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Vol. 14, No. 3, pp. 349-359, February 1, 2000
1 Imperial Cancer Research Fund (ICRF), Clare Hall
Laboratories, South Mimms, Hertfordshire EN6 3LD, UK;
2 Institut de Génétique et de Biologie
Moléculaire et Cellulaire (IGBMC), BP163 67404 Illkirch Cedex,
C.U. de Strasbourg, France; 3 Biocenter Oulu and Department of
Biochemistry, University of Oulu, FIN-90570 Oulu and Department of
Biology, University of Joensuu, FIN-80100 Joensuu, Finland;
4 Department of Veterinary Biochemistry, University of
Zurich-Irchel, CH-8057 Zürich, Switzerland
During human nucleotide excision repair, damage is recognized, two
incisions are made flanking a DNA lesion, and residues are replaced by
repair synthesis. A set of proteins required for repair of most lesions
is RPA, XPA, TFIIH, XPC-hHR23B, XPG, and ERCC1-XPF, but additional
components have not been excluded. The most complex and difficult to
analyze factor is TFIIH, which has a 6-subunit core (XPB, XPD, p44,
p34, p52, p62) and a 3-subunit kinase (CAK). TFIIH has roles both in
basal transcription initiation and in DNA repair, and several inherited
human disorders are associated with mutations in TFIIH subunits. To
identify the forms of TFIIH that can function in repair, recombinant
XPA, RPA, XPC-hHR23B, XPG, and ERCC1-XPF were combined with TFIIH
fractions purified from HeLa cells. Repair activity coeluted with the
peak of TFIIH and with transcription activity. TFIIH from cells with
XPB or XPD mutations was defective in supporting repair, whereas TFIIH from spinal muscular atrophy cells with a deletion of one p44 gene was
active. Recombinant TFIIH also functioned in repair, both a 6- and a
9-subunit form containing CAK. The CAK kinase inhibitor H-8 improved
repair efficiency, indicating that CAK can negatively regulate NER by
phosphorylation. The 15 recombinant polypeptides define the minimal set
of proteins required for dual incision of DNA containing a cisplatin
adduct. Complete repair was achieved by including highly purified human
DNA polymerase 
or 
, PCNA, RFC, and DNA ligase I in reaction
mixtures, reconstituting adduct repair for the first time with
recombinant incision factors and human replication proteins.
[Key Words: Transcription; DNA repair; cisplatin; kinase; DNA polymerase; xeroderma pigmentosum]
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