Purification and characterization of the SRS2 DNA helicase of the yeast Saccharomyces cerevisiae.
The SRS2 gene of Saccharomyces cerevisiae was identified through mutational analysis as a suppressor of radiation-sensitive mutations in the error-prone repair pathway and by a hyper-recombination phenotype. Comparison of the derived amino acid sequence revealed the gene to have high homology to the bacterial DNA helicases UvrD and Rep (Aboussekhra, A., Chanet, R., Zgaga, Z., Cassier-Chauvat, C., Heude, M., and Fabre, F. (1989) Nucleic Acids Res. 17, 7211-7219). We have purified the SRS2 protein from Escherichia coli extracts by tagging the SRS2 gene with 6 carboxyl-terminal histidine residues and overexpressing the tagged protein in a pET-3c vector. Extracts were passed over a metal-chelating affinity chromatography column followed by gel filtration to obtain an enriched protein fraction. Sephacryl gel filtration of pooled fractions containing the SRS2 protein yielded purified SRS2 protein by Coomassie Blue stain of SDS-polyacrylamide gel electrophoresis gels. The purified SRS2 protein was found to have in vitro DNA-dependent ATPase and DNA helicase activities. The polarity of the helicase activity was determined to be 3' to 5', the same polarity as that found for the UvrD and Rep proteins. The carboxyl-terminal region of the protein is shown to contain a sequence for nuclear localization. Expression of the SRS2 in yeast was examined and found to be extremely low.
Rong
L
L
Department of Biochemistry, New York University Medical Center, New York 10016.
Klein
H L
HL
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors; D001483:Base Sequence; D003001:Cloning, Molecular; D004265:DNA Helicases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005800:Genes, Fungal; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D000251:Adenosine Triphosphatases; D004265:DNA Helicases