Genomic characterization of the human DNA excision repair gene ERCC-1.

In this report the genomic characterization of the human excision repair gene ERCC-1 is presented. The gene consists of 10 exons spread over approximately 15 kb. By means of a transfection assay the ERCC-1 promoter was confined to a region of + 170 bp upstream of the transcriptional start site. Classical promoter elements like CAAT, TATA and GC-boxes are absent from this region. Furthermore, ERCC-1 transcription is not UV-inducible. A possible explanation is provided for the previously reported alternative splicing of exon VIII. Analysis of ERCC-1 cDNA clones revealed the occurrence of differential polyadenylation which gives ERCC-1 transcripts of 3.4 and 3.8 kb in addition to the major 1.1 kb mRNA. Apparent evolutionary conservation of differential polyadenylation of ERCC-1 transcripts suggests a possible role for this mode of RNA processing in the ERCC-1 repair function

Molecular characterization of the human excision repair gene ERCC-1: cDNA cloning and aminoacid homology with the yeast DNA repair gene RAD10.

The human excision repair gene ERCC-7 was cloned after DNA mediated gene transfer to the CHO mutant 43-38, which is sensitive to ultraviolet light and mitomycin-C. We describe the cloning and sequence analysis of the ERCC-7 cDNA and partial characterization of the gene. ERCC.1 has a size of 15 kb and is located on human chromosome 19. The ERCC.1 precursor RNA is subject to alternative splicing of an internal 72 bp coding exon. Only the cDNA of the larger 1.1 kb transcript, encoding a protein of 297 amino acids, was able to confer resistance to ultraviolet light and mitomycin-C on 43-38 cells. Significant amino acid sequence homology was found between the ERCC.7 gene product and the yeast excision repair protein RADIO. The most homologous region displayed structural homology with DNA binding domains of various polypeptides

Augmentation of protein production by a combination of the T7 RNA polymerase system and ubiquitin fusion: Overproduction of the human DNA repair protein, ERCC1, as a ubiquitin fusion protein in Escherichia coli.

This article presents the development of a set of new expression vectors for overproduction of proteins in Escherichia coli. The vectors, pETUBI-ES1, 2 and 3, allow in-frame cloning of any sequence with the ubiquitin gene driven by the strong T7f10 promoter. Combination of the T7 expression system with ubiquitin fusion appears to have a synergistic effect on protein overproduction. Large amounts of stable RNA are produced by T7 RNA polymerase, and fusion of ubiquitin to the N-terminus of target proteins seems to confer more efficient translation, better folding or protection against proteolytic degradation. The ubiquitin part can be utilized for purification of the fusion protein, after which it can be easily removed from the fusion product by ubiquitin-specific proteases. The advantage of combining both systems is demonstrated by the synthesis of large quantities (up to 40-50% of the total protein) of the human ERCC1 protein that hitherto was refractory to overproduction in various other E. coli and yeast expression systems

RAD25(SSL2), a yeast homolog of the human xeroderma pigmentosum group B DNA repair gene, is essential for viability.

Xeroderma pigmentosum (XP) patients are extremely sensitive to ultraviolet (UV) light and suffer from a high incidence of skin cancers, due to a defect in nucleotide excision repair. The disease is genetically heterogeneous, and seven complementation groups, A-G, have been identified. Homologs of human excision repair genes ERCC1, XPDC/ERCC2, and XPAC have been identified in the yeast Saccharomyces cerevisiae. Since no homolog of human XPBC/ERCC3 existed among the known yeast genes, we cloned the yeast homolog by using XPBC cDNA as a hybridization probe. The yeast homolog, RAD25 (SSL2), encodes a protein of 843 amino acids (M(r) 95,356). The RAD25 (SSL2)- and XPBC-encoded proteins share 55% identical and 72% conserved amino acid residues, and the two proteins resemble one another in containing the conserved DNA helicase sequence motifs. A nonsense mutation at codon 799 that deletes the 45 C-terminal amino acid residues in RAD25 (SSL2) confers UV sensitivity. This mutation shows epistasis with genes in the excision repair group, whereas a synergistic increase in UV sensitivity occurs when it is combined with mutations in genes in other DNA repair pathways, indicating that RAD25 (SSL2) functions in excision repair but not in other repair pathways. We also show that RAD25 (SSL2) is an essential gene. A mutation of the Lys392 residue to arginine in the conserved Walker type A nucleotide-binding motif is lethal, suggesting an essential role of the putative RAD25 (SSL2) ATPase/DNA helicase activity in viability

Localization of two human homologs, HHR6A and HHR6B, of the yeast DNA repair gene RAD6 to chromosome Xq24-25 and 5q23-31.

The chromosomal localizations of two closely related human DNA repair genes, HHR6A and HHR6B, were determined by in situ hybridization with biotinylated probes. HHR6A and HHR6B (human homolog of yeast RAD6) encode ubiquitin-conjugating enzymes (E2 enzymes), likely to be involved in postreplication repair and induced mutagenesis. The HHR6B gene was assigne

Postmeiotic transcription of X and Y chromosomal genes during spermatogenesis in the mouse.

During the meiotic prophase of spermatogenesis, the X and Y chromosomes form the heterochromatic sex body, showing little transcriptional activity. It has been suggested that transcription of the Xist gene is involved in this inactivation. After completion of the meiotic divisions, at least two Y chromosomal genes, Zfy and Sry, are transcribed in haploid spermatids. In contrast, postmeiotic transcription of X chromosomal genes has not been demonstrated. Using highly purified preparations of mouse pachytene spermatocytes, round spermatids, and

Expression of the ubiquitin-conjugating DNA repair enzymes HHR6A and B suggests a role in spermatogenesis and chromatin modification.

RAD6, a member of the expanding family of ubiquitin-conjugating (E2) enzymes, functions in the so-called "N-rule" protein breakdown pathway of Saccharomyces cerevisiae. In vitro, the protein can attach one or multiple ubiquitin (Ub) moieties to histones H2A and B and trigger their E3-dependent degradation. Rad6 mutants display a remarkably pleiotropic phenotype, implicating the protein in DNA damage-induced mutagenesis, postreplication repair, repression of retrotransposition, and sporulation. RAD6 transcription is strongly induced upon UV exposure and in meiosis, suggesting that it is part of a damage-induced response pathway and that it is involved in meiotic recombination. It is postulated that the protein exerts its functions by modulating chromatin structure. Previously, we have cloned two human homologs of this gene (designated HHR6A and HHR6B) and demonstrated that they partially complement the yeast defect. Here we present a detailed characterisation of their expression at the transcript and protein levels. Both HHR6 proteins, resolved by 2-dimensional immunoblot analysis, are expressed in all mammalian tissues and cell types examined, indicating that both genes are functional and constitutively expressed. Although the proteins are highly conserved, the UV induction present in yeast is not preserved, pointing to important differences in damage response between yeast and mammals. Absence of alterations in HHR6 transcripts or protein upon heat shock and during the cell cycle suggests that the proteins are not involved in stress response or cell cycle regulation. Elevated levels of HHR6 transcripts and proteins were found in testis. Enhanced HHR6 expression did not coincide with meiotic recombination but with the replacement of histones by transition proteins. Immunohistochemistry demonstrated that the HHR6 proteins are located in the nucleus, consistent with a functional link with chromatin. Electron microscopy combined with immunogold labeling revealed a preferential localisation of HHR6 in euchromatin areas, suggesting that the protein is associated with transcriptionally active regions. Our findings support the idea that both HHR6 genes have overlapping, constitutive functions related to chromatin confo