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J Biol Chem, Vol. 273, Issue 51, 34180-34189, December 18, 1998
From the The nucleotide excision repair (NER) pathway of
eukaryotes involves ~30 polypeptides. Reconstitution of this pathway
with purified components is consistent with the sequential assembly of
NER proteins at the DNA lesion. However, recent studies have suggested
that NER proteins may be pre-assembled in a high molecular weight
complex in the absence of DNA damage. To examine this model further, we
have constructed a histidine-tagged version of the yeast DNA damage
recognition protein Rad14. Affinity purification of this protein from
yeast nuclear extracts resulted in the co-purification of Rad1, Rad7,
Rad10, Rad16, Rad23, RPA, RPB1, and TFIIH proteins, whereas none of
these proteins bound to the affinity resin in the absence of
recombinant Rad14. Furthermore, many of the co-purifying proteins were
present in approximately equimolar amounts. Co-elution of these
proteins was also observed when the nuclear extract was fractionated by
gel filtration, indicating that the NER proteins were associated in a
complex with a molecular mass of >1000 kDa prior to affinity
chromatography. The affinity purified NER complex catalyzed the
incision of UV-irradiated DNA in an ATP-dependent reaction.
We conclude that active high molecular weight complexes of NER proteins
exist in undamaged yeast cells.
Affinity Purification and Partial Characterization of a Yeast
Multiprotein Complex for Nucleotide Excision Repair Using
Histidine-tagged Rad14 Protein
,,,
Department of Molecular Medicine, Institute
of Biotechnology, The University of Texas Health Science Center,
San Antonio, Texas 78245, the § Laboratory of Molecular
Pathology, Department of Pathology, The University of Texas
Southwestern Medical Center, Dallas, Texas 75225, and the
¶ Graduate Center for Toxicology, University of Kentucky,
Lexington, Kentucky 40536-0305
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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