Purification and characterization of Saccharomyces cerevisiae DNA damage-responsive protein 48 (DDRP 48).
A yeast protein was purified from wild type Saccharomyces cerevisiae (S. cerevisiae) to near homogeneity using an ethanolamine affinity chromatography procedure. The N-terminal amino acid sequencing and the amino acid composition analyses identified this protein as the product of the second open reading frame of S. cerevisiae DNA Damage-responsive gene 48 (DDR48) (Treger, J.M., and McEntee, K. (1990) Mol. Cell. Biol. 10, 3174-3184) The first methionine residue encoded by the translation starting codon was not present in the mature protein which is designated as DDRP 48. DDRP 48 was found to be a negatively charged and highly hydrophilic glycoprotein. The glycosidase cleavage analyses suggested that DDRP 48 was mainly N-link-glycosylated. The apparent molecular mass of DDRP 48 was estimated to be approximately 65 kilodaltons. DDRP 48 was found able to hydrolyze ATP and GTP yielding PPi. The Km values for ATP and GTP are 0.29 mM and 0.58 mM, respectively. The Western blot analysis demonstrated that DDRP 48 was expressed to various concentrations in different S. cerevisiae strains. Increased DDRP 48 abundance was observed after yeast cells carrying the wild type RAD 52 gene were exposed to either ethylmethane sulfonate or heat shock treatments. After similar DNA-damaging treatments, however, no significant inductions of DDRP 48 were found in a rad 52 mutant strain. These observations are consistent with the predictions resulting from previous studies on transcriptional regulation of the DDR 48 gene (Maga, J.A., McClanahan, T.A., and McEntee, K. (1986) Mol. & Gen. Genet. 205, 276-284; McClanahan, T., and McEntee, K. (1986) Mol. Cell. Biol. 6, 90-96).
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Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville 32610-0245.
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D000595:Amino Acid Sequence; D015153:Blotting, Western; D002846:Chromatography, Affinity; D004249:DNA Damage; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D005786:Gene Expression Regulation; D006031:Glycosylation; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid
C064200:DDR48 protein, S cerevisiae; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins