Affinity purification and partial characterization of a yeast multiprotein complex for nucleotide excision repair using histidine-tagged Rad14 protein

Karl Rodriguez, Jose Talamantez, Wen-Ya Huang, Simon H. Reed, Zhigang Wang, Ling Chen, William J. Feaver, Errol C. Friedberg, Alan E. Tomkinson

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish
Pages (from-to)34180-34189
Number of pages10
JournalJournal of Biological Chemistry
Volume273
Issue number51
DOIs
Publication statusPublished - 1998 Dec 18

Fingerprint

Multiprotein Complexes
Histidine
DNA Repair
Yeast
Purification
Repair
Nucleotides
Yeasts
Proteins
DNA
DNA Damage
Molecular Weight
Molecular weight
Affinity chromatography
Fungal Proteins
Molecular mass
Nuclear Proteins
Eukaryota
Affinity Chromatography
Gel Chromatography

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Rodriguez, Karl ; Talamantez, Jose ; Huang, Wen-Ya ; Reed, Simon H. ; Wang, Zhigang ; Chen, Ling ; Feaver, William J. ; Friedberg, Errol C. ; Tomkinson, Alan E. / Affinity purification and partial characterization of a yeast multiprotein complex for nucleotide excision repair using histidine-tagged Rad14 protein. In: Journal of Biological Chemistry. 1998 ; Vol. 273, No. 51. pp. 34180-34189.
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Affinity purification and partial characterization of a yeast multiprotein complex for nucleotide excision repair using histidine-tagged Rad14 protein. / Rodriguez, Karl; Talamantez, Jose; Huang, Wen-Ya; Reed, Simon H.; Wang, Zhigang; Chen, Ling; Feaver, William J.; Friedberg, Errol C.; Tomkinson, Alan E.

In: Journal of Biological Chemistry, Vol. 273, No. 51, 18.12.1998, p. 34180-34189.

Research output: Contribution to journalArticle

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AU - Rodriguez, Karl

AU - Talamantez, Jose

AU - Huang, Wen-Ya

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AU - Wang, Zhigang

AU - Chen, Ling

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AU - Friedberg, Errol C.

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