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O IX.2 Molecular cloning and functional characterization of the human XRCC2 DNA repair gene
S-IX : Radiat ion sensitivity, recombination and repair
S61
SESSION IX: Radiation sensitivity, recombination and repair ORALS
10 IX.II
Mutational analysis of the eempenents of DNA-PK
Penny Jeggo", Belinda Singleton·, Anne Priestley, Heather Beam ish', Guillermo Taccioli'', Dave GeUl , Steve Jad
10 IX.21
Molecular donlng and funttlonal tharacterlzatlon of the human XRCCZ DNA repair gene
John Thacker', Richard Cartwright', Cathryn Tambini', Andrew George', Johanna Rommens", Lap-Chee Tsui z, Stephen Scherer' . / MRC Radiation &; Genome Stability Unit. Harwell. Oxfordshire OX1 J ORD, UK; 1 Department of Genetics. Hosp ital for Sic/c Children, Toronto, Ontario, Canada Cell lines selected for sens itivity to DNA-damaging agents have recently proved to be invaluable for identifying human genes involved in the repair of DNA damage. The irs l line, isolated in our laboratory several years ago, is sensitive to a number of DNA-damaging agents including X-rays but especially to agents that cross-link DNA (e.g., mitomycin-C). Extensive complementation testing showed that this line is genetically dist inct from other rad iosensitive lines (Thacker & Wilkinson 1990 Mutation Res., 254, 135142). The human gene complementing irsl (provisionally named XRCC2) was first mapped to chromosome 7q36.1, following radiation-reduction of human/irsl fusion hybrids (Thacker et al. 1995 Human Mol. Genet., 4, 113120). The chr.7 content of hybrids (about 3-5 Mb) was further decreased by radiat ion reduction-fus ion, and one hybrid was found to contain only the marker D7S483. With this marker a YAC contig was ident ified, and
the YACs were fused individually to irsl cells to complement the defect. One YAC was found to complement, and was used to directly select for cDNAs by hybridization to libraries from several sources. The cDNAs were mapped to the YAC, and were then sequenced to find homologies to each other and to known DNA/protein sequences in the databases. The cDNAs ident ify at least 6 genes with in the YAC, one of which is the best candidate for XRCC2 . Smaller genomic segments, carried by PACs or cosmids, were isolated from the candidate region ; these were found to fully complement the mitomycin-C sensit ivity of irs I proving that the gene had been correctly ident ified. In agreement with the phenotype of irs[,the sequence of this gene pred icts homology to yeas t genes determining repa ir of severe forms of DNA damage (double -strand breaks, cross-links) by homologous recomb ination . The functional characterization of this gene will be important, since little is known about the operation of this repair pathway in human cells . Keyword(s): Recombination; gene cloning; XRCC2
10 IX.3!
Human SClDs with Increased lensltlvlty to Ionizing radIations and Impaired V(D)J rearrangements denne new genes Involved In DNA recombination/repaIr
Nathalie Nicolas, Marina Cavazzana-Calvo, Regina de Chasseval, Francoise Le Deist, Alain Fischer, Jean-Pierre de Villartay. INSERM U429. H6pital
Necker-Enfants Malades Paris. France The products of the RAG I and RAG2 genes initiate the lymphoid-specific phase of the V(D)J recomb ination by creating a DNA-double strand break (dsb), leaving hairpin-sealed coding ends. The next step employs the general DNA-repair machinery of the cells to resolve this dsb. Several genes involved in both V(D)J recombination and DNA repair have been identified through the analysis of in vitro mutants (CHO cells) and in vivo situations of severe combined immunodeficiency (murine and equ ine scid) . These stud ies lead to the description of the KulDNA-PK complex and the XRCC4 factor. A human SCID condition is characterized by an absence of Band T lymphocytes (T-BSCID) . Some patients also demonstrate an increas ed sensitivity to ionizing radiation of their fibroblasts and bone marrow precursor cells . In the latest, the V(D)J recombination is profoundly impaired with the lack of cod ingjoint formation while signal-jo ints are normal. The kinetics of DNA repair is however not affected in these pat ients and the KuSOIDNA-PK complex is present and functional. Finally, The XRCC4 eDNA sequence is normal. Altogether, functional and genetic studies distinguish the radiosensitive human T· B-SClDs from the murine scid condition and the other Recombination/Repair mutants. These patients therefore define a new group of mutants whose affected genets) is involved in sensitivity to ionizing radiation and V(D)J recombination. Keyword (s): dsb repair; scid; V(D)J Recombinaticn; DNA -PK
S61
SESSION IX: Radiation sensitivity, recombination and repair ORALS
10 IX.II
Mutational analysis of the eempenents of DNA-PK
Penny Jeggo", Belinda Singleton·, Anne Priestley, Heather Beam ish', Guillermo Taccioli'', Dave GeUl , Steve Jad
10 IX.21
Molecular donlng and funttlonal tharacterlzatlon of the human XRCCZ DNA repair gene
John Thacker', Richard Cartwright', Cathryn Tambini', Andrew George', Johanna Rommens", Lap-Chee Tsui z, Stephen Scherer' . / MRC Radiation &; Genome Stability Unit. Harwell. Oxfordshire OX1 J ORD, UK; 1 Department of Genetics. Hosp ital for Sic/c Children, Toronto, Ontario, Canada Cell lines selected for sens itivity to DNA-damaging agents have recently proved to be invaluable for identifying human genes involved in the repair of DNA damage. The irs l line, isolated in our laboratory several years ago, is sensitive to a number of DNA-damaging agents including X-rays but especially to agents that cross-link DNA (e.g., mitomycin-C). Extensive complementation testing showed that this line is genetically dist inct from other rad iosensitive lines (Thacker & Wilkinson 1990 Mutation Res., 254, 135142). The human gene complementing irsl (provisionally named XRCC2) was first mapped to chromosome 7q36.1, following radiation-reduction of human/irsl fusion hybrids (Thacker et al. 1995 Human Mol. Genet., 4, 113120). The chr.7 content of hybrids (about 3-5 Mb) was further decreased by radiat ion reduction-fus ion, and one hybrid was found to contain only the marker D7S483. With this marker a YAC contig was ident ified, and
the YACs were fused individually to irsl cells to complement the defect. One YAC was found to complement, and was used to directly select for cDNAs by hybridization to libraries from several sources. The cDNAs were mapped to the YAC, and were then sequenced to find homologies to each other and to known DNA/protein sequences in the databases. The cDNAs ident ify at least 6 genes with in the YAC, one of which is the best candidate for XRCC2 . Smaller genomic segments, carried by PACs or cosmids, were isolated from the candidate region ; these were found to fully complement the mitomycin-C sensit ivity of irs I proving that the gene had been correctly ident ified. In agreement with the phenotype of irs[,the sequence of this gene pred icts homology to yeas t genes determining repa ir of severe forms of DNA damage (double -strand breaks, cross-links) by homologous recomb ination . The functional characterization of this gene will be important, since little is known about the operation of this repair pathway in human cells . Keyword(s): Recombination; gene cloning; XRCC2
10 IX.3!
Human SClDs with Increased lensltlvlty to Ionizing radIations and Impaired V(D)J rearrangements denne new genes Involved In DNA recombination/repaIr
Nathalie Nicolas, Marina Cavazzana-Calvo, Regina de Chasseval, Francoise Le Deist, Alain Fischer, Jean-Pierre de Villartay. INSERM U429. H6pital
Necker-Enfants Malades Paris. France The products of the RAG I and RAG2 genes initiate the lymphoid-specific phase of the V(D)J recomb ination by creating a DNA-double strand break (dsb), leaving hairpin-sealed coding ends. The next step employs the general DNA-repair machinery of the cells to resolve this dsb. Several genes involved in both V(D)J recombination and DNA repair have been identified through the analysis of in vitro mutants (CHO cells) and in vivo situations of severe combined immunodeficiency (murine and equ ine scid) . These stud ies lead to the description of the KulDNA-PK complex and the XRCC4 factor. A human SCID condition is characterized by an absence of Band T lymphocytes (T-BSCID) . Some patients also demonstrate an increas ed sensitivity to ionizing radiation of their fibroblasts and bone marrow precursor cells . In the latest, the V(D)J recombination is profoundly impaired with the lack of cod ingjoint formation while signal-jo ints are normal. The kinetics of DNA repair is however not affected in these pat ients and the KuSOIDNA-PK complex is present and functional. Finally, The XRCC4 eDNA sequence is normal. Altogether, functional and genetic studies distinguish the radiosensitive human T· B-SClDs from the murine scid condition and the other Recombination/Repair mutants. These patients therefore define a new group of mutants whose affected genets) is involved in sensitivity to ionizing radiation and V(D)J recombination. Keyword (s): dsb repair; scid; V(D)J Recombinaticn; DNA -PK