Histones, the nucleosomal proteins that provide the structural framework for compacting the two to four meters of DNA into 90-180 mm of 30 nm diameter chromatin fibers in a typical mammalian cell, are now also known to provide essential functionalities to chromatin. We seek to elucidate some of these chromosomal functions of histones. The functionality of intense current interest is the phosphorylation of an evolutionarily conserved quadrapenultimate serine residue on many molecules of histone H2AX spatially and temporally coincident with each newly-formed DNA double strand break, be it accidental, incidental, or intentional. The phosphorylated form is named g-H2AX. The many molecules of g-H2AX are found as foci in nuclei at the site of the DNA DSB.We have identified which types of DNA DSBs induce g-H2AX formation. Published work from our group itself or in collaboraton with others have demonstrated g-H2AX formation at unplanned DNA DSBs sites due to ionizing radiation, radiomimetic agents such as bleomycin, Auger decay of 125I incorporated into DNA and topoisomerase I suicide complexes; and at planned DNA DSB sites due to V(D)J and class switch recombination, homologous recombination. We have completed an initial characterization of the parameters of g-H2AX formation. Foci are easily detected 1 minute after introduction of the DNA DSB, and attain maximal size by 30 minutes. Foci form at the sites of directed DNA DSBs. We have shown that one focus forms per disintegration and hence per DNA DSB. About 500-8000 g-H2AX molecules form per focus in mammals and about 40 in yeast in about 30 Mbp chromatin in mammals and 50 Kbp in yeast. In collaboration with Andre Nussenzweig's group, we have characterized mice lacking H2AX. H2AX-/- mice, although viable, are smaller than normal littermates and are sensitive to ionizing radiation. Primary MEFs isolated from them grow poorly; both MEFs and H2AX-/- stimulated B cell population have considerably numbers of cells with abnormal metaphases. Testes from H2AX-/- mice are smaller, lack sperm, and have high levels of apoptosis. Homologous autosome pair appears to be normal but the sex chromosomes often do not pair. H2AX-/- mice are competent for V(D)J recombination but not for class switch recombination. Foci of Nbs1, 53bp1, and Brca1, other proteins involved in DNA DSB repair form foci in normal mouse B lymphocytes, but not in H2AX-/- lymphocytes. However, Rad51 does form foci in H2AX-/- lymphocytes. This curious range of deficiencies may gives insight into relationships among various phenomena previously considered unrelated. We have generated and characterized yeast lacking the conserved serine residue phosphorylated in response to DNA DSBs. We have found a probable specific cause for their G2/M arrest in the finding that mutant yeast in the presence of CPT cannot completely duplicate their large chromosomes.We have also found a specific role for H2A serine 129 in the efficient repair of topoisomerase I-related lesions in yeast. In the manuscript, which is ready for submission, we demonstrate that H2A serine 129 is involved in top1 related damage, not only with CPT but also with other agents such as ionizing radiation.
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