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Drosophila brca2 is required for mitotic and meiotic DNA repair and efficient activation of the meiotic recombination checkpoint.

Klovstad M, Abdu U, Schüpbach T - PLoS Genet. (2008)

Bottom Line: Much about its precise function in the DNA damage responses is, however, not yet known.We have made mutations in the Drosophila homolog of BRCA2 and measured the levels of homologous recombination, non-homologous end-joining, and single-strand annealing in the pre-meiotic germline of Drosophila males.In addition, Brca2 co-immunoprecipitates with the checkpoint protein Rad9, suggesting a direct role for Brca2 in the transduction of the meiotic recombination checkpoint signal.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Molecular Biology, Princeton University, Princeton, New Jersey, United States of America.

ABSTRACT
Heterozygous mutations in the tumor suppressor BRCA2 confer a high risk of breast and other cancers in humans. BRCA2 maintains genome stability in part through the regulation of Rad51-dependent homologous recombination. Much about its precise function in the DNA damage responses is, however, not yet known. We have made mutations in the Drosophila homolog of BRCA2 and measured the levels of homologous recombination, non-homologous end-joining, and single-strand annealing in the pre-meiotic germline of Drosophila males. We show that repair by homologous recombination is dramatically decreased in Drosophila brca2 mutants. Instead, large flanking deletions are formed, and repair by the non-conservative single-strand annealing pathway predominates. We further show that during meiosis, Drosophila Brca2 has a dual role in the repair of meiotic double-stranded breaks and the efficient activation of the meiotic recombination checkpoint. The eggshell patterning defects that result from activation of the meiotic recombination checkpoint in other meiotic DNA repair mutants can be strongly suppressed by mutations in brca2. In addition, Brca2 co-immunoprecipitates with the checkpoint protein Rad9, suggesting a direct role for Brca2 in the transduction of the meiotic recombination checkpoint signal.

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brca2 Mutations Dramatically Decrease HR-h Repair, Increase SSA Repair, and Result in Increased Deletion Formation(A) The Rr3 assay measures the relative repair pathway usage for repair of I-SceI endonuclease-cut breaks in the pre-meiotic germline of Drosophila males. The Rr3 transgene contains an I-SceI site flanked by two partial copies of dsRed. One dsRed copy contains a 147 bp duplication. Repair by SSA reconstitutes dsRed and results in red fluorescent progeny. Repair by NHEJ modifies the I-SceI site and results in progeny that are dark regardless of the presence of the endonuclease. The EJ1 transgene is a variant of Rr3 which has a non-functional I-SceI site and a 16 bp deletion 156 bp from the I-SceI site. Progeny resulting from repair by HR-h are also dark even when the endonuclease is present and can be distinguished from NHEJ by PCR. PCR analysis also distinguishes between short and long tract HR-h; long-tract HR-h results in the inclusion of the deletion on EJ1 and a smaller product. Deletions are measured by the loss of the w+ marker.(B) In Cross 2, the EJ1 chromosome was present, and repair by HR-h could occur. Error bars represent the standard errors between individual males; P-values were calculated using a permutation test described in [31]. Number of males/total Rr3-carrying progeny counted for OreR, brca2, and okra, respectively: 72/3861, 72/3163, and 78/3486.(C) In Cross 1, the EJ1 chromosome was not present, and repair by HR-h was not available. Number of males/total Rr3-carrying progeny counted for OreR, brca2, and okra, respectively: 66/7103, 67/6054, and 65/5546.aThe deletion class was not isolated prior to sorting and therefore represents a small portion of the NHEJ class.
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pgen-0040031-g001: brca2 Mutations Dramatically Decrease HR-h Repair, Increase SSA Repair, and Result in Increased Deletion Formation(A) The Rr3 assay measures the relative repair pathway usage for repair of I-SceI endonuclease-cut breaks in the pre-meiotic germline of Drosophila males. The Rr3 transgene contains an I-SceI site flanked by two partial copies of dsRed. One dsRed copy contains a 147 bp duplication. Repair by SSA reconstitutes dsRed and results in red fluorescent progeny. Repair by NHEJ modifies the I-SceI site and results in progeny that are dark regardless of the presence of the endonuclease. The EJ1 transgene is a variant of Rr3 which has a non-functional I-SceI site and a 16 bp deletion 156 bp from the I-SceI site. Progeny resulting from repair by HR-h are also dark even when the endonuclease is present and can be distinguished from NHEJ by PCR. PCR analysis also distinguishes between short and long tract HR-h; long-tract HR-h results in the inclusion of the deletion on EJ1 and a smaller product. Deletions are measured by the loss of the w+ marker.(B) In Cross 2, the EJ1 chromosome was present, and repair by HR-h could occur. Error bars represent the standard errors between individual males; P-values were calculated using a permutation test described in [31]. Number of males/total Rr3-carrying progeny counted for OreR, brca2, and okra, respectively: 72/3861, 72/3163, and 78/3486.(C) In Cross 1, the EJ1 chromosome was not present, and repair by HR-h was not available. Number of males/total Rr3-carrying progeny counted for OreR, brca2, and okra, respectively: 66/7103, 67/6054, and 65/5546.aThe deletion class was not isolated prior to sorting and therefore represents a small portion of the NHEJ class.

Mentions: We used the Repair Reporter 3 (Rr3) assay [30] to simultaneously measure the relative levels of HR, NHEJ, and SSA repair in the pre-meiotic germline of individual males (Figure 1). The Rr3 assay monitors the repair of a DSB at an I-SceI endonuclease site flanked by partial copies of the reporter dsRed. There is a 147 base pair (bp) tandem duplication of dsRed flanking the break. I-SceI is ubiquitously expressed at the onset of zygotic transcription and the repair outcomes of DSBs in the male germline can be observed in the progeny (Figure 1A). Repair by SSA recreates a functional copy of dsRed and the resulting flies are fluorescent red even in the absence of the endonuclease. Repair by NHEJ creates small insertions or deletions at the I-SceI site and the resulting flies are dark even in the presence of endonuclease. These two classes were scored from populations either with or without endonuclease in order to distinguish them from the uncut reporter carrying flies, which are mosaic red and often undistinguishable from germline DsRed flies [30]. Repair by HR using the sister chromatid (HR-s) recreates the reporter, which can be re-cut until a terminal outcome is reached. HR repair using the homologous chromosome (HR-h) can occur when a Rr3 variant, EJ1, which contains a mutated I-SceI site is present on the homologous chromosome. Repair by HR-h results in dark flies even when the endonuclease is present and can be distinguished from NHEJ by PCR specific to the mutated I-SceI site.


Drosophila brca2 is required for mitotic and meiotic DNA repair and efficient activation of the meiotic recombination checkpoint.

Klovstad M, Abdu U, Schüpbach T - PLoS Genet. (2008)

brca2 Mutations Dramatically Decrease HR-h Repair, Increase SSA Repair, and Result in Increased Deletion Formation(A) The Rr3 assay measures the relative repair pathway usage for repair of I-SceI endonuclease-cut breaks in the pre-meiotic germline of Drosophila males. The Rr3 transgene contains an I-SceI site flanked by two partial copies of dsRed. One dsRed copy contains a 147 bp duplication. Repair by SSA reconstitutes dsRed and results in red fluorescent progeny. Repair by NHEJ modifies the I-SceI site and results in progeny that are dark regardless of the presence of the endonuclease. The EJ1 transgene is a variant of Rr3 which has a non-functional I-SceI site and a 16 bp deletion 156 bp from the I-SceI site. Progeny resulting from repair by HR-h are also dark even when the endonuclease is present and can be distinguished from NHEJ by PCR. PCR analysis also distinguishes between short and long tract HR-h; long-tract HR-h results in the inclusion of the deletion on EJ1 and a smaller product. Deletions are measured by the loss of the w+ marker.(B) In Cross 2, the EJ1 chromosome was present, and repair by HR-h could occur. Error bars represent the standard errors between individual males; P-values were calculated using a permutation test described in [31]. Number of males/total Rr3-carrying progeny counted for OreR, brca2, and okra, respectively: 72/3861, 72/3163, and 78/3486.(C) In Cross 1, the EJ1 chromosome was not present, and repair by HR-h was not available. Number of males/total Rr3-carrying progeny counted for OreR, brca2, and okra, respectively: 66/7103, 67/6054, and 65/5546.aThe deletion class was not isolated prior to sorting and therefore represents a small portion of the NHEJ class.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2233675&req=5

pgen-0040031-g001: brca2 Mutations Dramatically Decrease HR-h Repair, Increase SSA Repair, and Result in Increased Deletion Formation(A) The Rr3 assay measures the relative repair pathway usage for repair of I-SceI endonuclease-cut breaks in the pre-meiotic germline of Drosophila males. The Rr3 transgene contains an I-SceI site flanked by two partial copies of dsRed. One dsRed copy contains a 147 bp duplication. Repair by SSA reconstitutes dsRed and results in red fluorescent progeny. Repair by NHEJ modifies the I-SceI site and results in progeny that are dark regardless of the presence of the endonuclease. The EJ1 transgene is a variant of Rr3 which has a non-functional I-SceI site and a 16 bp deletion 156 bp from the I-SceI site. Progeny resulting from repair by HR-h are also dark even when the endonuclease is present and can be distinguished from NHEJ by PCR. PCR analysis also distinguishes between short and long tract HR-h; long-tract HR-h results in the inclusion of the deletion on EJ1 and a smaller product. Deletions are measured by the loss of the w+ marker.(B) In Cross 2, the EJ1 chromosome was present, and repair by HR-h could occur. Error bars represent the standard errors between individual males; P-values were calculated using a permutation test described in [31]. Number of males/total Rr3-carrying progeny counted for OreR, brca2, and okra, respectively: 72/3861, 72/3163, and 78/3486.(C) In Cross 1, the EJ1 chromosome was not present, and repair by HR-h was not available. Number of males/total Rr3-carrying progeny counted for OreR, brca2, and okra, respectively: 66/7103, 67/6054, and 65/5546.aThe deletion class was not isolated prior to sorting and therefore represents a small portion of the NHEJ class.
Mentions: We used the Repair Reporter 3 (Rr3) assay [30] to simultaneously measure the relative levels of HR, NHEJ, and SSA repair in the pre-meiotic germline of individual males (Figure 1). The Rr3 assay monitors the repair of a DSB at an I-SceI endonuclease site flanked by partial copies of the reporter dsRed. There is a 147 base pair (bp) tandem duplication of dsRed flanking the break. I-SceI is ubiquitously expressed at the onset of zygotic transcription and the repair outcomes of DSBs in the male germline can be observed in the progeny (Figure 1A). Repair by SSA recreates a functional copy of dsRed and the resulting flies are fluorescent red even in the absence of the endonuclease. Repair by NHEJ creates small insertions or deletions at the I-SceI site and the resulting flies are dark even in the presence of endonuclease. These two classes were scored from populations either with or without endonuclease in order to distinguish them from the uncut reporter carrying flies, which are mosaic red and often undistinguishable from germline DsRed flies [30]. Repair by HR using the sister chromatid (HR-s) recreates the reporter, which can be re-cut until a terminal outcome is reached. HR repair using the homologous chromosome (HR-h) can occur when a Rr3 variant, EJ1, which contains a mutated I-SceI site is present on the homologous chromosome. Repair by HR-h results in dark flies even when the endonuclease is present and can be distinguished from NHEJ by PCR specific to the mutated I-SceI site.

Bottom Line: Much about its precise function in the DNA damage responses is, however, not yet known.We have made mutations in the Drosophila homolog of BRCA2 and measured the levels of homologous recombination, non-homologous end-joining, and single-strand annealing in the pre-meiotic germline of Drosophila males.In addition, Brca2 co-immunoprecipitates with the checkpoint protein Rad9, suggesting a direct role for Brca2 in the transduction of the meiotic recombination checkpoint signal.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Molecular Biology, Princeton University, Princeton, New Jersey, United States of America.

ABSTRACT
Heterozygous mutations in the tumor suppressor BRCA2 confer a high risk of breast and other cancers in humans. BRCA2 maintains genome stability in part through the regulation of Rad51-dependent homologous recombination. Much about its precise function in the DNA damage responses is, however, not yet known. We have made mutations in the Drosophila homolog of BRCA2 and measured the levels of homologous recombination, non-homologous end-joining, and single-strand annealing in the pre-meiotic germline of Drosophila males. We show that repair by homologous recombination is dramatically decreased in Drosophila brca2 mutants. Instead, large flanking deletions are formed, and repair by the non-conservative single-strand annealing pathway predominates. We further show that during meiosis, Drosophila Brca2 has a dual role in the repair of meiotic double-stranded breaks and the efficient activation of the meiotic recombination checkpoint. The eggshell patterning defects that result from activation of the meiotic recombination checkpoint in other meiotic DNA repair mutants can be strongly suppressed by mutations in brca2. In addition, Brca2 co-immunoprecipitates with the checkpoint protein Rad9, suggesting a direct role for Brca2 in the transduction of the meiotic recombination checkpoint signal.

Show MeSH
Related in: MedlinePlus