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.
Highlights
The ability of a cell to recognize DNA damage and repair is an essential process for cell survival and genetic stability
Hybridoma clones were screened by enzyme-linked immunosorbent assay for specificity to the phospho-specific form of histone 2A variant (H2AV) and by immunocytological assays to address specificity to programmed meiotic double-strand breaks (DSBs) by screening meiotic tissue from DSB repair-deficient females
In the more mature oocytes we can see that the process of DNA repair is underway or complete as indicated by the removal of the g-H2AV modification
Summary
The ability of a cell to recognize DNA damage and repair is an essential process for cell survival and genetic stability. Polyclonal antibodies to human g-H2AX can detect Drosophila g-H2AV in Western blots (Rogakou et al 1999), studies have shown that these antibodies lack specificity in meiotic Drosophila tissue by immunocytological assays (Jang et al 2003; Mehrotra and McKim 2006).
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