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Molecular and Cellular Biology, April 2001, p. 2281-2291, Vol. 21, No. 7
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.7.2281-2291.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Strong Functional Interactions of TFIIH with XPC and XPG in Human DNA Nucleotide Excision Repair, without a Preassembled Repairosome

Sofia J. Araújo,1,2 Erich A. Nigg,3 and Richard D. Wood1,*

Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire EN6 3LD,1 and MRC Centre for Developmental Neurobiology, New Hunt's House, King's College London, Guy's Hospital Campus, London SE1 1UL,2 United Kingdom, and Department of Cell Biology, Max-Planck Institute for Biochemistry, D-82152 Martinsried, Germany3

Received 6 December 2000/Returned for modification 9 January 2001/Accepted 16 January 2001

In mammalian cells, the core factors involved in the damage recognition and incision steps of DNA nucleotide excision repair are XPA, TFIIH complex, XPC-HR23B, replication protein A (RPA), XPG, and ERCC1-XPF. Many interactions between these components have been detected, using different physical methods, in human cells and for the homologous factors in Saccharomyces cerevisiae. Several human nucleotide excision repair (NER) complexes, including a high-molecular-mass repairosome complex, have been proposed. However, there have been no measurements of activity of any mammalian NER protein complex isolated under native conditions. In order to assess relative strengths of interactions between NER factors, we captured TFIIH from cell extracts with an anti-cdk7 antibody, retaining TFIIH in active form attached to magnetic beads. Coimmunoprecipitation of other NER proteins was then monitored functionally in a reconstituted repair system with purified proteins. We found that all detectable TFIIH in gently prepared human cell extracts was present in the intact nine-subunit form. There was no evidence for a repair complex that contained all of the NER components. At low ionic strength TFIIH could associate with functional amounts of each NER factor except RPA. At physiological ionic strength, TFIIH associated with significant amounts of XPC-HR23B and XPG but not other repair factors. The strongest interaction was between TFIIH and XPC-HR23B, indicating a coupled role of these proteins in early steps of repair. A panel of antibodies was used to estimate that there are on the order of 105 molecules of each core NER factor per HeLa cell.

* Corresponding author. Present address: University of Pittsburgh Cancer Institute, S867 Scaife Hall, 3550 Terrace St., Pittsburgh, PA 15261. Phone: (412) 648-9248.

Molecular and Cellular Biology, April 2001, p. 2281-2291, Vol. 21, No. 7
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.7.2281-2291.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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