A HISTONE OCTAMER BLOCKS BRANCH MIGRATION OF A HOLLIDAY JUNCTION

The Holliday junction is a key intermediate in genetic recombination, Here, we examine the effect of a nucleosome core on movement of the Ho lliday junction in vitro by spontaneous branch migration. Histone octa mers consisting of H2A, H2B, H3, and H4 are reconstituted onto DNA dup lexes containing an artificial nucleosome-positioning sequence consist ing of a tandem array of an alternating AT-GC sequence motif, Characte rization of the reconstituted branch migration substrates by micrococc al nuclease mapping and exonuclease III and hydroxyl radical footprint ing reveal that 70% of the reconstituted octamers are positioned near the center of the substrate and the remaining 30% are located at the d istal end, although in both cases some translational degeneracy is obs erved. Branch migration assays with the octamer-containing substrates reveal that the Holliday junction cannot migrate spontaneously through DNA organized into a nucleosomal core unless DNA-histone interactions are completely disrupted, Similar results are obtained with branch mi gration substrates containing an octamer positioned on a naturally occ urring sequence derived from the yeast GLN3 locus, Digestion of Hollid ay junctions with T7 endonuclease I establishes that the junction is n ot trapped by the octamer but can branch migrate in regions free of hi stone octamers, Our findings suggest that migration of Holliday juncti ons during recombination and the recombinational repair of DNA damage requires proteins not only to accelerate the intrinsic rate of branch migration but also to facilitate the passage of the Holliday junction through a nucleosome.