Higher order chromatin structure presents a barrier to the recognition and repair of DNA damage. Double-strand breaks (DSBs) induce histone H2AX phosphorylation, which is associated with the recruitment of repair factors to damaged DNA. To help clarify the physiological role of H2AX, we targeted H2AX in mice. Although H2AX is not essential for irradiation-induced cell-cycle checkpoints, H2AX −/− mice were radiation sensitive, growth retarded, and immune deficient, and mutant males were infertile. These pleiotropic phenotypes were associated with chromosomal instability, repair defects, and impaired recruitment of Nbs1, 53bp1, and Brca1, but not Rad51, to irradiation-induced foci. Thus, H2AX is critical for facilitating the assembly of specific DNArepair complexes on damaged DNA.The first 120 amino acids of the H2AX and the H2A1/2 bulk isoprotein species exhibit a high degree of similarity, but H2AX carries a unique COOH-terminal tail that contains the * To whom correspondence should be addressed. andre_nussenzweig@nih.gov. HHS Public Access Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript consensus phosphatidyl inositol 3-kinase (PI-3 kinase) motif that is activated by DSBs (1, 2). Phosphorylation of H2AX (γ-H2AX) is induced by external genotoxic agents (2, 3) and is activated at physiological sites of recombination in lymphocytes (4, 5) and germ cells (6). Several essential DNA-repair factors implicated in homologous recombination (HR) (e.g., Brca1, Brca2, and Rad51) or that participate in both HR and nonhomologous end-joining (NHEJ) (e.g., Rad50, Mre11, Nbs1) form immunofluorescent foci that colocalize with γ-H2AX (7). However, the precise relation between focus formation and DNA repair is not understood.To determine the physiological role of H2AX in mammalian cells, we produced a targeted disruption of mouse H2AX (Web fig. 1A) (5,8). H2AX −/− mice were born at the expected frequency, and absence of H2AX protein was confirmed by two-dimensional gel electrophoresis and Western blotting (Web fig. 1, B to E) (8). Despite the loss of H2AX, treatment with γ-irradiation resulted in normal phosphorylation of Nbs1 (Web fig. 1E) (8).We conclude that H2AX is not essential for survival, or for irradiation-induced phosphorylation of Nbs1.H2AX −/− mice were growth retarded (Web fig. 2) (8), and H2AX −/− mouse embryo fibroblasts (MEFs) proliferated poorly in vitro (Fig. 1A). The difference in the growth of MEFs was partly due to a decrease in the number of dividing cells in H2AX −/− cultures as determined by incorporation of bromodeoxyuridine (BrdU) into DNA. During a 24-hour labeling period, only 44% of passage 1 H2AX −/− MEFs were actively cycling, compared with 72% for the controls, and the mitotic index of H2AX −/− MEFs was at least 50% lower than in wild-type cultures (see below; Fig. 1, D and F). By passage 4, H2AX −/− MEFS accumulated nondividing giant cells, suggesting premature entry into senescence. With continual passage, both H2AX −/− and wild-type MEFs went through crisis, after wh...

Lymphocyte polyclonal activation is a generalized mechanism of immune evasion among pathogens. In a mouse model of Trypanosoma cruzi infection (American trypanosomiasis), reduced levels of polyclonal lymphocyte responses correlate with resistance to infection and cardiopathy. We report here the characterization of a parasite protein with B-cell mitogenic properties in culture supernatants of infective forms, the cloning of the corresponding gene and the analysis of the biological properties of its product. We characterized the protein as a co-factor-independent proline racemase, and show that its expression as a cytoplasmic and/or membrane-associated protein is life-stage specific. Inhibition studies indicate that availability of the racemase active site is necessary for mitogenic activity. This is the first report to our knowledge of a eukaryotic amino acid racemase gene. Our findings have potential consequences for the development of new immune therapies and drug design against pathogens.

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