FIGURE 1.

High N/C ratio reduces DNA synthesis by reducing origin firing and slowing replication fork progression. A and B, interphase extracts were supplemented with [α-³²P]dATP and either 1,000, 2,000, 5,000, or 10,000 nuclei/μl. Extracts were incubated at 21 °C, and DNA synthesis was monitored at the times indicated. The time scale of ³²P incorporation varied somewhat between extracts, so the data shown are from individual experiments and are representative of multiple, independent experiments. C, to analyze replication fork density, bio-dUTP was added at 28 min to mark sites of ongoing replication. Total DNA was labeled with dig-dUTP. D, DNA fibers labeled as in C were prepared and stained as described under “Experimental Procedures.” The locations of replication forks at 28 min on two sample fibers are indicated by arrowheads. The bio-dUTP and dig-dUTP signals were photographed separately and merged at a slight vertical offset to facilitate inspection of each labeling pattern. E, a large number of fibers (at least 15 Mb of DNA) labeled as in C were analyzed for the presence of replication forks. Error bars, S.E. ****, significant at p < 0.0001. F, to determine fork velocity, bio-dUTP was added to extracts during early S phase and followed 10 min later with dig-dUTP to mark the extent of fork progression. G, sample fibers labeled as in F are shown. Digoxigenin-negative tracts used to determine fork progression are marked with brackets. H, fork progression, in nt/min, was determined for at least 260 forks at both low and high N/C ratios. Mean velocity ± S.E. is shown. ****, significant at p < 0.0001 (Student's t test). a.u., arbitrary units.