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 Werner's syndrome genes. We have studied the substrate specificity of a recombinant Sgs1 helicase (amino acid residues 400-1268 of the Sgs1 protein). Sgs1 shows a strong preference for binding branched DNA substrates, including duplex structures with a 3' single-stranded overhang and DNA junctions with 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 footprinting of the Sgs1-DNA complex shows that the protein binds specifically 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 backbone polarity of the 3' overhang is reversed in the junction region, but are unaffected if polarity reversal occurs four nucleotides away from the junction. These results indicate that the 3' to 5' polarity of unwinding by the recombinant Sgs1 protein is a direct consequence of the binding of the helicase to the single-stranded/double-stranded DNA junction and its recognition of the polarity of the single-stranded DNA at the junction. The recombinant Sgs1 also unwinds four-way junctions (synthetic Holliday junctions), a result that may be significant in terms of its role in suppressing DNA recombination in vivo.
Bennett
R J
RJ
Department of Molecular and Cellular Biology, Harvard University, 7 Divinity Avenue, Cambridge, MA, 02138, USA.
Keck
J L
JL
Wang
J C
JC
D001483:Base Sequence; D001816:Bloom Syndrome / Q000235:genetics; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D018983:DNA Footprinting; D004265:DNA Helicases / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D017665:Hydroxyl Radical; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D053484:RecQ Helicases; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity; D014898:Werner Syndrome / Q000235:genetics
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D017665:Hydroxyl Radical; D004247:DNA; C090472:SGS1 protein, S cerevisiae; D004265:DNA Helicases; D053484:RecQ Helicases