MAMMALIAN DNA NUCLEOTIDE EXCISION-REPAIR RECONSTITUTED WITH PURIFIED PROTEIN-COMPONENTS

Nucleotide excision repair is the principal way by which human cells r emove UV damage from DNA. Human cell extracts were fractionated to loc ate active components, including xeroderma pigmentosum (XP) and ERCC f actors. The incision reaction was then reconstituted with the purified proteins RPA, XPA, TFIIH (containing XPB and XPD), XPC, UV-DDB, XPG, partially purified ERCC1/XPF complex, and a factor designated IF7. UV- DDB (related to XPE protein) stimulated repair but was not essential. ERCC1- and XPF-correcting activity copurified with an ERCC1-binding po lypeptide of 110 kDa that was absent in XP-F cell extract. Complete re pair synthesis was achieved by combining these factors with DNA polyme rase epsilon, RFC, PCNA, and DNA ligase I. The reconstituted core reac tion requires about 30 polypeptides.