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The development of a monoclonal antibody recognizing the Drosophila melanogaster phosphorylated histone H2A variant (γ-H2AV).

Lake CM, Holsclaw JK, Bellendir SP, Sekelsky J, Hawley RS - G3 (Bethesda) (2013)

Bottom Line: The recognition of DNA double-strand breaks (DSBs) using a phospho-specific antibody to the histone 2A variant has become the gold standard assay for DNA damage detection.Here we report on the development of the first monoclonal antibody to the phospho-specific form of Drosophila H2AV and characterize the specificity of this antibody to programmed DSBs in oocytes and rereplication sites in endocycling cells by immunofluorescence assays and to DSBs resulting from irradiation in both cell culture and whole tissue by Western blot assays.These studies show that the antibody derived in the study is highly specific for this modification that occurs at DSB sites, and therefore will be a new useful tool within the Drosophila community for the study of DNA damage response, DSB repair, meiotic recombination and chemical agents that cause DNA damage.

View Article: PubMed Central - PubMed

Affiliation: Stowers Institute for Medical Research, Kansas City, Missouri 64110.

ABSTRACT
The recognition of DNA double-strand breaks (DSBs) using a phospho-specific antibody to the histone 2A variant has become the gold standard assay for DNA damage detection. Here we report on the development of the first monoclonal antibody to the phospho-specific form of Drosophila H2AV and characterize the specificity of this antibody to programmed DSBs in oocytes and rereplication sites in endocycling cells by immunofluorescence assays and to DSBs resulting from irradiation in both cell culture and whole tissue by Western blot assays. These studies show that the antibody derived in the study is highly specific for this modification that occurs at DSB sites, and therefore will be a new useful tool within the Drosophila community for the study of DNA damage response, DSB repair, meiotic recombination and chemical agents that cause DNA damage.

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Timing of DSB formation and repair as assayed by γ-H2AV staining in wild-type and mutant oocytes. (A) A schematic of the events in meiotic prophase in the Drosophila ovary showing the timing of DSB formation and repair in the germarium. In region 1 the germline stem cell (*) divides to produce a germline stem cell and a cystoblast. The cystoblast then undergoes four rounds of incomplete mitotic divisions to produce a 16-cell interconnected cyst. Meiosis initiates within several cells of the 16-cell cyst and synaptonemal complex (SC) (blue ribbon) can be visualized at this stage (region 2A). Programmed meiotic DSBs are induced (red dots) after SC forms in early region 2A. As the cyst matures and moves more posterior in the germarium the number of γ-H2AV foci within the pro-oocytes (marked by SC), and therefore the number of DSBs, decreases throughout region 2B (early/mid-pachytene). By region 3 (mid-pachytene) there are no foci. The removal of the γ-H2AV signal indicates that DSB repair is underway and/or complete. (B−D) Immunocytological analysis of oocytes from wild-type (B), DSB deficient mei-W68 (C), and DSB repair−deficient okra (d) females showing the timing of DSB formation as marked by Protein A purified mouse anti-γ-H2AV (green) and repair (the removal of γ-H2AV signal) in pro-oocytes (identified with SC by anti-C3G in red) and DAPI (blue). Selected pro-oocyte nuclei in each stage are shown in 2X magnification to the far right. The γ-H2AV signal seen in the 15 surrounding nurse cells that surround the oocyte in the region 3 cyst are caused by the initiation of the endoreduplication cycle. In 33% of wild-type germarium (n = 12) one to a few nuclei in the tip of germarium were positive for γ-H2AV staining (seen in B). The origin of these nuclei is unknown at this time. Scale bar: 5 µm.
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fig2: Timing of DSB formation and repair as assayed by γ-H2AV staining in wild-type and mutant oocytes. (A) A schematic of the events in meiotic prophase in the Drosophila ovary showing the timing of DSB formation and repair in the germarium. In region 1 the germline stem cell (*) divides to produce a germline stem cell and a cystoblast. The cystoblast then undergoes four rounds of incomplete mitotic divisions to produce a 16-cell interconnected cyst. Meiosis initiates within several cells of the 16-cell cyst and synaptonemal complex (SC) (blue ribbon) can be visualized at this stage (region 2A). Programmed meiotic DSBs are induced (red dots) after SC forms in early region 2A. As the cyst matures and moves more posterior in the germarium the number of γ-H2AV foci within the pro-oocytes (marked by SC), and therefore the number of DSBs, decreases throughout region 2B (early/mid-pachytene). By region 3 (mid-pachytene) there are no foci. The removal of the γ-H2AV signal indicates that DSB repair is underway and/or complete. (B−D) Immunocytological analysis of oocytes from wild-type (B), DSB deficient mei-W68 (C), and DSB repair−deficient okra (d) females showing the timing of DSB formation as marked by Protein A purified mouse anti-γ-H2AV (green) and repair (the removal of γ-H2AV signal) in pro-oocytes (identified with SC by anti-C3G in red) and DAPI (blue). Selected pro-oocyte nuclei in each stage are shown in 2X magnification to the far right. The γ-H2AV signal seen in the 15 surrounding nurse cells that surround the oocyte in the region 3 cyst are caused by the initiation of the endoreduplication cycle. In 33% of wild-type germarium (n = 12) one to a few nuclei in the tip of germarium were positive for γ-H2AV staining (seen in B). The origin of these nuclei is unknown at this time. Scale bar: 5 µm.

Mentions: As mentioned previously, studies using a polyclonal γ-H2AV antibody have precisely detailed the timing and repair of meiotically induced DSBs in Drosophila females (Mehrotra and McKim 2006). Therefore, we chose to first analyze the specificity of the monoclonal γ-H2AV in an immunocytological assay by comparing the timing of DSB formation and repair as indicated by the detection and removal of γ-H2AV signal during prophase (overview shown in Figure 2A) to the previously published data. To summarize, DSBs are induced in early region 2A (early pachytene), after synaptonemal complex formation. In region 2B (early-mid pachytene), the number of foci is reduced from that seen in region 2A. Once the oocyte reaches the end of the germarium, region 3 (mid-pachytene), the phosphorylated H2AV mark is completely removed.


The development of a monoclonal antibody recognizing the Drosophila melanogaster phosphorylated histone H2A variant (γ-H2AV).

Lake CM, Holsclaw JK, Bellendir SP, Sekelsky J, Hawley RS - G3 (Bethesda) (2013)

Timing of DSB formation and repair as assayed by γ-H2AV staining in wild-type and mutant oocytes. (A) A schematic of the events in meiotic prophase in the Drosophila ovary showing the timing of DSB formation and repair in the germarium. In region 1 the germline stem cell (*) divides to produce a germline stem cell and a cystoblast. The cystoblast then undergoes four rounds of incomplete mitotic divisions to produce a 16-cell interconnected cyst. Meiosis initiates within several cells of the 16-cell cyst and synaptonemal complex (SC) (blue ribbon) can be visualized at this stage (region 2A). Programmed meiotic DSBs are induced (red dots) after SC forms in early region 2A. As the cyst matures and moves more posterior in the germarium the number of γ-H2AV foci within the pro-oocytes (marked by SC), and therefore the number of DSBs, decreases throughout region 2B (early/mid-pachytene). By region 3 (mid-pachytene) there are no foci. The removal of the γ-H2AV signal indicates that DSB repair is underway and/or complete. (B−D) Immunocytological analysis of oocytes from wild-type (B), DSB deficient mei-W68 (C), and DSB repair−deficient okra (d) females showing the timing of DSB formation as marked by Protein A purified mouse anti-γ-H2AV (green) and repair (the removal of γ-H2AV signal) in pro-oocytes (identified with SC by anti-C3G in red) and DAPI (blue). Selected pro-oocyte nuclei in each stage are shown in 2X magnification to the far right. The γ-H2AV signal seen in the 15 surrounding nurse cells that surround the oocyte in the region 3 cyst are caused by the initiation of the endoreduplication cycle. In 33% of wild-type germarium (n = 12) one to a few nuclei in the tip of germarium were positive for γ-H2AV staining (seen in B). The origin of these nuclei is unknown at this time. Scale bar: 5 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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fig2: Timing of DSB formation and repair as assayed by γ-H2AV staining in wild-type and mutant oocytes. (A) A schematic of the events in meiotic prophase in the Drosophila ovary showing the timing of DSB formation and repair in the germarium. In region 1 the germline stem cell (*) divides to produce a germline stem cell and a cystoblast. The cystoblast then undergoes four rounds of incomplete mitotic divisions to produce a 16-cell interconnected cyst. Meiosis initiates within several cells of the 16-cell cyst and synaptonemal complex (SC) (blue ribbon) can be visualized at this stage (region 2A). Programmed meiotic DSBs are induced (red dots) after SC forms in early region 2A. As the cyst matures and moves more posterior in the germarium the number of γ-H2AV foci within the pro-oocytes (marked by SC), and therefore the number of DSBs, decreases throughout region 2B (early/mid-pachytene). By region 3 (mid-pachytene) there are no foci. The removal of the γ-H2AV signal indicates that DSB repair is underway and/or complete. (B−D) Immunocytological analysis of oocytes from wild-type (B), DSB deficient mei-W68 (C), and DSB repair−deficient okra (d) females showing the timing of DSB formation as marked by Protein A purified mouse anti-γ-H2AV (green) and repair (the removal of γ-H2AV signal) in pro-oocytes (identified with SC by anti-C3G in red) and DAPI (blue). Selected pro-oocyte nuclei in each stage are shown in 2X magnification to the far right. The γ-H2AV signal seen in the 15 surrounding nurse cells that surround the oocyte in the region 3 cyst are caused by the initiation of the endoreduplication cycle. In 33% of wild-type germarium (n = 12) one to a few nuclei in the tip of germarium were positive for γ-H2AV staining (seen in B). The origin of these nuclei is unknown at this time. Scale bar: 5 µm.
Mentions: As mentioned previously, studies using a polyclonal γ-H2AV antibody have precisely detailed the timing and repair of meiotically induced DSBs in Drosophila females (Mehrotra and McKim 2006). Therefore, we chose to first analyze the specificity of the monoclonal γ-H2AV in an immunocytological assay by comparing the timing of DSB formation and repair as indicated by the detection and removal of γ-H2AV signal during prophase (overview shown in Figure 2A) to the previously published data. To summarize, DSBs are induced in early region 2A (early pachytene), after synaptonemal complex formation. In region 2B (early-mid pachytene), the number of foci is reduced from that seen in region 2A. Once the oocyte reaches the end of the germarium, region 3 (mid-pachytene), the phosphorylated H2AV mark is completely removed.

Bottom Line: The recognition of DNA double-strand breaks (DSBs) using a phospho-specific antibody to the histone 2A variant has become the gold standard assay for DNA damage detection.Here we report on the development of the first monoclonal antibody to the phospho-specific form of Drosophila H2AV and characterize the specificity of this antibody to programmed DSBs in oocytes and rereplication sites in endocycling cells by immunofluorescence assays and to DSBs resulting from irradiation in both cell culture and whole tissue by Western blot assays.These studies show that the antibody derived in the study is highly specific for this modification that occurs at DSB sites, and therefore will be a new useful tool within the Drosophila community for the study of DNA damage response, DSB repair, meiotic recombination and chemical agents that cause DNA damage.

View Article: PubMed Central - PubMed

Affiliation: Stowers Institute for Medical Research, Kansas City, Missouri 64110.

ABSTRACT
The recognition of DNA double-strand breaks (DSBs) using a phospho-specific antibody to the histone 2A variant has become the gold standard assay for DNA damage detection. Here we report on the development of the first monoclonal antibody to the phospho-specific form of Drosophila H2AV and characterize the specificity of this antibody to programmed DSBs in oocytes and rereplication sites in endocycling cells by immunofluorescence assays and to DSBs resulting from irradiation in both cell culture and whole tissue by Western blot assays. These studies show that the antibody derived in the study is highly specific for this modification that occurs at DSB sites, and therefore will be a new useful tool within the Drosophila community for the study of DNA damage response, DSB repair, meiotic recombination and chemical agents that cause DNA damage.

Show MeSH
Related in: MedlinePlus