Binding specificity determines polarity of DNA unwinding by the Sgs1 protein of S-cerevisiae

Saccharomyces cerevisiae Sgs1 protein is a member of the RecQ DNA helicase family which also includes the products of the human Bloom's syndrome and W erner's syndrome genes. We have studied the substrate specificity of a reco mbinant Sgs1 helicase (amino acid residues 400-1268 of the Sgs1 protein). S gs1 shows a strong preference for binding branched DNA substrates, includin g duplex structures with a 3' single-stranded overhang and DNA junctions wi th multiple branches. Duplex DNA with a 5' rather than a 3' single-stranded tail is not recognized or unwound by Sgs1. DNase I and hydroxyl radical fo otprinting of the Sgs1-DNA complex shows that the protein binds specificall y to the junction of a double-stranded DNA and its 3' overhang. Binding and unwinding of duplex DNA with a 3' overhang are much reduced if the backbon e polarity of the 3' overhang is reversed in the junction region, but are u naffected if polarity reversal occurs four nucleotides away from the juncti on. These results indicate that the 3' to 5' polarity of unwinding by the r ecombinant Sgs1 protein is a direct consequence of the binding of the helic ase to the single-stranded/double-stranded DNA junction and its recognition of the polarity of the single-stranded DNA at the junction. The recombinan t Sgs1 also unwinds four-way junctions (synthetic Holliday junctions), a re sult that may be significant in terms of its role in suppressing DNA recomb ination in vivo. (C) 1999 Academic Press.