Skip to main content
. Author manuscript; available in PMC: 2010 May 8.
Published in final edited form as: J Mol Biol. 2009 Mar 13;388(3):431–442. doi: 10.1016/j.jmb.2009.03.014

Figure 3.

Figure 3

Activation of RFC on binding ATP and PCNA. (A) Sequential-mixing rapid-quench experiments, measuring [α-32P]ADP production, show that ptDNA triggers ATP hydrolysis; compare reactions with (● ) and without ptDNA (○ no PCNA, □ with PCNA). The data also verify slow step(s) during RFC, PCNA, ATP pre-incubation (Δt), before ptDNA-induced ATP hydrolysis; compare Δt = 2 s (green ●; kBurst = 35 ± 3 s−1) versus 0.02 s (red ●); final concentrations: 1 μM RFC, 500 μM ATP, 2.5 μM PCNA, 2.5 μM ptDNA. (B, C) Sequential-mixing stopped-flow experiments show gradual increase in ATP hydrolysis and Pi release with Δt (0.02 – 10 s), and maximal activity is detectable at Δt = 2 s with PCNA (B, light green trace) and at Δt = 7 s without PCNA (C, pink trace); final concentrations: same as above except for 0.5 μM RFC, 10 μM MDCC-PBP. kcat is 0.6 – 0.8 s−1 in all cases in the presence of ptDNA.