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

摘要: During human nucleotide excision repair, damage is recognized, two incisions are made flanking a DNA lesion, and residues replaced by repair synthesis. A set of proteins required for most lesions RPA, XPA, TFIIH, XPC-hHR23B, XPG, ERCC1-XPF, but additional components have not been excluded. The complex difficult to analyze factor which has 6-subunit core (XPB, XPD, p44, p34, p52, p62) 3-subunit kinase (CAK). TFIIH roles both in basal transcription initiation several inherited disorders associated with mutations subunits. To identify the forms that can function recombinant ERCC1-XPF were combined fractions purified from HeLa cells. Repair activity coeluted peak activity. cells XPB or XPD was defective supporting whereas spinal muscular atrophy deletion one p44 gene active. Recombinant also functioned 6- 9-subunit form containing CAK. CAK inhibitor H-8 improved efficiency, indicating negatively regulate NER phosphorylation. 15 polypeptides define minimal dual incision cisplatin adduct. Complete achieved including highly polymerase delta epsilon, PCNA, RFC, ligase I reaction mixtures, reconstituting adduct first time factors replication proteins.