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MutLα and Proliferating Cell Nuclear Antigen Share Binding Sites on MutSβ*

MutSβ (MSH2-MSH3) mediates repair of insertion-deletion heterologies but also triggers triplet repeat expansions that cause neurological diseases. Like other DNA metabolic activities, MutSβ interacts with proliferating cell nuclear antigen (PCNA) via a conserved motif (QXX(L/I)XXFF). We demonstrate that MutSβ-PCNA complex formation occurs with an affinity of ∼0.1 μm and a preferred stoichiometry of 1:1. However, up to 20% of complexes are multivalent under conditions where MutSβ 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 MutSβ not only abolishes PCNA binding, but unlike MutSα, also dramatically attenuates MutSβ-MutLα interaction, MutLα endonuclease activation, and bidirectional mismatch repair. As predicted by these findings, PCNA competes with MutLα for binding to MutSβ, an effect that is blocked by the cell cycle regulator p21CIP1. We propose that MutSβ-MutLα 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 MutSβ- and MutSα-initiated repair events differ in fundamental ways.

The mammalian mismatch repair system stabilizes the genome by correcting DNA biosynthetic errors, preventing illegitimate recombination events, and participating in the cellular response to certain types of DNA damage (reviewed in Ref. [1][2][3][4][5]. Mismatch repair deficiency is the cause of hereditary nonpolyposis colorectal cancer but may also be involved in the development of a subset of sporadic tumors (6).
The human mismatch recognition activities MutS␣ (MSH2-MSH6) and MutS␤ (MSH2 and MSH3) differ in their substrate specificities: MutS␣ recognizes base-base mismatches and some insertion-deletion (I/D) 2 mismatches, whereas MutS␤ predominantly processes I/D substrates (1)(2)(3)(4)(5). MutS␣ is also capable of recognizing certain types of DNA damage and participates in the checkpoint response to such lesions (7), whereas MutS␤ is believed to cooperate with the nucleotide excision repair machinery in the repair of interstrand cross-links (8,9). Thus, there is substantial overlap between the substrates recognized and processed by these two activities, but the determinants that govern whether a particular lesion is processed by MutS␣ or MutS␤ are not known.
Although MutS␣ and MutS␤ are generally regarded as genetic stabilization activities, both heterodimers have been implicated in the production of certain mutations. MutS␣ participates in the somatic hypermutation phase of immunoglobulin gene affinity maturation (10), and MutS␤ is required for the triplet repeat expansions that are responsible for a number of neurodegenerative diseases (11).
Heteroduplex repair reactions initiated by MutS␣ and MutS␤ have been reconstituted in a purified systems that also contain MutL␣ (MLH1-PMS2), exonuclease 1 (Exo1), RPA (replication protein A), PCNA, RFC, and DNA polymerase ␦ (12)(13)(14)(15)(16). Initiation of repair in the MutS␣-dependent system involves activation of a latent endonuclease of MutL␣ in a reaction that requires a mismatch, MutS␣, RFC, PCNA, and ATP (16). Action of the MutL␣ endonuclease is directed to the heteroduplex strand that contains a pre-existing break and is biased to the distal side of the mismatch to yield a molecule in which the mismatch is bracketed by strand breaks. This multiply incised intermediate serves as substrate for Exo1, which is activated by MutS␣ in a mismatch-dependent manner, leading to mismatch removal. The ensuing gap is filled by RPA and repaired by DNA polymerase ␦ in a reaction that also depends on PCNA and RFC. Although a MutS␤-dependent repair reaction has been reconstituted from purified components (15), it is not known whether activation of the MutL␣ endonuclease occurs in a MutS␤-dependent manner.
Coordination of these activities during the course of repair is presumably mediated by a temporally evolving set of proteinprotein and protein-DNA interactions. The most thoroughly studied of the multi-protein assemblies involved in mismatch repair have been the MutS␣-MutL␣ complex that assembles on heteroduplex DNA (17)(18)(19) and the MutS␣-PCNA complex that has been observed both in solution and on DNA (20,21). Although the former complex is generally believed to play an important role in the reaction (22), disruption of the MutS␣-PCNA interaction confers only a partial mismatch repair defect in vivo and in vitro (20,21,23). Although the MutS␤-PCNA and MutS␤-MutL␣ complexes have been the subject of only limited study (23)(24)(25)(26), yeast strains carrying mutations in the PCNA-binding motif of MSH3 display hypermutability similar to that of MSH3 null mutant (23). Also, little is known regarding the functional significance of the MutS␤-MutL␣ interaction. We demonstrate here that MutS␤ differs from MutS␣ in the manner that it interacts with PCNA and MutL␣.

EXPERIMENTAL PROCEDURES
MutS␤-expressing Baculovirus Constructs-The baculoviral donor plasmid pFastBacDual-MSH2-MSH3 (9) that harbors full-length MSH2 and MSH3 was modified by PCR mutagenesis to yield pFastBacDual-MSH2-MSH3⌬28, which contains intact MSH2 and an N-terminally truncated MSH3 gene encoding amino acids 29 -1137 of full-length MSH3 beginning with N-Met (see Fig. 1A). A second construct, pFastBacDual-MSH2-MSH3-F27A-F28A harboring MSH3 that contains Phe to Ala amino acid substitutions at positions 27 and 28 (see Fig.  1A), was also prepared by PCR mutagenesis of the full-length construct. High titer recombinant baculoviruses were prepared from the expression vectors and used to infect Sf9 cells for protein expression.
DNA Substrates and Proteins-Bacteriophages f1MR72 and f1MR73 were constructed by oligonucleotide mutagenesis of f1MR23 and f1MR24 (27), respectively, resulting in substitution of an EcoRV site for residues 5501-5506 as described for phages f1MR70 and f1MR71 (28). 6,440-bp dinucleotide insertion/deletion heteroduplex DNAs (-TG-) were constructed as described (27) from phages f1MR72 and f1MR73 and contained a site-specific nick in the complementary DNA strand 128 bp 3Ј or 5Ј to the mismatch as viewed along the shorter path in the circular molecules. The strand break for 5Ј -TG-was introduced by cleavage with Sau96I, whereas the strand break in 3Ј -TG-was introduced by cleavage with EcoRV (28). Substrates for analyses of DNA-protein assemblies by SPRS were 200 bp in length and were prepared as follows. Primers 5Ј-CCGCTA-CACTTGCCAGCGCCA-3Ј and 5Ј-biotin-GTTCAAAAAAC-CCCAGCTCC-3Ј were used to generate 200-and 202-bp PCR products from f1MR23 and f1MR24 (27), respectively. The strands were separated by denaturing high pressure liquid chromatography (29) and reannealed to generate a heteroduplex containing a centrally positioned -TG-loop, or an otherwise identical homoduplex.
MutS␤, MSH2-MSH3⌬28 (referred to as MutS␤⌬28), and MSH2-MSH3-F27A-F28A (referred to as MutS␤-F27A-F28A) were prepared from baculovirus-infected Sf9 cells by a procedure essentially identical to that described for native human MutS␤ (30). All other proteins were purified as described in the work of Iyer et al. (21) and the references listed therein. Concentrations of MutS␤ are expressed as heterodimer equivalents using an extinction coefficient of 136,690 M Ϫ1 cm Ϫ1 at 280 nm for MutS␤ (31).
Analyses of Protein-Protein and Protein-DNA Assemblies-Gel filtration chromatography was performed at 4°C as described (21). SPRS experiments were done on a Biacore 2000 (17,21) in a buffer composed of 25 mM HEPES-NaOH, pH 7.5, 150 mM NaCl (unless otherwise indicated), 5 mM MgCl 2 , 1 mM EDTA, and 0.1 mM dithiothreitol. Streptavidin sensor chips were derivatized with ϳ200 response units of a 200-bp -TG-I/D heteroduplex or an otherwise identical homoduplex DNA via a 5Ј biotin tag.
Far Western analyses were performed by spotting 0.25-4 pmol of the indicated proteins on a nitrocellulose membrane (Protran, Whatman) or by electrophoresing 2 pmol of protein through a 7% SDS-polyacrylamide gel, followed by transfer to nitrocellulose. After incubation in blocking buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.1% Triton X-100, and 5% milk solids) for 1 h at room temperature, the membrane was incubated overnight at 4°C with PCNA or MutL␣ in blocking buffer as indicated, followed by two buffer washes. The presence of bound MutL␣ or PCNA was detected immunochemically with mouse anti-MLH1 or anti-PCNA antibodies.
Small Angle X-ray Scattering (SAXS) Experiments-SAXS was performed on the Sibyls Beamline 12.3.1 at ALS, Berkeley. The scattering data were collected over a range of protein concentrations (10 -50 M) in a buffer containing 25 mM HEPES-KOH, pH 7.5, 150 mM KCl, 5 mM MgCl 2 , and 1 mM dithiothreitol, and data analyses were performed as described (21). For SAXS-based stoichiometry measurements, the scattering data were collected for mixtures of PCNA and MutS␤ (or MutS␤⌬28) in which PCNA concentration was varied (3.3-105 M) at a fixed concentration of MutS␤ (10, 20, or 35 M) or MutS␤⌬28 (10 M) in the buffer described above. Concentration-normalized scattering data were then used to derive forward scattering intensities I(0) (intensity at ϭ 0°) (32). The theoretical I(0) for a protein mixture was estimated as the sum of the expected I(0) contributions of each component of that mixture, the latter property being the product of a mass fraction of the component and the I(0) value corresponding to its molecular mass (supplemental Fig. S2B). Because protein concentrations were much higher than the K d for MutS␤-PCNA interaction (see Fig. 1), the limiting species was assumed to be present only in the (MutS␤) n -PCNA complex.
Mismatch Repair Assays-Bidirectional mismatch-provoked excision and repair assays were carried out at 37°C for 30 min by complementation of 100 g of RL95-2 (MSH2 Ϫ/Ϫ ) nuclear extracts with recombinant MutS␤ as indicated in 10-l reactions containing 20 mM Tris-HCl, pH 7.6, 110 mM KCl, 5 mM MgCl 2 , 1 mM reduced glutathione, 1.5 mM adenosine triphosphate, 0.05 mg/ml bovine serum albumin, and a 6440-bp -TG-I/D heteroduplex or homoduplex substrate (2.4 nM) (21). Mismatch repair in extracts was performed in a similar manner, except that the reactions also contained 0.1 mM each of dATP, dTTP, dCTP, and dGTP. Excision was scored by conversion of DNA to a NheI-resistant form (33), and repair was measured by restoration of XcmI sensitivity to the heteroduplex DNA (27). MutL␣ endonuclease assays (16) were carried out in a purified system composed of MutS␤ (22 nM), MutL␣ (50 nM), RFC (15 nM), PCNA (100 nM), and an I/D heteroduplex or homoduplex DNA substrate (1.2 nM) at 37°C for 10 min in a 20-l reaction in the buffer described above. The reaction products were resolved on alkaline agarose gels, and the extent of MutL␣-catalyzed incision was measured by indirect end labeling (16).

MutS␤ and PCNA Form a High
Affinity 1:1 Complex-MutS␤ interacts with PCNA via a conserved QXX(L/I)XXFF motif that resides near the N terminus of MSH3 (23,24,26), but the molecular nature of the MutS␤-PCNA complex has not been examined. To address the nature of this interaction as well as the functional consequences of its disruption, we constructed MSH3 variants in which the PCNA-binding motif (PIP box) was either deleted (MSH3⌬28) or altered by amino acid substitution mutation (MSH3-F27A-F28A) (Fig. 1A). These variants were co-expressed with MSH2 and purified as stable heterodimers referred to as MutS␤⌬28 and MutS␤-F27A-F28A. Both mutant heterodimers display Stokes radii comparable with the wild type protein as determined by gel filtration chromatography (Table 1). To determine whether these MSH3 mutations altered the mismatch recognition activity of MutS␤, we used SPRS to measure affinities of MutS␤, MutS␤⌬28, and MutS␤-F27A-F28A for a 200-bp -TG-dinucleotide I/D heteroduplex or an otherwise identical homoduplex. The apparent affinities and specificities of the MutS␤ variants did not differ significantly from that obtained with the wild type protein (Fig. 1B). Furthermore, wild type and mutant forms of MutS␤ dissociated with similar kinetics from heteroduplex DNA upon ATP challenge (not shown). The presence of 2.0 M PCNA, a saturating concentration for MutS␤-PCNA complex formation (see below), did not significantly alter the affinity or specificity of MutS␤ interaction with heteroduplex/homoduplex DNA (Fig. 1B).
The MutS␤-PCNA complex was characterized by the Hummel-Dreyer equilibrium gel filtration procedure that was previously employed to determine equilibrium parameters that govern formation of the MutS␣-PCNA complex (21). The Hummel-Dreyer elution profile for MutS␤ (Fig. 1C) in the presence of 0.75 M PCNA shows two distinct but overlapping peaks followed by a trough at ϳ1.35 ml that represents depletion of PCNA from the running buffer caused by complex formation with MutS␤. Because the later eluting peak (1.14 ml) corresponds to free MutS␤, we infer that the earlier peak (1.1 ml) represents the MutS␤-PCNA complex. By contrast, MutS␤⌬28 and MutS␤-F27A-F28A elute as single peaks (1.14 ml) corresponding to uncomplexed MutS␤. Furthermore, as judged by absence of a significant trough at 1.35 ml, the two mutant heterodimers fail to deplete PCNA from the equilibrating buffer, indicating that their ability to bind PCNA has been severely compromised. The extent of PCNA binding to MutS␤ was a hyperbolic function of PCNA concentration corresponding to an apparent K d of 0.10 M and a stoichiometry of 0.9 PCNA trimer/MutS␤ heterodimer (Fig.  1D). The formation of a complex with PCNA increases the Stokes radius of MutS␤ from 64 to 74 Å (Table 1).
PCNA binding to DNA-bound MutS␤ was evaluated by SPRS. As determined by this procedure, DNA-bound MutS␤ interacts with PCNA with an apparent K d of 0.020 M and a stoichiometry of 1.1 mol of PCNA trimer/mol of MutS␤ heterodimer (Fig. 1C). Although MutS␤⌬28 and MutS␤-F27A-F28A efficiently bind heteroduplex DNA (Fig. 1B), PCNA interaction with the heteroduplex-bound mutant proteins was almost undetectable (Fig. 1E). The apparent affinity of PCNA for DNA-bound MutS␤ as judged by SPRS is ϳ5-fold higher than that for free MutS␤ as determined by the Hummel-Dreyer method. A similar difference has been observed for affinities of the MutS␣-PCNA complex determined by the two methods (21) and is likely due to avidity or rebinding artifacts that are known to occur when multivalent species such as PCNA are present in the mobile phase of SPRS analysis (34).
MutS␤ PIP Box Mutants Are Defective in MutL␣ Interaction-Interaction between MutS and MutL homologs has been documented in several systems (3,4), but the nature of the human MutL␣-MutS␤ complex has not been addressed. We have therefore used SPRS to examine the capacity of MutS␤ and its PIP box mutants to support formation of a MutL␣-MutS␤-DNA ternary complex. As shown in Fig. 2A, in the presence of ATP-Mg ϩ2 MutS␤ forms specific but short-lived complexes with the 200-bp -TG-I/D heteroduplex described above. Inclusion of both MutL␣ and MutS␤ resulted in a substantial increase in DNA-bound mass ( Fig. 2A), indicative of ternary complex formation. This mass increase was not observed in the absence of ATP (not shown), and we were unable to detect DNA binding by MutL␣ alone (not shown). The latter results are consistent with previous findings that assembly of ternary complexes involving MutL and MutS homologs is ATP-dependent and that DNA binding by MutL homologs is limited at physiological ionic strength (17,25,(35)(36)(37)(38). The lifetime of the ternary complex is also short, with 80% dissociating with a t1 ⁄ 2 of 7 s. The apparent affinity of MutL␣ for DNA-bound MutS␤ was estimated from the MutL␣ dependence of the mass increase over and above that observed with MutS␤ alone (0.1 M). As shown in Fig. 2B, the results fit well to a rectangular hyperbola with a K d of 0.4 M.
To our surprise, substitution of MutS␤⌬28 or MutS␤-F27A-F28A ( Fig. 2A, middle and bottom panels) for MutS␤ abolished ternary complex formation, indicating that MSH3 PIP box mutations compromise the ability of DNA-bound MutS␤ to interact with MutL␣. By contrast, deletion of the PIP box motif of MutS␣, although severely attenuating the capacity of MutS␣ to associate with PCNA (21), does not alter the ability of the MSH2-MSH6 heterodimer to support ternary complex formation with MutL␣ and heteroduplex DNA (supplemental Fig. S1).
To assess the interaction of MutS␤ with MutL␣ by an independent method, we employed far Western analyses wherein proteins applied to a nitrocellulose membrane were incubated with PCNA or MutL␣ in solution, followed by immunochemical detection of membrane-bound PCNA or MutL␣. As shown in Fig. 2C (top panel), complexes of PCNA with MutS␣, MutS␤, and MutL␣ can be detected by this method, confirming previous findings (14,26). However, binding of PCNA to MutS␤⌬28 and MutS␤-F27A-F28A to PCNA was substantially reduced compared that of wild type MutS␤. When MutL␣ was in solution (Fig. 2C, bottom panel), robust complex formation with membrane-bound MutS␤ was observed. However, binding of MutL␣ to both MutS␤⌬28 and MutS␤-F27A-F28A was severely compromised. Although PCNA in solution is able to associate with membrane-bound MutL␣, such an interaction was not observed in the converse experiment wherein MutL␣ was in solution and PCNA was membrane-bound. The reason for this difference is not clear, but it is possible that PCNA binding to the membrane may occlude access to its MutL␣binding surface. An alternate possibility is that the ␣-MLH1 antibody used to probe for presence of PCNA-bound MutL␣ may compete with PCNA for a common binding site. MutL␣ in solution also interacts poorly with nitrocellulose-bound MutS␣, possibly because of membrane occlusion effects. Taken   TABLE 1

Biophysical properties of MutS␤ and MutS␤-PCNA derived from SAXS and gel filtration
The radii of gyration (R g ) were determined from Guinier plots (supplemental Fig. S2A, inset) (32) in which SAXS data collected over a range of protein concentration (supplemental Fig. S2C) were extrapolated to zero concentration. The maximum particle dimension (D max ) was estimated from the P(r) plots (49) shown in Fig. 5A. The Stokes radii were determined by gel filtration ("Experimental Procedures"). See also supplemental Fig. S2.

Response Units
MutSβ (  together with the SPRS ternary complex observations described above, these far Western results indicate that the MutS␤ motif responsible for its interaction, MutL␣, resides at least in part within the N-terminal 28 residues of MSH3 and includes Phe-27 and Phe-28. In fact, strong MutL␣ interaction with the MSH3 subunit of MutS␤ was directly demonstrable by far Western analysis of membrane transfers from SDS gel-resolved MutS␤ subunits, and this interaction was abolished by the PIP box mutations described above (Fig. 2D).
PCNA Competes with MutL␣ for Binding to MutS␤ but Not to MutS␣-The simplest interpretation of these findings is overlap of MSH3 binding sites for PCNA and MutL␣, an idea that predicts competition of the two proteins for complex formation with MutS␤. To test this possibility, we used SPRS to examine effects of PCNA on DNA-MutS␤-MutL␣ ternary complex formation. The addition of 2.0 M PCNA to a solution containing MutS␤ results in an increase in mass bound to the sensor surface consistent with MutS␤-PCNA complex formation (Fig.   3A, compare gray and green lines). The addition of the same amount of PCNA to a mixture of MutS␤ and MutL␣ results in a substantial decrease in chip-bound mass as compared with that in the absence of the clamp (Fig. 3A, compare red and black lines), indicating that PCNA inhibits the assembly of the DNA-MutS␤-MutL␣ ternary complex. PCNA inhibition of this reaction increases with increasing PCNA concentration (Fig. 3B), an effect that is reversed by the presence of a molar excess of the PCNA-binding cell cycle regulator p21 CIP1 (Fig. 3C).
As discussed above, unlike MutS␤ PIP box mutants, MutS␣⌬12, which lacks the MSH6 PCNA interaction motif, is proficient in assembly of the DNA-MutS␣-MutL␣ ternary complex (supplemental Fig. S1). It might therefore be expected that assembly of the MutS␣ ternary complex would be refractory to the inhibitory effect of PCNA. Indeed, the presence of PCNA in solutions containing MutS␣, MutL␣, and ATP resulted in a further increase in DNA-bound mass (Fig. 3D, compare red and black lines), indicating the formation of a DNA-MutS␣-PCNA-MutL␣ quarternary complex. Interestingly, the resultant mass increase is ϳ2-3-fold greater than that caused by PCNA association with the DNA-MutS␣ complex, which may indicate that MutL␣ promotes multiple MutS␣ loading events or that PCNA may interact with both the MutS␣ and MutL␣ components of the ternary complex (Fig. 2C) (13). By contrast, MutS␣⌬12 fails to support formation of a DNA-MutS␣⌬12-PCNA-MutL␣ quarternary complex (not shown), indicating that initial association of PCNA with the DNA-MutS␣-MutL␣ ternary complex occurs via multiply loaded MutS␣ molecules. These data suggest that in contrast to MutS␤, MutS␣ has distinct binding sites that can be simultaneously occupied by MutL␣ and PCNA.
The MSH3 PIP Box Is Required for MutL␣ Endonuclease Activation and Bidirectional Mismatch-provoked Excision and Repair-We have previously shown that MutS␣⌬12, although unable to associate with PCNA, retains mismatch recognition activity, supports MutL␣ endonuclease activation, and is as active as the wild type protein in mismatch-provoked excision. The mutant does, however, display a partial defect in 5Ј-but not 3Ј-directed mismatch repair (21). To determine the functional consequences of disruption of the MSH3 PIP box motif in MutS␤, we examined the activities of MutS␤ PIP box mutants in the nuclear extracts of MSH2 Ϫ/Ϫ RL95-2 cells and in a purified system that scores MutL␣ endonuclease activation. As judged by extract assay, MutS␤⌬28 displays a severe defect in 5Ј-and 3Ј-excision and repair as compared with the wild type protein (Fig. 4A). Unlike the wild type protein, the capacity of this truncation mutant to activate the MutL␣ endonuclease is also severely attenuated (Fig. 4B). The initial rates of MutS␤-dependent MutL␣ endonucleolytic activity supported by MutS␤-F27A-F28A are also ϳ3-4-fold lower than the wild type protein (data not shown). Thus, unlike MSH6, the integrity of the MSH3 PIP box is required for mismatch repair. Fig. S2A shows solution x-ray scattering data and linear portions of Guinier plots for MutS␤, MutS␤⌬28, PCNA, and an equimolar mixture of MutS␤ and PCNA. The corresponding pairwise interatomic distances distributions (P(r)) were derived from scattering profiles by indirect Fourier transform (Fig. 5A). Table 1 summarizes the model-independent structural parameters R g and D max obtained from these experiments. As observed previously for MutS␣ (21), the P(r) distributions for MutS␤ and MutS␤⌬28 are skewed toward larger r values, indicating that the conformations of these two heterodimers are significantly more elongated than the published structures of truncated forms of MutS homologs (supplemental Fig. S2D) (39,40). The addition of one PCNA trimer equivalent to MutS␤ results in a further skewing of the P(r) distribution toward higher values.

Solution Conformations of MutS␤ and the MutS␤-PCNA Complex-Supplemental
As noted above (Table 1), gel filtration studies showed an increase in Stokes radius for MutS␤ from 64 to 74 Å because of formation of the MutS␤-PCNA complex. The similar but dis-tinct conformational parameter, R g , measured by SAXS also increases when one PCNA equivalent is added to MutS␤, an effect that is not observed with MutS␤⌬28 (Table 1). The SAXS data also permit extraction of forward scattering intensity I(0), which is a linear function of molecular mass (41) (supplemental Fig. S2B). I(0) values determined for 1:1 mixtures of the MutS␤ and PCNA proteins are substantially greater than those for either of the individual molecules (supplemental Fig. S2B) and are consistent with an expected molecular mass of a 1:1 MutS␤-PCNA trimer complex (318 kDa). By contrast, an equimolar mixture of MutS␤⌬28 and PCNA yields a significantly lower value for I(0), indicating that the two proteins fail to interact (see below).
Given the trivalent nature of PCNA (42), it is potentially possible to assemble MutS␤-PCNA complexes of differing stoichiometries: MutS␤-PCNA, (MutS␤) 2 -PCNA, and (MutS␤) 3 -PCNA, with monovalent complexes favored under conditions of PCNA excess. As discussed above, the stoichiometry of this interaction determined by equilibrium gel filtration and SPRS is 1 MutS␤ heterodimer/PCNA homotrimer. Because complex formation in both of these procedures was measured under conditions where PCNA was in excess, these experiments are insensitive to the formation of potential (MutS␤) 2 -PCNA and (MutS␤) 3 -PCNA assemblies. To address this issue, we determined I(0) values for PCNA-MutS␤ mixtures as a function of molar ratio under conditions where the concentration of each protein was well above the K d for binary complex formation. As shown in Fig. 5B, the experimental maximum I(0) occurs at a PCNA:MutS␤ molar ratio of 0.5, indicative of the presence of multivalent species. Comparison of experimentally determined values with those calculated for mixtures of (MutS␤) 2 -PCNA and MutS␤-PCNA or (MutS␤) 3 -PCNA and MutS␤-PCNA demonstrated that the MutS␤-PCNA complex is the favored species at PCNA:MutS␤ molar ratios Ն1, but as many as 20% of the complexes are multivalent at lower values (supplemental Table S1). Furthermore, I(0) does not increase as a function of the MutS␤⌬28:PCNA ratio (Fig. 5B), a finding that independently confirms the PCNA interaction defect of this MutS␤ variant.
Ab Initio Shape Reconstructions for MutS␤ and the MutS␤-PCNA Complex from SAXS Data-Model-independent D max values (Table 1) (21) indicate that the MutS␤ conformation in solution (D max ϭ 165 Å) is more extended than MutS␣⌬341 (D max ϭ 140 Å) but more compact than MutS␣ (D max ϭ 202 Å). Because a crystal structure of MutS␤ is not available, the SAXS results described above were used to generate low resolution conformational models (43) of the heterodimer, as well as its complex with PCNA. The validity of the models was assessed using the crystal structures of the human MutS␣⌬341-DNA complex (40) and PCNA (42). Ab initio envelopes of MutS␤ and MutS␤⌬28 (Fig. 5C) accommodate superimposition of the crystal structure of the MutS␣⌬341-DNA complex and display additional mass that presumably corresponds to portions of MutS␤ that do not share sequence or conformational homology with MutS␣⌬341.
As noted above, the addition of one trimer equivalent of PCNA to MutS␤ results in a P(r) plot that is skewed toward higher interatomic distances by ϳ 55 Å, an effect that is mani- fested as a substantial increase in D max (Fig. 5A and Table 1). However, the maximum P(r) value for the MutS␤-PCNA complex occurs at an r value (56 Å) that is nearly identical to that for MutS␤ alone (54 Å). These results suggest that the MutS␤-PCNA complex adopts an "extended" end-to-end conformation rather than a stacked arrangement wherein the DNA-binding channels of the two proteins are juxtaposed, because the latter conformer would be expected to display a P(r) maximum at a substantially higher r value (21). In fact, ab initio shapes generated from SAXS data are consistent with an end-to-end association of MutS␤ and PCNA (Fig. 5D), with the ring shape of PCNA being clearly defined in the low resolution models along with substantial associated mass consistent with presence of a MutS homolog dimer equivalent. By contrast to the variety of extended ab initio shapes obtained for the MutS␣-PCNA complex (21), low resolution MutS␤-PCNA models are strik-ingly similar (Fig. 5D), suggesting that there is limited variability between individual MutS␤-PCNA conformers in solution.
Because MSH3, like MSH6, interacts with PCNA via a PIP box located near its N terminus, it might be expected that the MutS␤-PCNA complex may display similar characteristics. However, our results indicate that this is not the case. The affinity of MutS␤ for PCNA is ϳ8-fold higher than that of MutS␣, a difference that may be necessitated by the fact that the MutS␤ levels in human cells are 5-8-fold lower than that of the MSH2-MSH6 heterodimer (30, 44). Furthermore, although the stoichiometry of the MutS␣-PCNA complex is limited to 1:1 even when MutS␣ is in molar excess (21), as much as 20% of the MutS␤-PCNA complexes are multivalent under conditions of MutS␤ excess. This valency difference could reflect steric factors in that MutS␤ is significantly smaller than MutS␣. Despite these differences, complex formation between MutS␤ and PCNA does not significantly alter the affinity or specificity of MutS␤ for a -TG-insert, a property it shares with MutS␣.
However, the most striking difference between MutS␤ and MutS␣ is the finding described here that the modes of interaction of the two mismatch recognition activities with PCNA and MutL␣ differ dramatically. In contrast to MutS␣, which can interact independently with PCNA and MutL␣, interaction of these two proteins with MutS␤ occurs in an either/or fashion. As discussed above, MSH3 PIP box mutations compromise MutS␤ interaction with both PCNA and MutL␣. Furthermore, PCNA competes with MutL␣ for binding to MutS␤ and inhibits ATP-dependent assembly of the DNA-MutS␤-MutL␣ ternary complex, an effect that is reversed by p21, which is known to  Fig. S2B) are indicated on the right vertical axis. C, ab initio shape reconstructions of MutS␤ and MutS␤⌬28 were performed from SAXS data as described (21). The envelopes shown represent an average of 10 independent shape reconstructions. Because of nonavailability of a MutS␤ crystal structure, the MutS␣⌬341-DNA complex structure (40) is superimposed on the MutS␤ SAXS envelope for size reference. D, ab initio shapes of the MutS␤-PCNA complex were generated as described above from experimental scattering data collected for 1:1 molar mixtures of MutS␤ 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. interact strongly with PCNA (45). The simplest interpretation of these results is that the MutS␤ motif(s) involved in its interaction with MutL␣ partially overlap with the MSH3 PIP box responsible for the MutS␤-PCNA interaction. It is noteworthy in this regard that an RS(K/R)(Y/F)F peptide has been identified as a MutL␣ interaction motif in human Exo1 and BLM helicase (46). A similar highly conserved motif, LSRFF, overlaps with the MSH3 PIP box (QAVLSRFF) and may correspond to one component of the MutL␣-binding site within MutS␤. To our knowledge, this is the first instance where residues within the PCNA-binding motif of a protein are also employed for interaction with a second activity.
Recently, Fishel and co-workers (47) have described an interaction between human MLH1 and a polypeptide corresponding to the N-terminal 250 residues of human MSH3; interaction of MLH1 with MSH2 was not observed in this study. These findings are consistent with those described here. By contrast, studies in Saccharomyces cerevisiae (48) have implicated yMSH2 residues in the interaction of yMutS␣ with yMutL␣, although mutational alteration of the yMSH2 residues in question did not result in an interaction defect as severe as that described in our study. It is thus conceivable that MSH2 sequence elements contribute to MutS␤-MutL␣ interaction, but if this is the case, residues in the vicinity of the MSH3 PIP box must also be required.
Our finding that PCNA and MutL␣ interact in an either/or fashion with MutS␤ may reflect steric interference effects but could also be indicative of use of common interaction interfaces. For example, MutS␤ and MutL␣ interaction with a common PCNA motif would account for our findings, as would MutS␤ and PCNA interaction with a common interface on MutL␣.
Because ATP-dependent assembly of a ternary complex involving heteroduplex DNA and a MutS and MutL homolog is believed to be a key step in the initiation of mismatch repair (3,4), the inability of MutS␤ PIP box mutants to support MutL␣ endonuclease activation, mismatch-provoked excision, and repair might be attributed to the inability of these mutants to support ternary complex formation. However, it is also possible that in contrast to the MutS␣-initiated reaction, MutS␤ interaction with PCNA may be a key step in MutS␤-initiated repair events. The pleiotropic nature of these mutants does not permit distinction between these possibilities, although our results almost certainly indicate that MutS␣and MutS␤-initiated mismatch repair events proceed by distinct mechanisms.
Interestingly, S. cerevisiae studies have suggested that although PIP box integrity may be required for MSH3 function, it has only a limited role in MSH6 activity (20,23). In an msh3 null background, the rate of frameshift mutagenesis within a A 14 : T 14 run was ϳ 30-fold higher for an msh6⌬ allele (1.5 ϫ 10 Ϫ3 ) than for a msh6 PIP box mutant (5.2 ϫ 10 Ϫ5 ) (23). By contrast, deletion of msh3 in an msh6 null background resulted in a mutation rate (1.5 ϫ 10 Ϫ3 ) only ϳ2-3-fold higher than that observed upon inactivation of the MSH3 PIP box (6.5 ϫ 10 Ϫ4 ) (23). Our findings that MSH3 PIP box mutations are pleiotropic may explain these observations.
A potential mechanistic implication of our findings is that whereas PCNA is required for both MutS␣and MutS␤-depen-dent activation of MutL␣ endonuclease, a transient increase in local PCNA concentration could lead to specific destabilization of the MutL␣-MutS␤-DNA complex, thus aborting a MutS␤initiated event to allow a MutS␣-dependent reaction to proceed unhindered. Because some I/D mispairs are subject to either MutS␣or MutS␤-dependent repair (30), this type of PCNAmediated switch might function to control processing of such lesions by a particular pathway.