Mammalian DNA nucleotide excision repair reconstituted with purified protein components

• Published in: Cell Vol. 80; no. 6; pp. 859 - 868
• Main Authors: Aboussekhra, Abdelilah, Biggerstaff, Maureen, Shivji, Mahmud K.K, Vilpo, Juhani A, Moncollin, Vincent, Podust, Vladimir N, Protić, Miroslava, Hübscher, Ulrich, Egly, Jean-Marc, Wood, Richard D
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 24.03.1995; Elsevier
• Subjects: Animals; Biochemistry, Molecular Biology; DNA Damage; DNA Ligase ATP; DNA Ligases - metabolism; DNA Polymerase II; DNA Repair; DNA Replication; DNA-Binding Proteins - isolation & purification; DNA-Binding Proteins - metabolism; DNA-Directed DNA Polymerase - metabolism; Endonucleases; HeLa Cells; Humans; Life Sciences; Mammals; Plasmids; Proliferating Cell Nuclear Antigen - metabolism; Proteins - isolation & purification; Proteins - metabolism; Ultraviolet Rays; Xeroderma Pigmentosum Group A Protein
• ISSN: 0092-8674; 1097-4172
• DOI: 10.1016/0092-8674(95)90289-9

Nucleotide excision repair is the principal way by which human cells remove UV damage from DNA. Human cell extracts were fractionated to locate active components, including xeroderma pigmentosum (XP) and ERCC factors. 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 polypeptide of 110 kDa that was absent in XP-F cell extract. Complete repair synthesis was achieved by combining these factors with DNA polymerase ε, RFC, PCNA, and DNA ligase I. The reconstituted core reaction requires about 30 polypeptides.