Antibody recognition of the tumor-specific bcr-abl joining region in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is characterized by the presence of a 210-kD protein (P210bcr-abl) in the cytoplasm of leukemic cells, generated by the reciprocal translocation between chromosome 9 and chromosome 22. Due to this translocation, the abl oncogene is coupled to the bcr gene, forming a new determinant in this protein encoded by the bcr-abl joining region. In the joining region itself, either the bcr exon 2 is coupled to the abl exon 2 (b2-a2), or the bcr exon 3 is coupled to the abl exon 2 (b3-a2). Thus, these joining regions form by definition new tumor-specific determinants in the respective chimeric P210-bcr-abl molecules. This paper addresses the question as to whether these tumor-specific joining regions are exposed on the P210bcr-abl molecule in such a way that antibodies can be generated to detect these sites. To test this possibility a polyclonal antiserum, termed BP-1, was raised against a synthetic peptide representative for the b2-a2 joining region. The reactivity of BP-1 was analyzed in an ELISA system on various synthetic peptides. Peptide inhibition studies showed the presence of antibodies to different parts of the b2-a2 peptide in the polyvalent antiserum. The reactivity of BP-1 was then tested with native P210bcr-abl molecules in various CML cell lines (K562, LAMA-84, and BV173) using a protein kinase assay. In this context, the bcr-abl junctions were first analyzed at the DNA and RNA level. The present study indicates that BP-1 specifically recognizes the b2-a2 junction in native P210bcr-abl. Furthermore, BP-1 clearly discriminates between b2-a2 P210bcr-abl and b3-a2 P210bcr-abl. We conclude that the tumor-specific b2-a2 joining region is antigenically exposed on the native P210bcr-abl molecule.

Recombination and repair. Ku starts at the end.

Genetic evidence suggests that the Ku DNA-end-binding protein complex is central to the recombination-based repair of double-strand breaks that protects DNA from radiation and underlies antibody gene rearrangement.

Human and mouse homologs of the Saccharomyces cerevisiae RAD54 DNA repair gene: evidence for functional conservation.

BACKGROUND: Homologous recombination is of eminent importance both in germ cells, to generate genetic diversity during meiosis, and in somatic cells, to safeguard DNA from genotoxic damage. The genetically well-defined RAD52 pathway is required for these processes in the yeast Saccharomyces cerevisiae. Genes similar to those in the RAD52 group have been identified in mammals. It is not known whether this conservation of primary sequence extends to conservation of function. RESULTS: Here we report the isolation of cDNAs encoding a human and a mouse homolog of RAD54. The human (hHR54) and mouse (mHR54) proteins were 48% identical to Rad54 and belonged to the SNF2/SW12 family, which is characterized by amino-acid motifs found in DNA-dependent ATPases. The hHR54 gene was mapped to chromosome 1p32, and the hHR54 protein was located in the nucleus. We found that the levels of hHR54 mRNA increased in late G1 phase, as has been found for RAD54 mRNA. The level of mHR54 mRNA was elevated in organs of germ cell and lymphoid development and increased mHR54 expression correlated with the meiotic phase of spermatogenesis. The hHR54 cDNA could partially complement the methyl methanesulfonate-sensitive phenotype of S. cerevisiae rad54 delta cells. CONCLUSIONS: The tissue-specific expression of mHR54 is consistent with a role for the gene in recombination. The complementation experiments show that the DNA repair function of Rad54 is conserved from yeast to humans. Our findings underscore the fundamental importance of DNA repair pathways: even though they are complex and involve multiple proteins, they seem to be functionally conserved throughout the eukaryotic kingdom.

Molecular cloning of the human DNA excision repair gene ERCC-6.

The UV-sensitive, nucleotide excision repair-deficient Chinese hamster mutant cell line UV61 was used to identify and clone a correcting human gene, ERCC-6. UV61, belonging to rodent complementation group 6, is only moderately UV sensitive in comparison with mutant lines in groups 1 to 5. It harbors a deficiency in the repair of UV-induced cyclobutane pyrimidine dimers but permits apparently normal repair of (6-4) photoproducts. Genomic (HeLa) DNA transfections of UV61 resulted, with a very low efficiency, in six primary and four secondary UV-resistant transformants having regained wild-type UV survival. Southern blot analysis revealed that five primary and only one secondary transformant retained human sequences. The latter line was used to clone the entire 115-kb human insert. Coinheritance analysis demonstrated that five of the other transformants harbored a 100-kb segment of the cloned human insert. Since it is extremely unlikely that six transformants all retain the same stretch of human DNA by coincidence, we conclude that the ERCC-6 gene resides within this region and probably covers most of it. The large size of the gene explains the extremely low transfection frequency and makes the gene one of the largest cloned by genomic DNA transfection. Four transformants did not retain the correcting ERCC-6 gene and presumably have reverted to the UV-resistant phenotype. One of these appeared to have amplified an endogenous, mutated CHO ERCC-6 allele, indicating that the UV61 mutation is leaky and can be overcome by gene amplification.

Cloning of human and mouse genes homologous to RAD52, a yeast gene involved in DNA repair and recombination.

The RAD52 gene of Saccharomyces cerevisiae is required for recombinational repair of double-strand breaks. Using degenerate oligonucleotides based on conserved amino acid sequences of RAD52 and rad22, its counterpart from Schizosaccharomyces pombe, RAD52 homologs from man and mouse were cloned by the polymerase chain reaction. DNA sequence analysis revealed an open reading frame of 418 amino acids for the human RAD52 homolog and of 420 amino acid residues for the mouse counterpart. The identity between the two proteins is 69% and the overall similarity 80%. The homology of the mammalian proteins with their counterparts from yeast is primarily concentrated in the N-terminal region. Low amounts of RAD52 RNA were observed in adult mouse tissues. A relatively high level of gene expression was observed in testis and thymus, suggesting that the mammalian RAD52 protein, like its homolog from yeast, plays a role in recombination. The mouse RAD52 gene is located near the tip of chromosome 6 in region G3. The human equivalent maps to region p13.3 of chromosome 12. Until now, this human chromosome has not been implicated in any of the rodent mutants with a defect in the repair of double-strand breaks.

Structure and expression of the excision repair gene ERCC6, involved in the human disorder Cockayne's syndrome group B.

The human repair gene ERCC6--a presumed DNA (or RNA) helicase--has recently been found to function specifically in preferential nucleotide excision repair (NER). This NER subpathway is primarily directed towards repair of (the transcribed strand of) active genes. Mutations in the ERCC6 gene are responsible for the human hereditary repair disorder Cockayne's syndrome complementation group B, the most common form of the disease. In this report, the genomic organization and expression of this gene are described. It consists of at least 21 exons, together with the promoter covering a region of 82-90 kb on the genome. Postulated functional domains deduced from the predicted amino acid sequence, including 7 distinct helicase signatures, are--with one exception--encoded on separate exons. Consensus splice donor and acceptor sequences are present at all exon borders with the exception of the unusual splice donor at the end of exon VII. The 'invariable' GT dinucleotide in the consensus (C,A)AG/GTPuAGT is replaced by the exceptional GC. Based on 42 GC splice donor sequences identified by an extensive literature search we found a statistically highly significant better 'overall' match of the surrounding nucleotides to the consensus sequence compared to normal GT-sites. This confirms and extends the observation made recently by Jackson (Nucl. Acids Res., 19, 3795-3798 (1991)) derived from analysis of 26 cases. Analysis of ERCC6 cDNA clones revealed the occurrence of alternative polyadenylation, resulting in the (differential) expression of two mRNA molecules (which are barely detectable on Northern blots) of 5 and 7 kb in length.

Nucleotide excision repair syndromes: molecular basis and clinical symptoms.

The phenotypic consequences of a nucleotide excision repair (NER) defect in man are apparent from three distinct inborn diseases characterized by hypersensitivity of the skin to ultraviolet light and a remarkable clinical and genetic heterogeneity. These are the prototype repair syndrome, xeroderma pigmentosum (XP) (seven genetic complementation groups, designated XP-A to XP-G), Cockayne's syndrome (two groups: CS-A and CS-B) and PIBIDS, a peculiar photosensitive form of the brittle hair disease trichothiodystrophy (TTD, at least two groups of which one equivalent to XP-D). To investigate the mechanism of NER and to resolve the molecular defect in these NER deficiency diseases we have focused on the cloning and characterization of human DNA repair genes. One of the genes that we cloned is ERCC3. It specifies a chromatin binding helicase. Transfection and microinjection experiments demonstrated that mutations in ERCC3 are responsible for XP complementation group B, a very rare form of XP that is simultaneously associated with Cockayne's syndrome (CS). The ERCC3 protein was found to be part of a multiprotein complex (TFIIH) required for transcription initiation of most structural genes and for NER. This defines the additional, hitherto unknown vital function of the gene. This ERCC3 gene and several other NER genes involved in transcription initiation will be discussed.

ERCC6, a member of a subfamily of putative helicases, is involved in Cockayne's syndrome and preferential repair of active genes.

Cells from patients with the UV-sensitive nucleotide excision repair disorder Cockayne's syndrome (CS) have a specific defect in preferential repair of lesions from the transcribed strand of active genes. This system permits quick resumption of transcription after UV exposure. Here we report the characterization of ERCC6, a gene involved in preferential repair in eukaryotes. ERCC6 corrects the repair defect of CS complementation group B (CS-B). It encodes a protein of 1493 amino acids, containing seven consecutive domains conserved between DNA and RNA helicases. The entire helicase region bears striking homology to segments in recently discovered proteins involved in transcription regulation, chromosome stability, and DNA repair. Mutation analysis of a CS-B patient indicates that the gene is not essential for cell viability and is specific for preferential repair of transcribed sequences.

RAD26, the functional S. cerevisiae homolog of the Cockayne syndrome B gene ERCC6.

Transcription-coupled repair (TCR) is a universal sub-pathway of the nucleotide excision repair (NER) system that is limited to the transcribed strand of active structural genes. It accomplishes the preferential elimination of transcription-blocking DNA lesions and permits rapid resumption of the vital process of transcription. A defect in TCR is responsible for the rare hereditary disorder Cockayne syndrome (CS). Recently we found that mutations in the ERCC6 repair gene, encoding a putative helicase, underly the repair defect of CS complementation group B. Here we report the cloning and characterization of the Saccharomyces cerevisiae homolog of CSB/ERCC6, which we designate RAD26. A rad26 disruption mutant appears viable and grows normally, indicating that the gene does not have an essential function. In analogy with CS, preferential repair of UV-induced cyclobutane pyrimidine dimers in the transcribed strand of the active RBP2 gene is severely impaired. Surprisingly, in contrast to the human CS mutant, yeast RAD26 disruption does not induce any UV-, cisPt- or X-ray sensitivity, explaining why it was not isolated as a mutant before. Recovery of growth after UV exposure was somewhat delayed in rad26. These findings suggest that TCR in lower eukaryotes is not very important for cell survival and that the global genome repair pathway of NER is the major determinant of cellular resistance to genotoxicity.

Localization of the nucleotide excision repair gene ERCC6 to human chromosome 10q11-q21.

We have cloned the human DNA excision repair gene ERCC6 by virtue of its ability to correct the uv sensitivity of Chinese hamster overy cell mutant UV61. This mutant is a member of complementation group 6 of the nucleotide excision repair-deficient rodent mutants. By means of in situ hybridization and Southern blot analysis of mouse x human somatic cell hybrids, the gene was localized to human chromosome 10q11-q21. An RFLP detected within the ERCC6 locus can be helpful in linkage analysis.

Molecular analysis of mutations in the CSB (ERCC6) gene in patients with Cockayne syndrome.

Cockayne syndrome is a multisystem sun-sensitive genetic disorder associated with a specific defect in the ability to perform transcription-coupled repair of active genes after UV irradiation. Two complementation groups (CS-A and CS-B) have been identified, and 80% of patients have been assigned to the CS-B complementation group. We have analyzed the sites of the mutations in the CSB gene in 16 patients, to determine the spectrum of mutations in this gene and to see whether the nature of the mutation correlates with the type and severity of the clinical symptoms. In nine of the patients, the mutations resulted in truncated products in both alleles, whereas, in the other seven, at least one allele contained a single amino acid change. The latter mutations were confined to the C-terminal two-thirds of the protein and were shown to be inactivating by their failure to restore UV-irradiation resistance to hamster UV61 cells, which are known to be defective in the CSB gene. Neither the site nor the nature of the mutation correlated with the severity of the clinical features. Severe truncations were found in different patients with either classical or early-onset forms of the disease.

Disruption of mouse RAD54 reduces ionizing radiation resistance and homologous recombination.

Double-strand DNA break (DSB) repair by homologous recombination occurs through the RAD52 pathway in Saccharomyces cerevisiae. Its biological importance is underscored by the conservation of many RAD52 pathway genes, including RAD54, from fungi to humans. We have analyzed the phenotype of mouse RAD54-/- (mRAD54-/-) cells. Consistent with a DSB repair defect, these cells are sensitive to ionizing radiation, mitomycin C, and methyl methanesulfonate, but not to ultraviolet light. Gene targeting experiments demonstrate that homologous recombination in mRAD54-/- cells is reduced compared to wild-type cells. These results imply that, besides DNA end-joining mediated by DNA-dependent protein kinase, homologous recombination contributes to the repair of DSBs in mammalian cells. Furthermore, we show that mRAD54-/- mice are viable and exhibit apparently normal V(D)J and immunoglobulin class-switch recombination. Thus, mRAD54 is not required for the recombination processes that generate functional immunoglobulin and T cell receptor genes.

Sequence conservation of the rad21 Schizosaccharomyces pombe DNA double-strand break repair gene in human and mouse.

The rad21 gene of Schizosaccharomyces pombe is involved in the repair of ionizing radiation-induced DNA double-strand breaks. The isolation of mouse and human putative homologs of rad21 is reported here. Alignment of the predicted amino acid sequence of Rad21 with the mammalian proteins showed that the similarity was distributed across the length of the proteins, with more highly conserved regions at both termini. The mHR21(sp) (mouse homolog of Rad21, S. pombe) and hHR21(sp) (human homolog of Rad21, S. pombe) predicted proteins were 96% identical, whereas the human and S. pombe proteins were 25% identical and 47% similar. RNA blot analysis showed that mHR21sp mRNA was abundant in all adult mouse tissues examined, with highest expression in testis and thymus. In addition to a 3.1-kb constitutive mRNA transcript, a 2.2-kb transcript was present at a high level in postmeiotic spermatids, while expression of the 3.1-kb mRNA in testis was confined to the meiotic compartment. hHR21sp mRNA was cell cycle regulated in human cells, increasing in late S phase to a peak in G2 phase. The level of hHR21sp transcripts was not altered by exposure of normal diploid fibroblasts to 10 Gy ionizing radiation. In situ hybridization showed that mHR21sp resided on chromosome 15D3, whereas hHR21sp localized to the syntenic 8q24 region. Elevated expression of mHR21sp in testis and thymus supports a possible role for the rad21 mammalian homologs in V(D)J and meiotic recombination, respectively. Cell cycle regulation of rad21, retained from S. pombe to human, is consistent with a conservation of function between S. pombe and human rad21 genes.