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Published October 29, 2004 | Published
Journal Article Open

Histone H2AX Is Phosphorylated at Sites of Retroviral DNA Integration but Is Dispensable for Postintegration Repair

Abstract

The histone variant H2AX is rapidly phosphorylated (denoted {gamma}H2AX) 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 {gamma}H2AX in DNA repair; however, {gamma}H2AX formation is not required for V(D)J recombination. The latter finding has suggested a greater dependence on {gamma}H2AX 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 {gamma}H2AX functions. We found that integration promotes transient formation of {gamma}H2AX 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 {gamma}H2AX in chromatin repair.

Additional Information

© 2004 the American Society for Biochemistry and Molecular Biology. Received for publication, July 13, 2004 , and in revised form, August 10, 2004. Originally published In Press as doi:10.1074/jbc.M407886200 on August 11, 2004. We thank Dr. Samuel Litwin for performing the computer simulations summarized in Table I and the following Fox Chase Cancer Center Shared Facilities used in the course of this work: Cell Imaging Facility, Biostatistics Facility, and Research Secretarial Services. This work was supported by National Institutes of Health Grants AI40385, CA71515, CA98090, and CA06927, a Tobacco Formula Research Fund Grant from the Pennsylvania Department of Health, and by an appropriation from the Commonwealth of Pennsylvania. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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