The MutSalpha-proliferating cell nuclear antigen interaction in human DNA mismatch repair

Abstract

We have examined the interaction parameters, conformation, and functional significance of the human MutSalpha(.) proliferating cell nuclear antigen (PCNA) complex in mismatch repair. The two proteins associate with a 1:1 stoichiometry and a K(D) of 0.7 microm in the absence or presence of heteroduplex DNA. PCNA does not influence the affinity of MutSalpha for a mismatch, and mismatch-bound MutSalpha binds PCNA. Small angle x-ray scattering studies have established the molecular parameters of the complex, which are consistent with an elongated conformation in which the two proteins associate in an end-to-end fashion in a manner that does not involve an extended unstructured tether, as has been proposed for yeast MutSalpha and PCNA ( Shell, S. S., Putnam, C. D., and Kolodner, R. D. (2007) Mol. Cell 26, 565-578 ). MutSalpha variants lacking the PCNA interaction motif are functional in 3'- or 5'-directed mismatch-provoked excision, but display a partial defect in 5'-directed mismatch repair. This finding is consistent with the modest mutability conferred by inactivation of the MutSalpha PCNA interaction motif and suggests that interaction of the replication clamp with other repair protein(s) accounts for the essential role of PCNA in MutSalpha-dependent mismatch repair.

FIGURE 1.

Equilibrium formation of a 1:1 complex between the MutSα heterodimer and the PCNA trimer. A, interaction of MutSα and PCNA was analyzed by equilibrium gel filtration (see “Experimental Procedures”). Ten-μl samples containing 8 μm MutSα and 0.25-4 μm PCNA were loaded onto a Superdex 200 column equilibrated with 0.25 (black), 0.5 (blue), 1 (green), 1.5 (orange), or 4 (red) μm PCNA. The trough at 1.35 ml resulted from depletion of PCNA because of its association with MutSα. Formation of the MutSα·PCNA complex was associated with a decrease in the retention volume of the MutSα peak from 1.07 ml at 0.25 μm PCNA to 1.025 ml at 4 μm PCNA (inset). B, PCNA trimer binding to recombinant MutSα (•), HeLa cell MutSα (▴), or MutSαΔ12 (▪) was determined as described for A as a function of free PCNA concentration. Isotherms shown for MutSα-PCNA interaction were determined by nonlinear least squares fit of the data to a rectangular hyperbola. The increase in the apparent Stokes radius of the MutSα peak with increasing PCNA concentration in also shown (○). C, MutSα-PCNA interaction under conditions of MutSα excess was assessed by SPRS. Solutions of 0.2-2 μm MutSα were flowed over a Ni²⁺-NTA sensor chip derivatized with ∼200 response units of His₆-tagged PCNA. Molar stoichiometries were calculated assuming that 1 response unit of MutSα (258 kDa) corresponds to 0.33 response units for the PCNA trimer (86 kDa). Data were fit to a rectangular hyperbola. The inset shows sensorgram results of an experiment in which 1 μm recombinant p21 was flowed over a Ni²⁺-NTA chip containing 100 response units of His₆-PCNA.

FIGURE 2.

MutSαΔ12 and MutSαΔ341 are proficient in mismatch binding, but defective in PCNA interaction. A, the schematic depicts the domain organization of MSH6, MSH6Δ12, and MSH6Δ341, all of which contain domains I-V (14). The N-terminal PCNA-binding motif (gray box) is lacking in MSH6Δ12 and MSH6Δ341. The N-terminal amino acid sequences are shown for full-length MSH6 and MSH6Δ12. Recombinant full-length MSH6 used in this study contained a Gly at position 2 that was introduced during cloning of the gene (S. Gradia and R. Fishel, personal communication). B, MutSαΔ12 (9 μm in column load) was subjected to equilibrium gel filtration on a column equilibrated with 1 μm (solid line) or 4 μm (dotted line) PCNA as described in the legend to Fig. 1. C, shown are the results from SDS-PAGE analysis of PCNA binding to MutSα variants bound to magnetic bead-linked 41-bp G-T heteroduplex or A·T homoduplex DNA (see “Experimental Procedures”).

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.

FIGURE 4.

SAXS of MutSα, MutSαΔ341, and PCNA. A, scattering intensities versus Q are shown for MutSα (blue line), MutSαΔ12 (green line), MutSαΔ341 (red line), MutSαΔ341 complexed to a 15-bp G-T heteroduplex (brown line), and PCNA (black line). Linear portions of Guinier plots (inset) determined from scattering profiles were used to determine R_g values (shown in Table 1). B, normalized pairwise interatomic distances (P(r)) were derived from solution scattering intensities (see “Experimental Procedures”). The color scheme is as described for A. C, ab initio shape reconstructions of MutSαΔ341, MutSαΔ341 bound to a 15-bp G-T heteroduplex, and PCNA were obtained from SAXS data (see “Experimental Procedures”). SUPCOMB (28) was used to superimpose and align the crystal structure of PCNA (39) or the MutSαΔ341·DNA complex (14) on SAXS envelopes.

FIGURE 5.

Stoichiometry and ab initio analysis of the MutSα·PCNA complex. A, P(r) plots for MutSα (solid blue line) and an equimolar mixture of MutSα and PCNA (red) were determined from composite scattering profiles (inset) as described in the legend to Fig. 4. The P(r) plot for MutSαΔ341 (dotted blue line) and the PCNA scattering profile (inset, black line) are shown for comparison. B, forward scattering intensities (I(0)) (intensity at θ = 0°) of mixtures of MutSα and PCNA were obtained (see “Experimental Procedures”) as a function of the PCNA/MutSα molar ratio for native MutSα (○), MutSαΔ12 (▴), and MutSαΔ341 (▪). Because I(0) is a linear function of molecular mass (supplemental Fig. 2) (38), we calculated the expected volume average dependence of this parameter (23) for models that assume different modes of MutSα-PCNA interaction: (i) stoichiometric formation of a 1:1 complex between MutSα and the PCNA trimer (red circles); (ii) interaction stoichiometry of two molecules of MutSα/PCNA trimer (blue circles); and (iii) stoichiometry of two molecules of MutSα/PCNA trimer when MutSα is in excess, but disproportionating to a 1:1 complex when PCNA is in excess (green line). The predicted dependence of I(0) for a PCNA mixture with MutSαΔ12 (red triangles) or MutSαΔ341 (red squares) assuming no interaction is also shown. C, ab initio shape reconstructions are shown. Low resolution models for MutSα and the 1:1 MutSα·PCNA complex were obtained from experimental scattering data as described under “Experimental Procedures.” The molecular envelope for MutSα (gray spheres) represents the average of 10 independent shape reconstructions, and the crystal structure of MutSαΔ341 was positioned manually by PyMOL for comparison of dimensions. For the MutSα·PCNA complex, a single representative shape reconstruction is shown (gray spheres), with the crystal structures of MutSαΔ341 and PCNA manually placed to indicate their possible locations within the complex. Although the solution scattering profile of the MutSα·PCNA complex is consistent with an extended conformation, the low resolution of the reconstruction does not permit assignment of the relative orientation of the two proteins about the long axis of the molecular envelope. (Additional independent shape reconstructions for MutSα·PCNA are shown in supplemental Fig. 3.)

FIGURE 6.

The MSH6 PCNA-binding motif is not required for mismatch-provoked excision and MutLα endonuclease activation, but is necessary for optimum 5′-directed mismatch repair. A, excision on 3′ (▿, ▵, ▾, and ▴) or 5′ (○, □, •, and ▪) heteroduplex (open symbols) or homoduplex (closed symbols) DNA (see “Experimental Procedures”) was scored in the absence of exogenous dNTPs in nuclear extracts of MSH6^-/- HCT-15 cells supplemented with 780 fmol of MutSα (○, ▿, •, and ▾) or MutSαΔ12 (□, ▵, ▪, and ▴). Data shown are averages of three experiments, with S.D. values ranging from 0.01 to 0.2 fmol. Excision in the absence of exogenous MutSα was not detectable. B, MutSα and MutSαΔ12 (780 fmol) were scored for their ability to support MutLα endonuclease activation (32) and 3′-directed (9) or 5′-directed (31) mismatch-provoked excision in purified systems (see “Experimental Procedures”). C, repair of 5′ (• and ▪) and 3′ (○ and □) G-T heteroduplexes in MSH6-deficient HCT-15 extracts was scored as a function of exogenous MutSα (• and ○) or MutSαΔ12 (▪ and □). Reactions were as described for A but contained added dNTPs. Data are the average of three experiments with S.D. values indicated by error bars.