doi: 10.1093/embo-reports/kvf037. Epub 2002 Feb 15.
Disruption of Brca2 increases the spontaneous mutation rate in vivo: synergism with ionizing radiation
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- PMID:11850397
- PMCID: PMC1084010
- DOI: 10.1093/embo-reports/kvf037
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Disruption of Brca2 increases the spontaneous mutation rate in vivo: synergism with ionizing radiation
Andrew N J Tutt et al. EMBO Rep.2002 Mar.
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Abstract
The breast cancer predisposition gene BRCA2 encodes a protein involved in the repair of DNA double-strand breaks, which arise spontaneously and following exposure to ionizing radiation (IR). To develop a mouse model that examines the effect of BRCA2 mutation and IR exposure on in vivo somatic mutation acquisition, we crossed mice with targeted disruption of Brca2 with a LacZ transgenic mutation reporter strain. Loss of both wild-type Brca2 alleles caused a 2.3-fold increase, equivalent to an extra 100 mutations per cell, in the in vivo acquisition of spontaneous somatic mutation by 2 weeks gestation. IR (4 Gy) had a disproportionate effect on animals homozygous for Brca2 disruption, inducing 3.4-fold more mutations compared with wild-type animals. These data provide the first evidence that loss of Brca2 increases in vivo somatic mutation acquisition and synergizes with IR exposure, with potential attendant implications for mammographic screening and therapeutic IR in mutation carriers.
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Fig. 1. Schematic diagram of aLacZ plasmid transgenic mouse model for analysis of the effect ofBrca2 loss of function onin vivo somatic mutation frequency. Mice are shown of the three potentialBrca2 genotypes either treated or untreated with IR. These mice are also homozygous for two pUR288LacZ plasmid concatamer chromosome integration sites on chromosomes 3 and 4 (marked as black chromosomes). Each concatamer contains ∼10 copies of pUR288. The ampicillinr gene is shown as a blue box, the bacterial origin of replication as a yellow box and theLacZ reporter gene in red.HindIII sites between the plasmids are marked (H). Flanking regions of genomic DNA are represented by the green line. Individual linear plasmids are shown after release from genomic DNA byHindIII digestion. The central plasmid has a point mutation withinLacZ represented by a green dot. The plasmid on the right-hand side has suffered a deletion or genomic rearrangement removing the 3′LacZ sequence with a break point extending into flanking genomic DNA. This removes the originalHindIII site and thusHindIII digestion recovers aLacZ mutant plasmid of different size to the wild type. Single reporter plasmids are recovered from genomic DNA, transformed intoE. coli and mutation frequency determined by comparing colony numbers on mutant selection (P-gal) and non-selective rescue efficiency (X-gal) plates.
Fig. 2. Analysis of the effect of loss ofBrca2 onin vivo somatic mutation frequency and mutation type. (A) Scatter plot showing the somaticLacZ mutation frequency in the pUR288 reporter inBrca2Wt/Wt (n = 4),Brca2Wt/Tr (n = 5) andBrca2Tr/Tr (n = 3) day E14.5 embryos. Data points represent individual animals each based on >105 recovered reporter plasmids. Horizontal bars represent the mean. (B)AvaI–PstI restriction fragments from recoveredLacZ mutant pUR288 plasmids run as an example of Size-change and No-change mutants compared with the wild-type pUR288 restriction digest pattern. (C) The bar chart shows the frequency of no-change, size-change and mouse genome rearrangementLacZ mutants inBrca2Wt/Wt (n = 4),Brca2Wt/Tr (n = 5) andBrca2Tr/Tr (n = 3) day E14.5 embryos. Error bars represent the standard error of the mean (SEM).
Fig. 3. Analysis of the effect of IR onin vivo somatic mutation frequency and mutation type. (A) The bar chart shows the somatic mutation frequency in theLacZ pUR288 reporter in 4 Gy irradiatedBrca2Wt/Wt (n = 8),Brca2Wt/Tr (n = 9) andBrca2Tr/Tr (n = 6) day E14.5 embryos compared with the unirradiated embryos of the same genotype. Error bars represent SEM. (B) Scatter plot showing the frequency of somaticLacZ mutation in the pUR288 reporter induced over the spontaneous background in 4 Gy-irradiatedBrca2Wt/Wt (n = 8),Brca2Wt/Tr (n = 9) andBrca2Tr/Tr (n = 6) day E14.5 embryos. (C) The bar chart shows the frequency of size-change and no-changeLacZ mutants in 4 Gy-irradiatedBrca2Wt/Wt (n = 8),Brca2Wt/Tr (n = 9) andBrca2Tr/Tr (n = 6) day E14.5 embryos. Error bars represent the SEM.
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