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Molecular and Cellular Biology, April 2001, p. 2281-2291, Vol. 21, No. 7
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.
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
*
Corresponding author. Present address: University of
Pittsburgh Cancer Institute, S867 Scaife Hall, 3550 Terrace St.,
Pittsburgh, PA 15261. Phone: (412) 648-9248.
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