A ubiquitin carrier protein from wheat germ is structurally and functionally similar to the yeast DNA repair enzyme encoded by RAD6.
The RAD6 gene from the yeast Saccharomyces cerevisiae encodes a ubiquitin carrier protein (E2) required for a variety of cellular processes including DNA repair, induced mutagenesis, and sporulation. Here we identify an E2 from a higher plant, wheat, that is similar to RAD6 with respect to both structure and in vitro substrate specificity. The protein was purified from wheat germ by a combination of ubiquitin covalent affinity chromatography and anion-exchange HPLC and has an apparent molecular mass of 23 kDa [referred to as E2(23 kDa)]. E2(23 kDa) was capable of binding ubiquitin by means of a thiol ester linkage in an ATP-dependent and ubiquitin-activating enzyme-dependent reaction. In the presence of a variety of target proteins, E2(23 kDa), like the RAD6 gene product, formed covalent ubiquitin-protein conjugates in vitro only with histones in a ubiquitin protein ligase-independent reaction. E2(23 kDa) recognized both core and linker histones with an apparent order of preference of H2A greater than or equal to H1 greater than H2B greater than H3 greater than H4. This E2 protein was approximately 17-fold more effective at conjugating ubiquitin to histones than three other purified wheat germ E2 proteins tested. Mouse anti-E2(23 kDa) antibodies were used to isolate E2(23 kDa) DNA sequences from a wheat cDNA expression library. Antibody-positive clones were confirmed by amino acid identity of the sequence deduced from the cDNA to the peptide sequence of an E2(23 kDa) tryptic fragment. Protein expressed in Escherichia coli by the E2(23 kDa) cDNA was capable of both thiol ester adduct formation and conjugation of ubiquitin to histones. Analysis of the E2(23 kDa) cDNA shows that it encodes a protein with considerable amino acid sequence similarity to the yeast RAD6 gene product. Similarities exist at the amino terminus, the region surrounding the putative ubiquitin binding site, and at the carboxyl terminus, which is unusually acidic. Based on both the structural and enzymatic similarities to the RAD6 gene product, E2(23 kDa) may represent the first DNA repair enzyme identified in higher plants.
Sullivan
M L
ML
Department of Horticulture, University of Wisconsin-Madison 53706.
Vierstra
R D
RD
D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D004260:DNA Repair; D005800:Genes, Fungal; D017343:Genes, Plant; D008025:Ligases / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010944:Plants / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D012689:Sequence Homology, Nucleic Acid; D014908:Triticum / Q000235:genetics / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes; D014452:Ubiquitins / Q000378:metabolism
D002352:Carrier Proteins; D029701:Saccharomyces cerevisiae Proteins; D014452:Ubiquitins; C046288:ubiquitin carrier proteins; C471606:RAD6 protein, S cerevisiae; D044763:Ubiquitin-Conjugating Enzymes; D008025:Ligases