Figure 3.

Stimulation of DNA-dependent ATPase activity of RFC by mutant clamps. (A-D) Results of enzyme-coupled ATPase assay (each reaction contained 50 nM RFC, 100 nM PCNA, 100 nM DNA, and 0.5 μM ATP; see Methods). The ATPase rate of each reaction is displayed relative to the ATPase rate of RFC in the presence of PCNA and DNA-30, which is scaled to a value of one. Error bars represent standard deviation of multiple trials. DNA-30 has 30 base pairs in the duplex region and a 10-base 5' overhang on the primer strand; DNA-25 has 25 base pairs in the duplex region, etc. DNA-13/overhang is identical to DNA-13 but contains a 17-base 3' overhang on the template strand. (A) Wild-type PCNA stimulates RFC ATPase activity in the presence of DNA. (B) The point mutations in PCNA that result in the largest effects on ATPase activity in the presence of DNA are R80A and R149A, which have deficiencies of only 20-25% relative to wild-type PCNA. (C) Simultaneous mutation of PCNA residues K20A, K77A, R80A, and R149A results in an inability of PCNA to stimulate DNA-dependent ATPase activity. (D) PCNA K20A/K77A/R80A/R149A is deficient in stimulating ATPase activity when the primer-template DNA constructs present are long enough to extend through the clamp during loading, but its activity approaches that of wild-type PCNA in the presence of short DNA that cannot reach into the clamp.