We seek to understand the roles that histones perform in chromosome function. In eukaryotes, the DNA is associated with histone proteins to form compact complexes called nucleosomes. These are in turn arranged in various higher order structures to form interphase chromatin and metaphase chromosomes. As a result, the two to four meters of DNA double-helix in a typical mammalian cell is present as 90-180 mm of 30 nm diameter fibers in the interphase nucleus and 120 microns of 700 nm diameter chromosome arms during G2 and metaphase. The nucleosome is a complex of about 145 bp DNA and eight histone proteins, two from each of four core histone protein families, H4, H3, H2B and H2A. There is at least another 20 bp of DNA stretching between adjacent nucleosomes which may be complexed with the linker histone H1. Indications that histones may provide more than just a static monofunctional framework for DNA compaction came from our original discovery in mammals that the H2A histone family subsumes three subfamilies whose members contain characteristic sequence elements that have been conserved independently throughout eukaryotic evolution. Our current interest centers on histone H2AX since we showed that it is extensively phosphorylated within minutes of the introduction of DNA double-strand breaks into cells, with hundreds to several thousand H2AX molecules becoming phosphorylated per DNA double-strand break in mammals. We named this modified H2AX form gamma-H2AX. The phosphorylated residue is a serine 4 residues from the C-terminus. This serine residue is conserved throughout evolution from yeasts with the sequence KATKASQEL* (* signifies the C-terminus) to human with KATQASQEY*.During this reporting period, we prepared a gamma-H2AX antibody and showed that gamma-H2AX is present in discrete foci inside nuclei within minutes of irradiation. The number of foci is consistent with the expected number of DNA double-strand breaks, indicating that gamma-H2AX forms en masse at the sites of DNA double-strand breaks. With the antibody we also showed that the phosphorylation of the serine residue in SQXX* in response to ionizing radiation is conserved throughout eukaryotic evolution, occurring not only in mammalian species but also in the non- mammalian species, X. laevis, D. melanogaster, and S. cerevisiae. We also made significant methodological improvements in two areas. The first area pertains to targeted DNA damage in vivo. By combining the known sensitivity of BrdU containing DNA to UVA light in the presence of a dye with the ability of a UVA laser microbeam to be focused to 0.5 micron in diameter, we are able to place DNA double-strand breaks into targeted regions of nuclei. This improvement brings many types of nuclear local response experiments within the range of most laboratories since it eliminates the requirement for expensive and restricted facilities for ionizing radiation. The second area pertains to histone gel analysis. We improved our earlier procedures to permit direct loading of histone 0.5 N HCl extracts onto polyacrylamide gels, eliminating a number of steps and opportunities for oxidation artifacts. - Chromatin Structure, DNA Rejoining, Histone H2AX, Ionizing Radiation, DNA Double-Strand Breaks, - Neither Human Subjects nor Human Tissues
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