Table 2.

Functional activity of gp45 clamps in the polymerase holoenzyme strand displacement assay

Sliding clamp	+DTT	% Activity	+DTT	% Activity
gp45	218	100	218	100
gp45-K82C;S166C	203	93	195  ±  7.8	89
gp45-DPDPB	35  ±  3	16	196  ±  5.3	90
gp45-BMH	36	17	42  ±  0.7	19

Values are presented in active wt gp45 concentration units (nM), with the dynamic range and linearity of the scale determined by calibration with a standard curve of 0, 55, 109, and 218 nM wt gp45. The relative active concentrations of the gp45 variants then were determined after back-extrapolation from the amount of strand displacement with each modified gp45. %Activity was calculated relative to 218 nM wt gp45, which was defined as 100% active. This parameter can be calculated in this way because the assay is linear over the entire (0–218 nM) gp45 concentration range examined. Data listed as +DTT were obtained from reactions in which all of the clamp-loader proteins, rather than just gp45-DPDPB, were incubated with DTT.