polymerase. The human ortholog, PolQ, has the same arrangement (7), and the polymerase domain has been sho...

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