MutLalpha and proliferating cell nuclear antigen share binding sites on MutSbeta

Abstract

MutSbeta (MSH2-MSH3) mediates repair of insertion-deletion heterologies but also triggers triplet repeat expansions that cause neurological diseases. Like other DNA metabolic activities, MutSbeta interacts with proliferating cell nuclear antigen (PCNA) via a conserved motif (QXX(L/I)XXFF). We demonstrate that MutSbeta-PCNA complex formation occurs with an affinity of approximately 0.1 microM and a preferred stoichiometry of 1:1. However, up to 20% of complexes are multivalent under conditions where MutSbeta is in molar excess over PCNA. Conformational studies indicate that the two proteins associate in an end-to-end fashion in solution. Surprisingly, mutation of the PCNA-binding motif of MutSbeta not only abolishes PCNA binding, but unlike MutSalpha, also dramatically attenuates MutSbeta-MutLalpha interaction, MutLalpha endonuclease activation, and bidirectional mismatch repair. As predicted by these findings, PCNA competes with MutLalpha for binding to MutSbeta, an effect that is blocked by the cell cycle regulator p21(CIP1). We propose that MutSbeta-MutLalpha interaction is mediated in part by residues ((L/I)SRFF) embedded within the MSH3 PCNA-binding motif. To our knowledge this is the first case where residues important for PCNA binding also mediate interaction with a second protein. These findings also indicate that MutSbeta- and MutSalpha-initiated repair events differ in fundamental ways.

FIGURE 1.

Formation of a 1:1 equilibrium complex between MutSβ and PCNA is mediated by a PCNA-binding motif near the N terminus of MSH3. A, domain structure of human MSH3 as predicted by sequence alignment with MSH6. The PCNA-binding motif (conserved residues shown in blue) near the N terminus of full-length MSH3 is deleted in MSH3Δ28 and altered by Ala substitution for the conserved Phe residues in MSH3-F27A-F28A. B, DNA affinities of MutSβ, MutSβΔ28, and MutSβ-F27A-F28A were determined by SPRS (“Experimental Procedures”) by flowing the proteins over a sensor chip derivatized with a 200-bp homoduplex or a -TG- I/D heteroduplex DNA. Mass response units at saturation were recorded as a function of MutSβ concentration and fit to a rectangular hyperbola to yield apparent affinities shown. PCNA effects on MutSβ-DNA interaction were assessed by titration with MutSβ in the presence of 2.0 μm PCNA. C, interaction of PCNA with MutSβ in the absence of DNA was evaluated by equilibrium gel filtration (“Experimental Procedures”). Ten-μl samples containing 1 μm MutSβ (or 1.14 μm MutSβΔ28 or 1.0 μm MutSβ-F27A-F28A) and 0.75 μm PCNA were loaded onto a 2.4-ml Superdex 200 column equilibrated with 0.75 μm PCNA, and the column was developed isocratically at 0.01 ml/min. The protein elution profiles as detected by absorbance at 230 nm are shown for MutSβ (blue), MutSβΔ28 (red), and MutSβ-F27A-F28A (green). D, extents of PCNA trimer binding to MutSβ (closed circles), MutSβΔ28 (closed squares), or MutSβ-F27A-F28A (open triangles) were determined from trough areas as a function of free PCNA concentration. Binding isotherms shown were determined by nonlinear least squares fit to a rectangular hyperbola, which yielded a K_d of 0.10 μm and a stoichiometry of 0.8 PCNA homotrimer/wild type MutSβ heterodimer. Formation of the MutSβ-PCNA complex is associated with an increase in apparent Stokes radius (open circles). E, interaction parameters of PCNA with DNA-bound MutSβ were determined by SPRS. Solutions containing 0.10 μm MutSβ (or MutSβΔ28 or MutSβ-F27A-F28A) and 0–0.50 μm PCNA were allowed to flow over a sensor chip derivatized with a 200-bp -TG- I/D heteroduplex. The amount of PCNA bound to the sensor surface was determined as a function of PCNA concentration for MutSβ (circles), MutSβΔ28 (squares), or MutSβ-F27A-F28A (open triangles) by subtracting the mass response units recorded for each MutSβ variant alone from that determined in the presence of PCNA. Molar stoichiometries were calculated assuming that 1 response unit of MutSβ (232 kDa) corresponds to 0.37 response unit for the PCNA trimer (86 kDa). The data were fit to a rectangular hyperbola, yielding an apparent K_d of 0.02 μm, and a stoichiometry of 1 mol of PCNA/mol of MutSβ.

FIGURE 2.

MutSβ PIP box mutants are defective in MutLα interaction. A, ATP-dependent assembly of the MutLα-MutSβ-DNA ternary complex was scored by SPRS using a 200 bp -TG- I/D heteroduplex (solid lines) or homoduplex (dashed lines) DNA. The top panel shows mass bound upon flow of 0.10 μm MutSβ alone (gray) or a mixture of 0.10 μm MutSβ and 0.24 μm MutLα (black) over heteroduplex or homoduplex in the presence of 1 mm ATP. Similar analyses were performed with MutSβΔ28 (middle panel) and MutSβ-F27A-F28A. B, apparent affinity of MutLα for MutSβ (circles), MutSβΔ28 (squares), or MutSβ-F27A-F28A (triangles) was determined from SPRS experiments like those described above but in which the concentration of MutLα was varied as shown in the presence of 0.10 μm MutSβ. The data were fit to a hyperbola by nonlinear least squares regression to yield an apparent K_d of 0.40 μm on heterduplex DNA (closed symbols). Complex formation on homoduplex DNA (open symbols) was not saturable. C, interaction of MutSβ variants with PCNA and MutLα was also assessed independently by far Western analysis (“Experimental Procedures”). The indicated amount of each protein was spotted on a nitrocellulose membrane and incubated with 0.18 μm of either PCNA or MutLα at 4 °C overnight. PCNA and MutLα were detected immunochemically. D, interaction of MutLα with separated MSH2 or MSH3 subunits of MutSβ or its variants (bovine serum albumin (BSA) served as a negative control) was assayed by far Western analysis after subunit resolution by SDS-PAGE (“Experimental Procedures”). The membrane treatment was as in C except that incubation was with 0.09 μm of MutLα. See also supplemental Fig. S1.

FIGURE 3.

PCNA and MutLα compete for binding to MutSβ but not to MutSα. The effect of PCNA on formation of DNA-MutSβ-MutLα and DNA-MutSα-MutLα ternary complexes was evaluated by SPRS using a sensor chip derivatized with 200-bp -TG- I/D heteroduplex (solid lines) or control homoduplex (dashed lines) DNA. A, sensorgram profiles show mass response units upon flow of solutions containing 1 mm ATP and 0.10 μm MutSβ (gray); 0.10 μm MutSβ and 0.24 μm MutLα (black); 0.10 μm MutSβ and 2.0 μm PCNA (green); and 0.10 μm MutSβ, 0.24 μm MutLα and 2.0 μm PCNA (red). B, inhibition of ternary complex formation as a function of PCNA concentration was measured by monitoring mass bound when solutions containing 0.050 μm MutSβ (gray) or a mixture composed of 0.050 μm MutSβ, 0.050 μm MutLα, and one of the following were allowed to flow over the sensor chip in the presence of 0.25 mm ATP and 125 mm KCl: 0 (black), 0.50 (green), 1.0 (blue), or 2.0 (red) μm PCNA. C, effect of p21 on PCNA-dependent inhibition of DNA-MutSβ-MutLα ternary complex formation was assessed as in B by flowing a mixture of 0.050 μm MutSβ, 0.050 μm MutLα, and 0.25 mm ATP (black) or the same mixture supplemented with one of the following: 1.0 μm PCNA (blue), 0.50 μm PCNA (green), 1.0 μm PCNA and 6.0 μm p21 (red), or 0.50 μm PCNA and 6.0 μm p21 (orange). D, SPRS experiments were as in A but with 0.20 μm MutSα (gray); 0.20 μm MutSα and 0.20 μm MutLα (black); 0.20 μm MutSα and 1.6 μm PCNA (green); and 0.20 μm MutSα, 0.20 μm MutLα and 1.6 μm PCNA (red).

FIGURE 4.

MSH3 PIP box mutants are defective in MutLα endonuclease activation, mismatch-provoked excision, and mismatch repair. A, top panel, repair of 5′- (closed symbols) or 3′-dinucleotide (open symbols) I/D heteroduplex (“Experimental Procedures”) was scored in nuclear extracts of MSH2^−/− RL95-2 cells as a function of exogenously added MutSβ (circles) or MutSβΔ28 (squares). Bottom panel, excision on 5′- and 3′- substrates was scored by NheI-resistant gap formation assay (33) in RL95-2 extracts in the absence of exogenous dNTPs as a function of added MutSβ and MutSβΔ28. B, MutSβ (black bars) and MutSβΔ28 (white bars) (100 ng or 430 fmol) were compared for their ability to support MutLα endonuclease activation (16) on 5′ and 3′ -TG- I/D heteroduplex or A-T homoduplex DNAs (“Experimental Procedures”). The results shown are corrected for background observed in the absence of MutSβ, MutLα, RFC, and PCNA.

FIGURE 5.

Small angle x-ray scattering studies of MutSβ and the MutSβ-PCNA complex. A, normalized pair distribution (P(r)) plots for MutSβ (blue), MutSβΔ28 (red), an equimolar mixture of MutSβ and PCNA (green), and PCNA alone (black, reproduced from Ref. 21) were derived by indirect Fourier transform (49) of solution scattering data (supplemental Fig. S2A). B, stoichiometry of the MutSβ-PCNA interaction was determined (“Experimental Procedures”) from experimentally determined forward scattering intensities I(0) plotted as a function of PCNA:MutSβ (black circles) or PCNA:MutSβΔ28 (black squares) molar ratio. The theoretical dependence of I(0) on PCNA:MutSβ molar ratio was calculated for scenarios that assume formation of the stoichiometric complexes MutSβ-PCNA (318 kDa) (red circles), (MutSβ)₂-PCNA (550 kDa) (blue circles), or (MutSβ)₃-PCNA (782 kDa) (green circles) (“Experimental Procedures”). The expected I(0) values for PCNA mixtures with MutSβΔ28 (red squares) are also shown with the assumption of no interaction. Molecular masses corresponding to I(0) values (supplemental Fig. S2B) are indicated on the right vertical axis. C, ab initio shape reconstructions of MutSβ and MutSβΔ28 were performed from SAXS data as described (21). The envelopes shown represent an average of 10 independent shape reconstructions. Because of nonavailability of a MutSβ crystal structure, the MutSαΔ341-DNA complex structure (40) is superimposed on the MutSβ SAXS envelope for size reference. D, ab initio shapes of the MutSβ-PCNA complex were generated as described above from experimental scattering data collected for 1:1 molar mixtures of MutSβ and PCNA. Eight independent ab initio shapes in multiple colors are shown manually superimposed on each other. Despite the low resolution of these models, a central channel of dimensions similar to that of PCNA is clearly defined and was used to align the individual reconstructions. See also supplemental Fig. S2 and Table S1.