Nucleotide excision repair II: From yeast to mammals

An intricate network of repair systems safeguards the integrity of genetic material, by eliminating DNA lesions induced by numerous environmental and endogenous genotoxic agents. Nucleotide excision repair (NER) is one of the most versatile DNA repair systems. Deficiencies in this process give rise to the classical human DNA repair disorders xeroderma pigmentosum (XP) and Cockayne's syndrome (CS), and to a recently recognized disease called PIBIDS, a photosensitive form of the brittle hair disorder trichothiodystrophy. This is the second of a two-part review on NER. Part I (in the previous issue of TIG) concentrated on the main characteristics of the NER pathway of E. coli and yeast. Part II compares the mammalian and yeast systems, and attempts to integrate current knowledge on the eukaryotic pathway to suggest an outline for the reaction mechanism

DNA Repair: Nucleotide excision–repair in the test tube

AbstractThe eukaryotic nucleotide excision–repair pathway has been reconstituted in vitro, an achievement that should hasten the full enzymological characterization of this highly complex DNA-repair pathway

Nucleotide excision repair I: from E.coli to yeast.

Genetic information is constantly deteriorating, mainly as a consequence of the action of numerous genotoxic agents. In order to cope with this fundamental problem, all living organisms have acquired a complex network of DNA repair systems to safeguard their genetic integrity. Nucleotide excision repair (NER), one of the most important of these, is a complex multi-enzyme reaction that removes a remarkably wide range of lesions. This is the first of a series of two reviews on this repair process. Part I focuses on the main characteristics of the NER pathway in E. coli and yeast. Part II, to appear in the next issue of TIG, deals with NER in mammals and compares it with the process in yeast

After surviving cancer, what about late life effects of the cure?

The widely used chemotherapeutic cisplatin causes ototoxicity as late-term side effect. In this issue, Benkafadar et al () decipher the mechanism of cisplatin-induced ototoxicity and provide evidence that transient inhibition of p53 ameliorates ototoxicity without influencing chemotherapeutic efficacy. These findings may open exciting perspectives for reducing (late-term) side effects of cancer treatment

DNA structural elements required for ERCC1-XPF endonuclease activity

The heterodimeric complex ERCC1-XPF is a structure-specific endonuclease responsible for the 5' incision during mammalian nucleotide excision repair (NER). Additionally, ERCC1-XPF is thought to function in the repair of interstrand DNA cross-links and, by analogy to the homologous Rad1-Rad10 complex in Saccharomyces cerevisiae, in recombination between direct repeated DNA sequences. To gain insight into the role of ERCC1-XPF in such recombinational processes and in the NER reaction, we studied in detail the DNA structural elements required for ERCC1-XPF endonucleolytic activity. Recombinant ERCC1-XPF, purified from insect cells, was found to cleave stem-loop substrates at the DNA junction in the absence of other proteins like replication protein A, showing that the structure-specific endonuclease activity is intrinsic to the complex. Cleavage depended on the presence of divalent cations and was optimal in low Mn2+ concentrations (0.2 mM). A minimum of 4-8 unpaired nucleotides was required for incisions by ERCC1-XPF. Splayed arm and flap substrates were also cut by ERCC1-XPF, resulting in the removal of 3' protruding single-stranded arms. All incisions occurred in one strand of duplex DNA at the 5' side of a junction with single-stranded DNA. The exact cleavage position varied from 2 to 8 nucleotides away from the junction. One single-stranded arm, protruding either in the 3' or 5' direction, was necessary and sufficient for correct positioning of incisions by ERCC1-XPF. Our data specify the engagement of ERCC1-XPF in NER and allow a more direct search for its specific role in recombination

The isolation of plasmids containing DNA complementary to messenger RNA for variant surface glycoproteins of Trypanosoma brucei.

We have isolated poly(A)+ RNA from four antigenic variants (117, 118, 121, 221) of one clone of Trypanosoma brucei. Translation of these poly(A)+ RNAs in a rabbit reticulocyte lysate gave rise to proteins that could be precipitated with antisera against homologous variant surface glycoprotein, the protein responsible for antigenic variation in trypanosomes. From the electrophoretic mobility of these in vitro products in sodium dodecyl sulphate (SDS) gels we infer that variant surface glycoproteins (VSGs) are made as pre-proteins, which require trimming to yield mature VSGs. The total translation products from the four poly(A)+ RNAs produced a complex set of bands on SDS gels, which only differed in the region where the variant pre-glycoproteins migrated. The only detectable variation in the messenger RNA populations of these variants is, therefore, in the messenger RNA for variant pre-glycoproteins. We have made duplex DNA copies of these poly(A)+ RNAs, linked the complementary DNA to plasmid pBR322 by GC tailing and cloned this recombinant DNA in Escherichia coli. Colony hybridization with complementary DNA made on poly(A)+ RNA showed that 7--10% of the colonies contained DNA that hybridized only with the homologous probe. Plasmid DNA was isolated from ten such colonies (two or three of each variant complementary DNA), bound to diazobenzyloxymethyl-cellulose (DBM) paper and used to select complementary messenger RNA from total poly(A)+ RNA by hybridization. In eight cases the RNA recovered from the filter gave variant pre-glycoprotein as the predominant product of in vitro translation. Poly(A)+ RNA from each of the variants only hydridized to the homologous complementary DNA in filter hybridizations. Each trypanosome variant, therefore, contains no detectable messenger RNAs for the three heterologous variant-specific glycoproteins tested. We conclude from this lack of cross-hybridization that antigenic diversity in trypanosomes, unlike antibody diversity in mammals, does not involve the linkage of a repertoire of genes for the variable N-terminal half to a single gene for the C-termina

Unscheduled DNA synthesis in xeroderma pigmentosum cells after microinjection of yeast photoreactivating enzyme.

Photoreactivating enzyme (PRE) from yeast causes a light-dependent reduction of UV-induced unscheduled DNA synthesis (UDS) when injected into the cytoplasm of repair-proficieint human fibroblasts (Zwetsloot et al., 1985). This result indicates that the exogenous PRE monomerizers UV-induced dimers in these cells competing with the endogenous excision repair. In this paper we present the results of the injection of yeast PRE on (residual) UDS in fibroblasts from different excision-deficient XP-strains representing complementation groups A, C, D, E, F, H and I (all displaying more than 10% of the UDS of wild-type

Induction of a mutant phenotype in human repair proficient cells after overexpression of a mutated human DNA repair gene.

Antisense and mutated cDNA of the human excision repair gene ERCC-1 were overexpressed in repair efficient HeLa cells by means of an Epstein-Barr-virus derived CDNA expression vector. Whereas antisense RNA did not influence the survival of the transfected cells, a mutated cDNA generating an ERCC-1 protein with two extra amino acids in a conserved region of its C-terminal part resulted in a significant sensitization of the HeLa transfectants to mitomycin C-induced damage. These results suggest that overexpression of the mutated ERCC-1 protein interferes with proper functioning of the excision repair pathway in repair proficient cells and is compatible with a model in which the mutated ERCC-1 protein competes with the wildtype polypeptide for a specific step in the repair process or for occupation of a site in a repair complex. Apparently, this effect is more pronounced for mitomycin C induced crosslink repair than for UV-induced DNA damage