FIGURE 11.

DNA/RNA duplex length affects primase activity but not affinity. A, the effect of RNA primer length on activity of primase-PolαΔcat. Lanes 1–3, T1-P1; lanes 4 and 5, T6-P4; lanes 6 and 7, T7-P4; lanes 8 and 9, T5-P3. Reactions corresponding to lanes 2, 4, 6, and 8 were conducted in the presence of [α-³³P]GTP alone to show the position of the first product of primer extension. Reactions corresponding to lanes 3, 5, 7, and 9 were supplemented with 0.1 mm each UTP, CTP, and ATP. Reactions were run for 2 min at 35 °C, and products were resolved by 20% urea-PAGE. B, 10-bp DNA/RNA duplex (T7-P4) efficiently competes with a 7-bp duplex (T4-P2) for binding with primase-PolαΔcat. All reactions contained 0.2 μm T4-P2; reactions corresponding to lanes 2–7 contained 0.2 μm primase-PolαΔcat. Samples were resolved by 5% native PAGE and visualized using the Typhoon 9410 imager. T4 is labeled with Cy3 at the 5′-end.