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 γH2AX) 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 γH2AX in DNA repair; however, γH2AX formation is not required for V(D)J recombination. The latter finding has suggested a greater dependence on γH2AX 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 γH2AX functions. We found that integration promotes transient formation of γH2AX 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 γH2AX in chromatin repair.