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We have examined interaction parameters, conformation, and functional significance of the human MutSα·PCNA complex in mismatch repair. The two proteins associate with a 1:1 stoichiometry and a KD of 0.7 μM in absence or presence of heteroduplex DNA. PCNA does not influence the affinity of MutSα for a mismatch, and mismatch-bound MutSα binds PCNA. Small angle X-ray scattering studies have established molecular parameters of the complex and are consistent with an elongated conformation in which the two proteins associate in end-to-end fashion in a manner that does not involve an extended unstructured tether as has been proposed for yeast MutSα and PCNA (Shell et al. (2007) Mol. Cell 26, 565-578). MutSα 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 MutSα PCNA interaction motif and suggests that interaction of the replication clamp with other repair protein(s) accounts for the essential role of PCNA in mismatch repair.
Mismatch repair rectifies DNA biosynthetic errors, mediates several recombination-associated phenomena, and in mammalian cells participates in an early step of the cellular response to certain classes of DNA damage (reviewed in (1,2)). Defects in the human pathway are the cause of hereditary non-polyposis colorectal cancer and have been implicated in the development of a subset of sporadic tumors.
A strand-specific mismatch repair reaction that is presumably responsible for replication error correction has been studied in mammalian cell extracts and in several purified systems. A number of activities have been implicated in this pathway including MutSα (MSH2·MSH6 heterodimer), MutSβ (MSH2·MSH3), MutLα (MLH1·PMS2), Exo1, RPA, HMGB1, PCNA, RFC, and DNA polymerase δ. This reaction can be conceptually divided into three steps: mismatch recognition, excision, and repair DNA synthesis/ligation. The replication clamp PCNA functions at several stages of this multi-step reaction. It participates in the DNA synthesis phase of repair (3), consistent with its function as a cofactor for DNA polymerase δ (4). PCNA is also necessary during an early step of the reaction (5) and has been shown to interact with activities implicated in early steps of repair (6-11). However, the significance of these various PCNA interactions is not clear. Analysis of PCNA involvement in mismatch repair has emphasized the MutSα-PCNA complex, which has been suggested to be a key intermediate in mismatch repair. A conserved QxxL/IxxFF motif near the N-terminus of MSH6 has been implicated in this interaction (6-8). A human MutSα variant lacking the MSH6 N-terminal 77 amino acids including the QxxL/IxxFF motif is severely compromised in its ability to support mismatch repair in vitro (8), but alanine substitution of conserved residues within the PCNA interaction motif of yeast MSH6 results in only a modest increase in mutability (6,7). The implications of the PCNA·MutSα interaction for the mechanism of mismatch repair are also uncertain. Biochemical analyses have indicated that yeast PCNA enhances the specific affinity of yeast MutSα for a mismatch (7), but a subsequent study concluded that while PCNA, MutSα and homoduplex DNA form a stable ternary complex, binding to a mismatch leads to disruption of the PCNA·MutSα interaction (12). These two conclusions appear to be at odds with thermodynamic expectations: if PCNA increases the affinity of MutSα for a mispair, then a mismatch should increase the affinity of PCNA for MutSα.
This study further clarifies the nature and function of the human MutSα-PCNA interaction. We show that MutSα binds PCNA with a stoichiometry of 1:1, adopting an elongated conformation in solution, and that the affinity of this interaction is not significantly affected by mismatch binding. Abrogation of the MutSα-PCNA interaction has little effect on mismatch-provoked 5′- or 3′-directed excision. However, 5′-directed repair is partially attenuated under these conditions.
A baculoviral expression vector that expresses full length MSH2 and an N-terminal truncated form of MSH6 (MSH6ΔN12) was prepared by PCR mutagenesis from the previously described pFastbacDual-MSH2-MSH6 (13). The resulting pFastbacDual-MSH2-MSH6ΔN12, encodes amino acids 13-1361 of full-length MSH6, beginning with N-Met-Pro-Lys-Ser, and was used to generate a baculovirus according to the protocol of the manufacturer (Invitrogen). Recombinant virus was plaque-purified and high titer stocks were produced, which were used to infect Sf9 insect cells for protein expression.
Circular heteroduplex DNAs contained a single G-T mismatch (A·T in homoduplex controls) and a strand discontinuity located either 3′ or 5′ to the mismatch (9). DNAs for surface plasmon resonance spectroscopy (SPRS), magnetic bead assays, and equilibrium gel filtration were 21, 41 or 201 bp duplexes containing a centrally located G-T mismatch (or A·T base pair) flanked by sequences corresponding to those in the circular substrates above (13). For nitrocellulose filter binding assays, synthetic 41 bp hetero- and homoduplex DNAs were prepared by annealing HPLC purified 5′-32P-labeled T-containing strand with an equimolar amount of unlabeled purified A or G containing strand. Substrates for SPRS and magnetic bead assays contained a 5′ biotin tag on the T-containing strand.
Recombinant MutSα, MSH2·MSH6ΔN12 (referred to below as MutSαΔ12), and MutLα were isolated according to Blackwell et al. (13), and MSH2·MSH6ΔN341 (MutSαΔ341) according to Warren et al. (14). MutSα isolated from HeLa cell nuclear extracts (15) was used in some experiments. Isolates of MutSα and its variants typically contained ~1 mole of ADP per mole of heterodimer. Exo1 and RPA (16), RFC (9), native PCNA (17), His6-PCNA (18), and p21 (19) were isolated as described. MutSα and PCNA concentrations, which are expressed as heterodimer and homotrimer equivalents, respectively, were determined by the method of von Hippel (20), which yielded extinction coefficients at 280 nm of 208,465 (MuSα), 206,975 (MutSαΔ12), 171,650 (MutSαΔ341), and 50,040 (PCNA) M-1 cm-1.
Gel filtration at 4°C employed a 2.4 ml Superdex 200 PC 3.2 column on an AKTA Purifier 10 HPLC system (GE Healthcare) equilibrated with 25 mM HEPES-NaOH, pH 7.5 (or 10 mM Tris-HCl, pH 7.6), 5 mM MgCl2, 125 mM NaCl, and 0.1 mM dithiothreitol (DTT). The column was calibrated using blue dextran (excluded volume) and standards of known Stokes radii (GE Healthcare): apoferritin (67.1 Å), aldolase (48.1 Å) and albumin (35.5 Å). For equilibrium gel filtration, the Superdex 200 column was equilibrated with PCNA as indicated (or a mixture of PCNA and a 21 bp G-T heteroduplex). A 10 μl sample of MutSα in equilibration buffer was injected, and the column developed at 0.02 ml/min. The eluate was monitored at 280, 260 and 230 nm as indicated, and/or collected in 0.025 ml fractions, which were analyzed by SDS-PAGE.
Bead-linked DNA assay was used to score PCNA-MutSα complexes on DNA. A slurry of M280 streptavidin-coated magnetic beads (1 ml, Invitrogen) was derivatized by resuspension in 1 ml of 10 mM Tris-HCl, pH 7.5, 500 mM KCl, and 1 mM EDTA containing 1 nmol of 41 bp homo- or G-T heteroduplex DNA. For binding assays, 18 μl of derivatized beads were washed and resuspended in 90 μl of 25 mM HEPES-KOH, pH 7.5, 5 mM MgCl2, 100 mM KCl containing 400 nM MutSα and 1 μM PCNA. After incubation for 10 min at 4°C, beads were collected, washed with assay buffer, and bound protein eluted with 25 μl of 25 mM HEPES-KOH, pH 7.5, 5 mM MgCl2, 400 mM NaCl followed by SDS-PAGE analysis.
MutSα binding to 41 bp homoduplex and heteroduplex DNAs was scored by the nitrocellulose-DEAE paper double-filter method (21). Reactions (50 μl) contained 25 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1 nM 32P-labeled 41bp G-T heteroduplex or A·T homoduplex DNA, and MutSα and PCNA as indicated. After 20 minutes at 4°C, the solution was applied to a stacked filter set, which had been pre-washed twice with 100 μl of binding buffer, and drawn through under 30 kPa of vacuum. Nitrocellulose and DEAE filters were dried, and radioactivity bound to each quantitated by phosphorimager.
SPRS was performed on a BIAcore 2000. Interaction of MutSα or p21 with PCNA in the absence of DNA was analyzed in 25 mM HEPES-NaOH, pH 7.5, 125 mM NaCl, and 1 mM EDTA using Ni+2-chelated nitrilotriacetic acid chip derivatized with His6-PCNA (100-200 RU). Interaction of PCNA with DNA-bound MutSα was evaluated by two phase protein flow (20 μl/min) over a streptavidin chip derivatized with biotin-tagged 201 bp homoduplex or heteroduplex DNA in 25 mM HEPES-NaOH, pH 7.5, 5 mM MgCl2, 150 mM NaCl, 0.02% surfactant P-20, and 1 mM DTT. In the first phase, 200 nM MutSα was allowed to flow for 5 minutes which was followed immediately by a second phase wherein 200 nM MutSα supplemented by 0.025 -2.5 μM PCNA was allowed to flow for an additional 5 min.
SAXS was performed on the Sibyls beamline 12.3.1 at the Advanced Light Source. Protein samples were dialyzed against 25 mM HEPES-KOH, pH 7.5, 5 mM MgCl2, 150 mM KCl, and 2 mM DTT. Scattering intensities (I) were determined at multiple protein concentrations (10 - 50 μM) as a function of the scattering vector Q = 4πsinθ/λ, where 2θ is the scattering angle, and λ is the wavelength of the incident beam, and corrected for background scattering by dialysis buffer. Forward scattering intensities I(0) were calculated from Guinier plots (ln I vs Q2) (22) of scattering data normalized for concentration (mass/volume). Radii of gyration (Rg) were determined by Guinier plot from scattering curves extrapolated to zero concentration using the program PRIMUS (23).
Composite scattering curves were generated with PRIMUS by scaling and merging background corrected high Q region data from higher concentration samples (20 - 50 μM) with low Q region 0-extrapolated data. Scattering curves were subjected to an indirect Fourier transform using the GNOM program (24) to yield the pair distribution function (P(r)), from which maximum particle dimensions (Dmax) and Rg were derived (25). GNOM output files were also used to generate low-resolution ab initio models of the proteins using the dummy atom approach as implemented in DAMMIN (26) or GASBOR (27). Unless noted otherwise, at least ten independent shape reconstructions were performed for each data set. Individual models were aligned by SUPCOMB (28) and averaged by DAMAVER (29) to yield the most probable conformation.
Mismatch repair and excision assays in nuclear extracts were performed as described (16,30). Mismatch-provoked 5′- (31) or 3′- (9) directed excision in purified systems, and MutSα-, RFC-, and PCNA-dependent MutLα endonuclease activation (32) were scored as described.
The interaction of human MutSα with PCNA has been qualitatively documented (3,8,9), but quantitative features of the interaction have not been addressed. To determine the stoichiometry of the MutSα-PCNA interaction, we attempted to isolate a MutSα·PCNA complex by gel filtration. Purified preparations of MutSα and PCNA eluted from Superdex 200 gel with retention times corresponding to Stokes radii of 67 Å and 40 Å, respectively (Table 1), in agreement with previous findings (15,33). However, preincubation of MutSα (1 - 8 μM) with PCNA (1 - 16 μM) followed by gel filtration of the mixture failed to indicate interaction of the two proteins as judged by UV elution profile, and we were unable to detect PCNA in column fractions containing MutSα by SDS gel electrophoresis (not shown).
These findings suggested that the MutSα-PCNA interaction is characterized by a modest affinity and/or a short lifetime. We therefore examined this interaction by equilibrium gel filtration (34). A Superdex 200 column pre-equilibrated with uniform concentration of PCNA was injected with a known amount of MutSα. Association of MutSα and PCNA results in depletion of PCNA at its retention volume, which is manifested as a trough in UV absorbance. The amount of PCNA bound to MutSα was determined from the area of the trough and extinction coefficients of the two proteins. Fig. 1A shows elution profiles for filtration of a 10 μl sample of 8 μM MutSα on a column pre-equilibrated with 0.25 - 1.5 μM PCNA. Trough areas at the PCNA elution volume (~1.35 ml) were used to determine the number of moles of PCNA bound per mole of MutSα. A plot of these values against PCNA concentration fit well to a hyperbola with a KD of 0.7 μM and a stoichiometry of 1.0 PCNA trimer per recombinant MutSα heterodimer (Fig. 1B). Essentially identical values (0.8 μM and 1: 0.94) were obtained using MutSα isolated from HeLa cells. These results were verified by surface plasmon resonance spectroscopy (SPRS) analyses in which excess recombinant MutSα was flowed over a Ni2+-NTA chip containing immobilized His6-PCNA (Fig. 1C). Such experiments yielded a KD of 0.5 μM, and a stoichiometry of one MutSα heterodimer per PCNA trimer. By contrast, analysis of recombinant p21 binding to a His6-PCNA chip at a concentration several hundred times the KD yielded a stoichiometry of three molecules of p21 per PCNA trimer (Fig. 1C, inset), consistent with previous findings (35).
The MSH6 polypeptide contains distinct structural domains (Fig. 2A), and harbors a PCNA interaction motif (QxxL/IxxFF) near the N-terminus that has been implicated PCNA-MutSα interaction (6-8). To evaluate the contributions of this motif to interaction affinity and biological function, we have prepared MutSα derivatives, MutSαΔ341 (14) and MutSαΔ12 (Experimental Procedures), lacking this motif. Both form stable heterodimers, and the structure of MutSαΔ341 is known (14). The Stokes’ radii of MutSαΔ12 and MutSαΔ341 were determined by gel filtration chromatography to be 67 Å and 60 Å, respectively (Table I), consistent with the previously determined Stokes’ radius of native MutSα (15). This finding, coupled with the results of small angle X-ray scattering (SAXS) and DNA binding studies described below suggest that deletion of the PCNA-binding motif does not significantly alter the conformation or stability of MutSα.
The capacity of MutSαΔ12 to bind PCNA was also examined by equilibrium gel filtration (Fig. 2B). Interaction of the two proteins was not detectable by this method, indicating that deletion of the N-terminal PCNA-binding motif of MSH6 reduces the interaction affinity of the two proteins by at least 10-fold (Fig. 1C). The PCNA binding defects of MutSαΔ12 and MutSαΔ341 were also assessed by pull down assay using magnetic beads derivatized with G-T heteroduplex or A·T homoduplex DNA (Fig 2C). Binding of PCNA to A·T or G-T beads in the absence of other proteins was not detectable (lanes 1 and 2). However, PCNA was captured by G-T beads if MutSα was present (lane 4), and was also retained at low levels on A·T beads in the presence of MSH2·MSH6 (lane 3). Although the extent of retention of MutSαΔ341 and MutSαΔ12 by the bead-linked DNAs was comparable to that of native MutSα, PCNA was not detectably retained in the presence of either of the truncated proteins (lanes 5-8).
The influence of DNA binding by MutSα on its ability to associate with PCNA was also assessed by equilibrium gel filtration and SPRS. Fig. 3A shows elution profiles for filtration of a 10 μl sample of 4 μM MutSα through a column pre-equilibrated with 21 bp G-T-heteroduplex (800 nM) and variable concentrations of PCNA. Absorbance profiles showed the presence of two troughs representing simultaneous depletion of PCNA and DNA, due to their association with MutSα. Measurement of trough areas revealed that 1 heteroduplex was bound per MutSα heterodimer. Binding of PCNA to the MutSα·DNA complex was hyperbolic with respect to PCNA concentration, characterized by a KD of 0.6 μM and a stoichiometry of 1:1:1 for the PCNA·MutSα·DNA complex (Fig. 3B).
PCNA interaction with DNA-bound MutSα was also examined by SPRS using 201 bp G-T and A·T DNAs (Supplemental Fig.1A). Supplementation of a 200 nM MutSα flow with 2.5 μM PCNA over a heteroduplex-derivatized sensor chip resulted in an increase in bound mass above that observed with MutSα alone. This mass increase displayed saturation behavior with increasing PCNA concentration, with a limiting stoichiometry corresponding to 1 PCNA trimer per MutSα heterodimer (Supplemental Fig.1B) and an apparent KD of 100 nM. Mismatch dependence of MutSα binding under these conditions was 3-4 fold, and no significant PCNA binding to the DNA chip was observed in the absence of MutSα (Supplemental Fig.1A). The KD for PCNA binding to DNA-bound MutSα observed in these experiments is significantly less than that obtained by equilibrium gel filtration. This may be due to the restricted order of ternary complex assembly in the SPRS experiments (PCNA binding to DNA bound MutSα as opposed to the gel filtration experiments where MutSα can bind either PCNA or DNA prior to ternary complex assembly). However, the KD difference could also be due to avidity and/or rebinding effects in SPRS experiments. Such effects, which have been previously observed in biosensor studies involving flow of a multivalent biomolecule like PCNA over a surface derivatized with a monovalent binding partner, result in anomalously high apparent affinities (36).
A previous study has indicated that yeast PCNA increases the affinity of yeast MutSα for a mismatch (7). However, it has also been reported that while yPCNA forms a complex with homoduplex-bound yMutSα, the trimeric clamp is excluded from the yMutSα·mismatch complex (12). Since these two conclusions are at odds with thermodynamic expectations and because we have found that human PCNA readily binds to the MutSα·mismatch complex, we have used a nitrocellulose membrane assay to evaluate the effects of human PCNA on the heteroduplex and homoduplex affinities of human MutSα. As shown in Fig. 3C, affinities and mismatch dependence of DNA binding by MutSα are comparable when determined in the absence or presence of a large excess of the replication clamp.
We have used SAXS to probe solution conformations of native MutSα and the MutSα·PCNA complex. Fig. 4A shows solution X-ray scattering profiles (I versus Q) for MutSα, MutSαΔ12, MutSαΔ341, PCNA, and MutSαΔ341 complexed to a 15 bp G-T heteroduplex. The corresponding distributions of pairwise interatomic vectors (P(r)) were obtained from scattering profiles by indirect Fourier transform (Fig. 4 B). The model-independent conformational parameters Rg (radius of gyration) and Dmax (maximum particle dimension) obtained from these analyses are summarized in Table 1. For PCNA and the MutSαΔ341·DNA complex, scattering profiles (Supplemental Fig. 2A), as well as Rg and Dmax parameters were calculated from available crystal structures, with the resulting values in good agreement with those determined by SAXS. As observed previously for Sulfolobus solfataricus PCNA, (37), the P(r) profile for human PCNA is bimodal. A second noteworthy feature of the P(r) functions shown in Fig. 4B is that in contrast to that for MutSαΔ341, the P(r) distributions for MutSα and MutSαΔ12 are highly skewed toward larger r values. This suggests that the conformations of these two heterodimers are significantly more elongated than that of MutSαΔ341, which contains 1,953 of the 2,294 residues present in native MutSα.
We also analyzed X-ray scattering by equimolar mixtures of MutSα and the PCNA trimer (Fig. 5A). The Rg value and the forward scattering intensity I(0) determined for 1:1 mixtures of the two proteins are significantly greater than those for either of the individual components (Table 1, Supplemental Fig. 2B). The latter parameter is a linear function of molecular mass (38), and as shown in Supplemental Fig. 2B, the I(0) value observed for the equimolar mixture is consistent with the 344 kDa molecular mass expected for the 1:1 MutSα·PCNA trimer complex that we have observed using other methods (Figs. 11 and and3).3). Because PCNA is potentially trivalent with respect to its partner interactions (35), SAXS data were also collected at variable [PCNA]/[MutSα] ratios. As shown in Fig. 5B, corresponding I(0) values increase until the ratio achieves a value of 1 and then decrease. This is the behavior expected for an interaction model that assumes stoichiometric formation of a 1:1 complex between the two proteins. By contrast, experimental I(0) values differed markedly from those predicted by models that invoke multivalent interaction with the PCNA trimer (Fig. 5B). Furthermore, no increase in I(0) was observed upon PCNA titration of MutSαΔ12 or MutSαΔ341, confirming the severe nature of the interaction defect conferred by deletion of the MSH6 PCNA interaction motif.
The gel filtration studies described above demonstrated an increase in Stokes’ radius from 67 Å for MutSα to 80 Å upon formation of the MutSα·PCNA complex (Fig. 1B), and a similar increase was observed upon PCNA binding to the complex of MutSα with a 21 bp G-T heteroduplex (Fig. 3B, ~ 69 to 83 Å). Corresponding increases were also observed in the similar, but non-identical parameter Rg determined from SAXS measurements. As summarized in Table 1, Stokes’ radii and Rg values determined by these two different methods correlate well.
We have employed the SAXS data described above for construction of low-resolution models of the repair proteins considered here (27). The application of SAXS in these cases is facilitated by the availability of crystal structures for the human MutSαΔ341·DNA complex (14) and PCNA (39), which provide useful controls for assessing validity of the models (40). Fig. 4C summarizes shape reconstructions for MutSαΔ341, the MutSαΔ341·DNA complex, and PCNA. Surface envelopes for the two former proteins are shown superimposed on the crystal structure of the MutSαΔ341·DNA complex, while the latter is superimposed on the PCNA structure. Agreement in each case is good. As can be seen, shape reconstructions for MutSαΔ341 and the MutSαΔ341·DNA complex are similar, and as summarized in Table 1, binding of a 15 bp G-T heteroduplex to MutSαΔ341 is associated with only a 2 - 3 Å decrease in Rg.
As expected, the Dmax value of 202 Å for full length MutSα determined by SAXS is substantially greater than the 130 Å value calculated from the crystal structure of the human MutSαΔ341·DNA complex (14) (Table 1). A low-resolution model determined from SAXS data for the full length protein is shown in the left panel of Fig. 5C. Comparison with the ab initio SAXS structure of MutSαΔ341 (Fig. 4C) reveals a common structural feature, the dimensions of which readily accommodate the MutSαΔ341 crystal structure. In addition, the MutSα SAXS envelope displays additional mass that presumably corresponds to the MSH6 N-terminal 341 residues that are absent in MutSαΔ341.
Two limiting conformations of the MutSα-PCNA complex can be imagined: a stacked complex wherein the DNA binding channels of the two proteins are aligned and an extended conformation in which the two proteins associate in an end-to-end manner with their DNA binding channels orthogonal to the long axis of the complex. Although MutSαΔ341 is unable to bind PCNA, we have utilized the crystal structures of PCNA and the MutSαΔ341·DNA complex to illustrate these two models and have calculated their expected P(r) functions. As shown in Supplemental Fig. 2C, the P(r) function for the stacked complex displays pseudo-symmetry and the function maximum is shifted to a higher r value than the P(r) function calculated from MutSαΔ341 crystal structure. By contrast, the P(r) function predicted for the end-to-end complex is bimodal, but the P(r) maximum is achieved at an r value only slightly greater than that for MutSαΔ341. P(r) functions were calculated for two different isomers of the end-to-end complex that differ with respect to a 90° rotation of the two proteins about the long axis of the complex; as shown in Supplemental Fig. 2C, the calculated P(r) functions for the two isomers were indistinguishable. Experimental X-ray scattering results obtained with the 1:1 complex of PCNA with native MutSα are similar to those predicted for the end-to-end models. The P(r) function determined from MutSα·PCNA SAXS data displays a distinct shoulder at r values of ~100 - 200 Å, but achieves its maximum at a value of r that is essentially identical to that for native MutSα and only slightly greater than that for MutSαΔ341. Low-resolution shape reconstructions are also indicative of an extended conformation. Fig. 5C (left panel) shows the results of a single shape reconstruction for the MutSα·PCNA complex, with the results of seven other independent reconstructions shown in Supplemental Fig. 3. Unlike the other low-resolution conformations described above, this set of reconstructions was not averaged. While all reconstructions were consistent with an extended conformation, placement of MutSαΔ341 and PCNA crystal structures within the set of envelopes was possible only if the rotational orientation of the two proteins about the long axis of the complex was allowed to vary. This is consistent with model calculations described above indicating that P(r) is insensitive to the rotational orientation of the two proteins in the extended complex.
To assess the functional significance of the MutSα·PCNA interaction, we compared the activities of MutSα and MutSαΔ12 in mismatch repair and several partial reactions implicated in overall repair. As shown in Fig. 6A, the two heterodimers do not differ significantly in their ability to restore mismatch-provoked excision to nuclear extracts prepared from MSH6-/- HCT-15 tumor cells, and similar results have been obtained with extracts prepared from MSH2-/- RL95-2 cells (not shown). MutSα and MutSαΔ12 were also compared in several purified systems. The two proteins are similarly active in their ability to support MutSα-, RFC-, and PCNA-dependent activation of the MutLα endonuclease (32) and are comparable in their ability to support 5′-directed mismatch-provoked excision in a purified system comprised of MutSα, MutLα, Exo1, and RPA (31) or 3′-directed excision in the presence of MutSα, MutLα, Exo1, RFC, PCNA, and RPA (9) (Fig. 6B). Similar results were obtained with MutSαΔ341 in reconstituted excision reactions (not shown). However, the MutSαΔ12 mutation confers a partial, but significant and reproducible defect in the overall mismatch repair reaction. While MutSα and MutSαΔ12 are similarly active in restoration of 3′-directed mismatch repair to nuclear extracts of MSH6-/- HCT-15 cells, MutSαΔ12 is only about half as active as the wild type protein in its ability to restore 5′-directed repair (Fig 6C). Essentially identical results were obtained with extracts derived from MSH2-/- RL95-2 (not shown), and we have previously shown that MutSαΔ341 displays a similar partial defect that is restricted to the 5′-directed mismatch repair reaction (14). Thus, while dispensable for 3′- or 5′-directed excision steps, the N-terminal PCNA binding motif of MSH6 plays a significant role in 5′-directed mismatch repair when the overall reaction is scored.
PCNA, the trimeric replication sliding clamp is believed to coordinate various DNA metabolic processes via its interactions with components of replication, repair, and recombination systems. In human mismatch repair, PCNA has been implicated at two stages in the reaction: it is required for activation of the MutLα endonuclease (32) but is also necessary for the repair synthesis step of the reaction (3), functions that presumably depend on its known ability to interact with multiple mismatch repair factors, including MutSα, MutLα, Exo1 and DNA polymerase δ. However, the conformational nature of these complexes and the contributions of individual interactions to various steps of the reaction remain largely uncertain. We have addressed these issues for the human MutSα-PCNA complex. Previous studies with yeast proteins led to the suggestion that MutSα and PCNA associate to form an active mispair recognition complex, which has a higher affinity for heteroduplex DNA, and that PCNA is released upon mismatch binding (7,12). By contrast, we observe that human MutSα binds heteroduplex DNA with comparable affinity and specificity in the absence or presence of PCNA. Consistent with these observations, mismatch-bound MutSα has an affinity for PCNA that is very similar to that of free MutSα.
A key function of PCNA in mismatch repair is its role in activation of the latent endonuclease of MutLα (32). This endonuclease, which also depends on MutSα and RFC for its activation, introduces additional breaks into the discontinuous strand of a nicked heteroduplex that serve as entry sites for Exo1. Despite the severe nature of the PCNA interaction defect conferred by MutSαΔ12 and MutSαΔ341 mutations, MutSαΔ12 supports MutLα activation and both mutants are essentially fully active in their abilities to support mismatch-provoked excision in a reconstituted system. Furthermore, MutSαΔ12 supports normal levels of mismatch-provoked excision upon supplementation of MutSα-deficient nuclear extracts. Thus, the interaction between MutSα and PCNA is not critical for early steps of mismatch repair, suggesting that PCNA interaction with other activities such as MutLα and/or Exo1 account for involvement of the replication clamp in early steps of the reaction. Indeed, a recent study has suggested an important effector role for PCNA in function of the MutLα endonuclease (41).
While removal of the PCNA interaction motif from MutSα has little if any effect on MutLα endonuclease activation or mismatch-provoked excision steps of the reaction, MutSαΔ12 and MutSαΔ341 both display a limited but reproducible defect in 5′-, but not 3′-directed mismatch repair. Because we have been unable to detect a corresponding defect in 5′-directed mismatch-provoked excision (scored under conditions of DNA synthesis block), we infer that this partial 5′-repair defect is manifested at the repair synthesis step. This defect, which reduces the rate of the reaction ~50%, may indicate MutSα participation in the DNA synthesis step of 5′-directed repair. It is also noteworthy that the nature of this defect is consistent with the significant but limited mutability increase that has been observed upon alanine substitution of conserved residues within the PCNA interaction motif of MSH6 (6,7). In contrast to our results with Δ12 and Δ341 MutSα variants, removal of the 77 N-terminal residues of human MSH6 has been shown to substantially reduce the mismatch repair activity MutSα in human cell extracts (8). It is possible that the MSH6Δ77 mutation interferes with the interaction of MutSα with mismatch repair factors other than PCNA or compromises function of the protein in a manner unrelated to its inability to interact with PCNA.
The nature of PCNA lends itself to models wherein a single homotrimeric ring can interact with up to three molecules of a binding partner. In fact, human PCNA is capable of binding 3 molecules of p21 or Fen1 (35,42). However, the several different experimental approaches used here demonstrate that the interaction stoichiometry of human MutSα with the PCNA trimer is restricted to 1:1, even under conditions of large MutSα excess. Given the trivalent nature of PCNA, the 1:1 stoichiometry limitation may be due to steric factors, although it is important to note that while the MutSα·PCNA complex cannot bind a second molecule of MutSα, the PCNA component of the complex might be capable of simultaneous interaction with another repair or replication activity.
Our low-resolution SAXS models reveal that MutSα and PCNA associate in an end-to-end fashion in solution to form an elongated complex in which the DNA binding channels of the two proteins are not aligned, but rather are orthogonal to the long axis of the complex. While contrary to expectation, a similar extended conformation has been demonstrated for the Sulfolobus solfataricus ligase·PCNA complex in solution (37). Because MSH6 residues 1-341 are not present in MutSαΔ341, for which the crystal structure has been determined (14), the structure of the N-terminal MSH6 domain has been uncertain. However, recent NMR studies have demonstrated that residues 89-194 of human MSH6 adopt a globular conformation (Sophie Zinn-Justin, personal communication; PDB accession 2GFU). Our SAXS reconstructions with native MutSα and the MutSα·PCNA complex are also consistent with the idea that the N-terminal domain of MSH6 has globular features and interacts with PCNA to form a complex with significantly defined conformational character. By contrast, SAXS studies of an N-terminal fragment of yeast MSH6 (residues 1-304) have indicated that this polypeptide exists in an extended unstructured conformation, leading Shell et al. (43) to conclude that yeast MutSα interacts with PCNA by virtue of a highly extended unstructured tether. While the Shell et al. study included SAXS data collection on full-length yeast MutSα in the absence or presence of PCNA, surface reconstructions for these polypeptide complexes were not described. It is also noteworthy that while the yeast MSH6 N-terminal segment supports PCNA interaction with 3:1 stoichiometry (43), native yeast MutSα, probably interacts with the yeast PCNA trimer with 1:1 stoichiometry (7). The significance of these apparent differences in the nature of the human and yeast MutSα·PCNA complexes thus remain to be resolved.
We thank Joshua Warren for many helpful discussions and Sophie Zinn-Justin for sharing unpublished results.
*This study was supported in part by R01 GM45190 and P01 CA92584 from the National Institutes of Health. P.M. is an Investigator of the Howard Hughes Medical Institute.