Characterization of genes and proteins involved in excision repair of human cells.
To extend our knowledge of the excision repair system in mammalian cells we have focussed on the isolation of genes and proteins involved in this process. For the purification and characterization of human repair proteins the microneedle injection assay technique is utilized. This system is based on the transient correction of the excision repair defect of xeroderma pigmentosum (XP) fibroblasts (scored as increase of ultraviolet (u.v.)-induced unscheduled DNA synthesis (UDS) upon microinjection of crude extracts from complementing XP or normal cells. Specific correction is observed in fibroblasts of all (9) excision-deficient XP complementation groups. The XP-A and G correcting factors were found to be proteins and several purification steps (including (NH4)2SO4 fractionation, chromatography of phosphocellulose, heparin and u.v.-irradiated DNA-cellulose) have been worked out for the XP-A correcting protein. The microinjection system was also used for the introduction of (partially) purified repair enzymes of lower organisms. Micrococcus luteus endonuclease and bacteriophage T4 endonuclease V were able to correct all XP complementation groups tested, in marked contrast to the more sophisticated Escherichia coli uvrABC complex injected with uvrD. Photoreversal of dimers could be registered after introduction of the yeast photoreactivating enzyme in repair-competent, XP-variant, XP-C and XP-I fibroblasts (monitored as decrease of (residual) UDS). Remarkably, no effect was noticed in XP-A, D, E and H, suggesting that something prevents dimers in these cells from being monomerized by the injected enzyme. Using DNA-mediated gene transfer we have cloned a human gene (designated ERCC-1) that compensates for the excision defect of the u.v. and mitomycin C-sensitive Chinese hamster ovary cell (CHO) mutant 43-3B (complementation group 2). Characterization of this gene and its cDNA revealed the following features: (1) ERCC-1 corrects the full spectrum of repair deficiencies in mutants of complementation group 2. No correction is observed in mutants of the other CHO complementation groups. (2) The ERCC-1 gene has a size of 15 X 10(3) base-pairs (bp) and consists of 10 exons, one of which appears to be differentially spliced. (3) It encodes two largely identical mRNAs, which differ in the presence or absence of a 72 bp coding exon, situated in the 3' half of the mRNA. Only the cDNA of the large transcript is able to confer repair proficiency to 43-3B cells. No effect of u.v. treatment is found at the level of ERCC-1 transcription in HeLa cells.(ABSTRACT TRUNCATED AT 400 WORDS)
Hoeijmakers
J H
JH
Department of Cell Biology and Genetics, Erasmus University, Rotterdam, The Netherlands.
D000595:Amino Acid Sequence; D001483:Base Sequence; D004247:DNA / Q000528:radiation effects; D004260:DNA Repair; D004720:Endonucleases; D005347:Fibroblasts; D005796:Genes; D006367:HeLa Cells; D006801:Humans; D008845:Microinjections; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D011506:Proteins; D014466:Ultraviolet Rays
D011506:Proteins; D004247:DNA; D004720:Endonucleases