Figure 2

H2A and H2A.X-F S1 phosphorylation is independent of DNA replication and the cell cycle. (A) Sperm chromatin was incubated in egg extract with the addition of BSA (control) or the addition of 150 ng of Geminin to inhibit DNA replication. The reactions were flash-frozen at 0, 30, or 90 min and chromatin was isolated through a sucrose cushion and immunoblotted as shown. The migration positions of H2A.X-F, H2A, and H4 are indicated. (B) Double-stranded DNA bound to streptavidin beads was incubated in egg extract for 0, 15, 30, or 60 min, then isolated and washed. Precipitated protein was immunoblotted for H2A, H3, H4, and S1ph as indicated. (C) Sperm chromatin was incubated in cycling egg extract and aliquots were flash-frozen every 15 min, from 0 to 180 min. Chromatin was isolated through a sucrose cushion and immunoblotted as shown. Interphase and mitosis were observed by DAPI-stained chromatin and noted at the top of the panel.