PURIFICATION AND CHARACTERIZATION OF THE SGS1 DNA HELICASE ACTIVITY OF SACCHAROMYCES-CEREVISIAE

The yeast Saccharomyces cerevisiae Sgs1 protein is a member of a famil y of DNA helicases that include the Escherichia coli RecQ protein and the products of human Bloom's syndrome and Werner's syndrome genes. To study the enzymatic characteristics of the protein, a recombinant Sgs 1 fragment (amino acids 400-1268 of the 1447-amino acid full-length pr otein) was overexpressed in yeast and purified to near homogeneity. Th e purified protein exhibits an ATPase activity in the presence of sing le-or double-stranded DNA. In the presence of ATP or dATP, unwinding o f duplex DNA or a DNA-RNA heteroduplex by the recombinant Sgs1 fragmen t was readily observed. Similar to the E. coli RecQ helicase, displace ment of the DNA strand occurs in the 3' to 5' direction with respect t o the single-stranded DNA flanking the duplex. The efficiency of unwin ding was found to correlate inversely with the length of the duplex re gion and was enhanced by the presence off. coli single-stranded DNA bi nding protein. In addition, the recombinant Sgs1 fragment was found to bind more tightly to a forked DNA substrate than to either single-or double-stranded DNA.