Affinity purification and partial characterization of a yeast multiprotein complex for nucleotide excision repair using histidine-tagged Rad14 protein.
The nucleotide excision repair (NER) pathway of eukaryotes involves approximately 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.
Rodriguez
K
K
Department of Molecular Medicine, Institute of Biotechnology, The University of Texas Health Science Center, San Antonio, Texas 78245, USA.
Talamantez
J
J
Huang
W
W
Reed
S H
SH
Wang
Z
Z
Chen
L
L
Feaver
W J
WJ
Friedberg
E C
EC
Tomkinson
A E
AE
D002846:Chromatography, Affinity; D004260:DNA Repair / Q000528:radiation effects; D045643:DNA Repair Enzymes; D004271:DNA, Fungal / Q000235:genetics / Q000528:radiation effects; D004278:DNA, Superhelical / Q000235:genetics / Q000528:radiation effects; D004307:Dose-Response Relationship, Radiation; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006639:Histidine; D010641:Phenotype; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism* / Q000528:radiation effects; D029701:Saccharomyces cerevisiae Proteins; D014466:Ultraviolet Rays
D004271:DNA, Fungal; D004278:DNA, Superhelical; D005656:Fungal Proteins; C072975:RAD14 protein, S cerevisiae; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D006639:Histidine; D045643:DNA Repair Enzymes