Primer synthesis contributes strongly to Chk1 phosphorylation. (A) Schematic of purified ssM13 structures. Biotinylated ends are represented by a dot. (B) Purified ssM13 structures were coupled to streptavidin and added at 6 ng/µl to NPE containing 300 µM aphidicolin. Samples were taken after 20 min and blotted for the proteins indicated. Chk1 phosphorylation was quantitated using Photoshop. (C) Purified ssM13 and 6–80-mer structures were added to NPE containing 300 µM aphidicolin (aph). The indicated concentration of the 6–80 mer is shown, and ssM13 was added so the total concentration of DNA was 144 ng/µl. Sperm chromatin (2,500 nuclei/µl) was also replicated in HSE and NPE containing 15 µM aphidicolin. Samples were analyzed at 20 (structures) or 60 min (chromatin) for Chk1 phosphorylation and quantitated as in Fig. 6 B. (B and C) Error bars indicate standard error (n = 3).