Radiation-induced HPRT mutations resulting from misrejoined DNA double-strand breaks
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Radiation-induced HPRT mutations resulting from misrejoined DNA double-strand breaks. / Rothkamm, Kai; Gunasekara, Kut; Warda, Salam A; Krempler, Andrea; Löbrich, Markus.
in: RADIAT RES, Jahrgang 169, Nr. 6, 06.2008, S. 639-48.Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung
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TY - JOUR
T1 - Radiation-induced HPRT mutations resulting from misrejoined DNA double-strand breaks
AU - Rothkamm, Kai
AU - Gunasekara, Kut
AU - Warda, Salam A
AU - Krempler, Andrea
AU - Löbrich, Markus
PY - 2008/6
Y1 - 2008/6
N2 - DNA double-strand breaks (DSBs) are the most severe lesions induced by ionizing radiation, and unrejoined or misrejoined DSBs can lead to cell lethality, mutations and the initiation of tumorigenesis. We have investigated X-ray- and alpha-particle-induced mutations that inactivate the hypoxanthine guanine phosphoribosyltransferase (HPRT) gene in human bladder carcinoma cells and in hTERT-immortalized human fibroblasts. Fifty to 80% of the mutants analyzed exhibited partial or total deletions of the 9 exons of the HPRT locus. The remaining mutants retained unaltered PCR products of all 9 exons but often displayed a failure to amplify the HPRT cDNA. Hybridization analysis of a 2-Mbp NotI fragment spanning the HPRT gene with a probe 200 kbp distal to the HPRT locus indicated altered fragment sizes in most of the mutants with a wild-type PCR pattern. These mutants likely contain breakpoints for genomic rearrangements in the intronic sequences of the HPRT gene that allow the amplification of the exons but prevent HPRT cDNA amplification. Additionally, mutants exhibiting partial and total deletions of the HPRT exons also frequently displayed altered NotI fragments. Interestingly, all mutations were very rarely associated with interchromosomal exchanges analyzed by FISH. Collectively, our data suggest that intrachromosomal genomic rearrangements on the Mbp scale represent the prevailing type of radiation-induced HPRT mutations.
AB - DNA double-strand breaks (DSBs) are the most severe lesions induced by ionizing radiation, and unrejoined or misrejoined DSBs can lead to cell lethality, mutations and the initiation of tumorigenesis. We have investigated X-ray- and alpha-particle-induced mutations that inactivate the hypoxanthine guanine phosphoribosyltransferase (HPRT) gene in human bladder carcinoma cells and in hTERT-immortalized human fibroblasts. Fifty to 80% of the mutants analyzed exhibited partial or total deletions of the 9 exons of the HPRT locus. The remaining mutants retained unaltered PCR products of all 9 exons but often displayed a failure to amplify the HPRT cDNA. Hybridization analysis of a 2-Mbp NotI fragment spanning the HPRT gene with a probe 200 kbp distal to the HPRT locus indicated altered fragment sizes in most of the mutants with a wild-type PCR pattern. These mutants likely contain breakpoints for genomic rearrangements in the intronic sequences of the HPRT gene that allow the amplification of the exons but prevent HPRT cDNA amplification. Additionally, mutants exhibiting partial and total deletions of the HPRT exons also frequently displayed altered NotI fragments. Interestingly, all mutations were very rarely associated with interchromosomal exchanges analyzed by FISH. Collectively, our data suggest that intrachromosomal genomic rearrangements on the Mbp scale represent the prevailing type of radiation-induced HPRT mutations.
KW - Alpha Particles
KW - Cell Line, Tumor
KW - Chromosome Mapping
KW - DNA Breaks, Double-Stranded
KW - DNA Primers/chemistry
KW - DNA Repair
KW - DNA, Complementary/metabolism
KW - Dose-Response Relationship, Radiation
KW - Fibroblasts/metabolism
KW - Humans
KW - Hypoxanthine Phosphoribosyltransferase/genetics
KW - In Situ Hybridization, Fluorescence
KW - Mutation
KW - Nucleic Acid Hybridization
KW - X-Rays
U2 - 10.1667/RR1185.1
DO - 10.1667/RR1185.1
M3 - SCORING: Journal article
C2 - 18494542
VL - 169
SP - 639
EP - 648
IS - 6
ER -