FIGURE 3.

MutSα-PCNA interaction occurs on heteroduplex DNA. A, interaction of MutSα and PCNA in the presence of heteroduplex DNA was analyzed by equilibrium gel filtration as described in the legend to Fig. 1 except that the column was equilibrated with a mixture of 800 nm DNA and 0.25 (black line), 0.5 (blue line), 1.5 (orange line), or 4 (red line) μm PCNA. The column was loaded with 10-μl samples containing 4μm MutSα, 800 nm DNA, and 0.25, 0.5, 1.5, or 4 μm PCNA, and eluate absorbance was monitored at 230 and 260 nm. Troughs at 1.35 and 1.61 ml are the result of depletion of free DNA and free PCNA, respectively, because of their association with MutSα. In the absence of PCNA (gray line), a single trough corresponding to DNA depletion due to MutSα binding was observed. B, PCNA (•) and DNA (○) bound per mol of MutSα were determined as a function of free PCNA concentration from the experiments shown in A by measurement of trough areas at 230 (PCNA) and 260 (DNA) nm. Data for interaction of the MutSα·DNA complex with PCNA were fit to a rectangular hyperbola as described in the legend to Fig. 1. Formation of the ternary complex was also evident as a decrease in retention volume of the MutSα·DNA peak (A), corresponding to an increase in the apparent Stokes radius (□). C, PCNA effects on MutSα affinities for 41-bp G-T heteroduplex (open symbols) or A·T homoduplex (closed symbols) DNA were evaluated by nitrocellulose filter binding assay (see “Experimental Procedures”). Reactions contained MutSα (○ and •), MutSαΔ12 (□ and ▪), or 10 μm PCNA plus MutSα (▵ and ▴) as indicated.