Nucleotide excision repair of DNA with recombinant human proteins: definition of the minimal set of factors, active forms of TFIIH, and modulation byCAK

During human nucleotide excision repair, damage is recognized, two incision s are made flanking a DNA lesion, and residues are replaced by repair synth esis. A set of proteins required for repair of most lesions is RPA, XPA, TF IIH, XPC-hHR23B, XPG, and ERCC1-XPF, but additional components have not bee n excluded. The most complex and difficult to analyze factor is TFIIH, whic h has a B-subunit core (XPB, XPD, p44, p34, p52, p62) and a 3-subunit kinas e (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 rep air, recombinant XPA, RPA, XPC-hHR23B, XPG, and ERCC1-XPF were combined wit h TFIIH fractions purified from HeLa cells. Repair activity coeluted with t he peak of TFIIH and with transcription activity. TFIIH from cells with XPB or XPD mutations was defective in supporting repair, whereas TFIIH from sp inal muscular atrophy cells:with a deletion of one p44 gene was active. Rec ombinant TFIIH also functioned in repair, both a 6- and a 9-subunit form co ntaining CAK. The CAK kinase inhibitor H-8 improved repair efficiency, indi cating that CAK can negatively regulate NER by phosphorylation. The 15 reco mbinant polypeptides define the minimal set of proteins required for dual i ncision of DNA containing a cisplatin adduct, Complete repair was achieved by including highly purified human DNA polymerase delta or epsilon, PCNA, R FC, and DNA ligase I in reaction mixtures, reconstituting adduct repair for the first time with recombinant incision factors and human replication pro teins.