Abstract
The histone variant H2AX is rapidly phosphorylated (denoted gammaH2AX) in large chromatin domains (foci) flanking double strand DNA (dsDNA) breaks that are produced by ionizing radiation or genotoxic agents and during V(D)J recombination. H2AX-deficient cells and mice demonstrate increased sensitivity to dsDNA break damage, indicating an active role for gammaH2AX in DNA repair; however, gammaH2AX formation is not required for V(D)J recombination. The latter finding has suggested a greater dependence on gammaH2AX for anchoring free broken ends versus ends that are held together during programmed breakage-joining reactions. Retroviral DNA integration produces a unique intermediate in which a dsDNA break in host DNA is held together by the intervening viral DNA, and such a reaction provides a useful model to distinguish gammaH2AX functions. We found that integration promotes transient formation of gammaH2AX at retroviral integration sites as detected by both immunocytological and chromatin immunoprecipitation methods. These results provide the first direct evidence for the association of newly integrated viral DNA with a protein species that is an established marker for the onset of a DNA damage response. We also show that H2AX is not required for repair of the retroviral integration intermediate as determined by stable transduction. These observations provide independent support for an anchoring model for the function of gammaH2AX in chromatin repair.
Highlights
The evolutionarily conserved histone H2AX comprises approximately 2–25% of the histone H2A pool in mammalian cells and is incorporated randomly into nucleosomes [1]
To find out whether H2AX phosphorylation is required for postintegration repair, we performed transduction experiments using mouse embryonic fibroblasts (MEFs) lines obtained from H2AX knock-out mice and derivatives [24, 25], and the ASV-EGFP vector
We show that retroviral infection induces the formation of histone ␥H2AX foci, and chromatin immunoprecipitation assays confirmed that H2AX phosphorylation occurs at sites of retroviral DNA integration
Summary
Vol 279, No 44, Issue of October 29, pp. 45810 –45814, 2004 Printed in U.S.A. Histone H2AX Is Phosphorylated at Sites of Retroviral DNA Integration but Is Dispensable for Postintegration Repair*. Tended C-terminal tail of H2AX contains a serine (Ser-139) embedded in an invariant SQE motif that is a target for phosphorylation by the phosphatidylinositol 3-kinase-related DNAPK, ataxia-telangiectasia-mutated (ATM), and ATM and Rad3related (ATR) protein kinases [2,3,4] This H2AX serine residue is massively and rapidly phosphorylated at sites of double strand breaks (DSBs) and stalled replication forks [3, 5, 6], forming microscopically visible foci on staining with a specific antibody. As a consequence of integrase-mediated joining, the host cell DNA suffers a DSB, but the ends are held together by single strand links to viral DNA (Fig. 1B) Postintegration repair of this intermediate (Fig. 1B) is essential for the maintenance of host DNA integrity as well as the stable association of retroviral DNA with host chromosomes. We asked whether H2AX is phosphorylated at sites of retroviral DNA integration and whether this response is essential for repair of this complex lesion as determined by survival of stably transduced cells
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