Reference "A ubiquitin carrier protein from wheat germ is structurally and functionally similar to the yeast DNA repair enzyme encoded by RAD6"

Reference ID

9212

Title

A ubiquitin carrier protein from wheat germ is structurally and functionally similar to the yeast DNA repair enzyme encoded by RAD6

Source

Proceedings of the National Academy of Sciences of the United States of America, 1989, vol. 86, pp. 9861-9865

Authors (2)

Sullivan-M-L

Vierstra-R-D

Abstract

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.