Figure 4.
Cooperative and stoichiometric assembly of PCNA by RFC. (a) FRET versus time after adding RFC of indicated concentration to a solution containing 20 nM Cy3-PCNA, 20 nM Cy5-PCNA, and 0.5 mM ATP. (b) Amplitude of FRET change (obtained by single exponential fits to the FRET versus time curve) versus RFC concentration. (c) Average lifetime of RFC-induced PCNA assembly as measured in panel b as a function of RFC concentration. (d) Proposed kinetic mechanism of PCNA trimer assembly templated by RFC. A free RFC (R) and an RFC bound by a PCNA monomer (RP) are in rapid equilibrium. RP undergoes a conformational change to form the activated complex (RP∗), and two additional monomers bind to form R(P)3. (e) Average lifetime of the PCNA assembly reaction versus PCNA concentration at 200 nM RFC.