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 epsilon, RFC, PCNA, and DNA ligase I. The reconstituted core reaction requires about 30 polypeptides.

Nucleotide excision repair, which is defective in xeroderma pigmentosum (XP), involves incision of a DNA strand on each side of a lesion. We isolated a human gene homologous to yeast Rad1 and found that it corrects the repair defects of XP group F as well as rodent groups 4 and 11. Causative mutations and strongly reduced levels of encoded protein were identified in XP-F patients. The XPF protein was purified from mammalian cells in a tight complex with ERCC1. This complex is a structure-specific endonuclease responsible for the 5' incision during repair. These results demonstrate that the XPF, ERCC4, and ERCC11 genes are equivalent, complete the isolation of the XP genes that form the core nucleotide excision repair system, and solve the catalytic function of the XPF-containing complex.

Interstrand DNA cross-link damage is a severe challenge to genomic integrity. Nucleotide excision repair plays some role in the repair of DNA cross-links caused by psoralens and other agents. However, in mammalian cells there is evidence that the ERCC1-XPF nuclease has a specialized additional function during interstrand DNA cross-link repair, beyond its role in nucleotide excision repair. We placed a psoralen monoadduct or interstrand cross-link in a duplex, 4 -6 bases from a junction with unpaired DNA. ERCC1-XPF endonucleolytically cleaved within the duplex on either side of the adduct, on the strand having an unpaired 3 tail. Crosslinks that were cleaved only on the 5 side were purified and reincubated with ERCC1-XPF. A second cleavage was then observed on the 3 side. Relevant partially unwound structures near a cross-link may be expected to arise frequently, for example at stalled DNA replication forks. The results show that the single enzyme ERCC1-XPF can release one arm of a cross-link and suggest a novel mechanism for interstrand cross-link repair.An important structure-specific DNA nuclease family in eukaryotes is represented by the ERCC1-XPF complex in mammalian cells and by the Rad1-Rad10 complex in Saccharomyces cerevisiae. These enzymes specifically cleave DNA near junctions between single-stranded and duplex DNA in cases where the single strand has a 5Ј33Ј polarity as it moves away from the junction (1-3). The ability of these enzymes to cut such bubble, flap, and Y structures is necessary for many DNA transactions. In mammalian cells, the heterodimeric ERCC1-XPF nuclease is formed by tight association of ERCC1 and XPF subunits (2, 4). Nucleotide excision repair (NER) 1 uses this nuclease to cleave a damaged DNA strand on the 5Ј side of an opened "bubble" intermediate formed around a lesion. Some strategies for repair of DNA double-stranded breaks in S. cerevisiae involve the use of Rad1-Rad10, sometimes in a complex with Msh2 and Msh3 (5). In these cases the nuclease aids in resolution of recombination intermediates by clipping off nonhomologous 3Ј single-stranded tails. In Drosophila, the XPF homolog mei9 is implicated in meiotic recombination (6). In the fission yeast Schizosaccharomyces pombe, the XPF homolog rad16 (swi9) and ERCC1 homolog swi10 are involved in the recombination events that lead to mating-type switching (7).NER-defective cells are sensitive to agents such as psoralen, which causes interstrand DNA cross-links (8). Beyond its participation in NER, however, ERCC1-XPF appears to have an additional role in the repair of interstrand DNA cross-links. It is known, for example, that ERCC1-defective and many XP group F cells are much more sensitive to DNA cross-linking agents than are other NER mutants (9, 10). Very little is known about the mechanism of repair of interstrand DNA cross-links. A number of pathways for cross-link repair can be envisaged. One possibility would mirror the model of Cole (11) for crosslink repair in Escherichia coli, which involves sequential NER and homologous...

Complementation group A of xeroderma pigmentosum (XP) represents one of the most prevalent and serious forms of this cancer‐prone disorder. Because of a marked defect in DNA excision repair, cells from individuals with XP‐A are hypersensitive to the toxic and mutagenic effects of ultraviolet light and many chemical agents. We report here the isolation of the XP‐A DNA repair protein by complementation of cell extracts from a repair‐defective human XP‐A cell line. XP‐A protein purified from calf thymus migrates on denaturing gel electrophoresis as a doublet of 40 and 42 kilodaltons. The XP‐A protein binds preferentially to ultraviolet light‐irradiated DNA, with a preference for damaged over nondamaged nucleotides of approximately 10(3). This strongly suggests that the XP‐A protein plays a direct role in the recognition of and incision at lesions in DNA. We further show that this protein corresponds to the product encoded by a recently isolated gene that can restore excision repair to XP‐A cells. Thus, excision repair of plasmid DNA by cell extracts sufficiently resembles genomic repair in cells to reveal accurately the repair defect in an inherited disease. The general approach described here can be extended to the identification and isolation of other human DNA repair proteins.

The mammalian ERCC1‐encoded polypeptide is required for nucleotide excision repair of damaged DNA and is homologous to Saccharomyces cerevisiae RAD10, which functions in repair and mitotic intrachromosomal recombination. Rodent cells representing repair complementation group 1 have nonfunctional ERCC1. We report that repair of UV‐irradiated DNA can be reconstituted by combining rodent group 1 cell extracts with correcting protein from HeLa cells. Background repair was minimized by employing fractionated rodent cell extracts supplemented with human replication proteins RPA and PCNA. Group 1‐correcting activity has a native molecular mass of 100 kDa and contains the 33 kDa ERCC1 polypeptide, as well as complementing activities for extracts from rodent group 4 and xeroderma pigmentosum group F (XP‐F) cells. Extracts of group 1, group 4 or XP‐F cells do not complement one another in vitro, although they complement extracts from other groups. The amount of ERCC1 detectable by immunoblotting is reduced in group 1, group 4 and XP‐F extracts. Recombinant ERCC1 from Escherichia coli only weakly corrected the group 1 defect. The data suggest that ERCC1 is part of a functional protein complex with group 4 and XP‐F correcting activities. The latter two may be equivalent to one another and analogous to S. cerevisiae RAD1.