Conformational trapping of Mismatch Recognition PNAS PLUS Complex MSH2/MSH3 on repair-resistant DNA loops

Walter H. Langa,1,2, Julie E. Coatsb,1, Jerzy Majkaa, Greg L. Huraa, Yuyen Linb, Ivan Rasnikb,3, and Cynthia T. McMurraya,c,d,3

aLawrence Berkeley National Laboratory, Life Sciences Division, 1 Cyclotron Road, Berkeley, CA 94720; cDepartment of Molecular Pharmacology and Experimental Therapeutics; dDepartment of Biochemistry and Molecular Biology, Mayo Foundation, 200 First Street, Rochester, MN 55905; and bDepartment of Physics, Emory University, 400 Dowman Drive, MSC N214, Atlanta, GA 30322

Edited* by Peter H. von Hippel, Institute of Molecular Biology, Eugene, OR, and approved August 3, 2011 (received for review April 6, 2011)

Insertion and deletion of small heteroduplex loops are common which fails to effectively couple DNA binding with downstream mutations in DNA, but why some loops are prone to mutation and repair signaling. We envision that conformational regulation of others are efficiently repaired is unknown. Here we report that the small loop repair occurs at the level of the junction dynamics. mismatch recognition complex, MSH2/MSH3, discriminates between a repair-competent and a repair-resistant loop by sensing the con- Results formational dynamics of their junctions. MSH2/MSH3 binds, bends, Conformational Integrity of MSH2/MSH3 and the DNA “Junction” Tem- and dissociates from repair-competent loops to signal downstream plates. We characterized the DNA-binding affinity and nucleotide repair. Repair-resistant Cytosine-Adenine-Guanine (CAG) loops adopt binding properties of MSH2/MSH3 bound to looped templates, a unique DNA junction that traps nucleotide-bound MSH2/MSH3, either a ðCAÞ4 loop or CAG hairpin loops of either 7 or 13 and inhibits its dissociation from the DNA. We envision that junction (ðCAGÞ7 and ðCAGÞ13) triplet repeats (Fig. 1A). Both loop and dynamics is an active participant and a conformational regulator hairpin templates were constructed from two single-stranded oli- of repair signaling, and governs whether a loop is removed by gonucleotides (Fig. 1A). Neither the ðCAÞ4 loop nor the CAG MSH2/MSH3 or escapes to become a precursor for mutation. stem had complementary sequences within the duplex portion of the template. Thus, the junction templates folded into stable DNA repair ∣ mismatch repair ∣ smFRET ∣ trinucelotide expansion extrahelical loops, which have been previously characterized in solution (26, 31). Folding of the CAG loops creates A/A mis- BIOCHEMISTRY nsertion or deletion of small extrahelical loops is one of the matches every third in the stem, for a total of three ð Þ Imost common mutations in human cancers (1–3), but the me- mismatches in the CAG 7 template or a total of six mismatches chanism by which they occur is unknown. Small loops, bulges, in the ðCAGÞ13 template. DNA templates were analyzed by gel or kinked DNA occur frequently in DNA, and provide signals electrophoresis to (i) ensure the absence of any traces of single- for p53 recognition (4–7), recombination (8, 9), and/or most often stranded DNA and (ii) that the DNA loops were intact, as judged removal by the mismatch repair system (10–14). Two hetero- by an increase in the loop size and gel mobility (Fig. 1B). Unless dimeric mismatch recognition complexes, MSH2/MSH6 and specifically noted, all DNA templates were synthesized contain- MSH2/MSH3, operate in mammals with distinct, but overlapping ing a duplex base of 18 bases (Fig. 1A). specificities (12–14). The crystal structure (15–18), Atomic Force The purified human MSH2/MSH3 protein (hereafter referred Microscopy (AFM) (19), and single molecule fluorescence reso- to as MSH2/MSH3) was also of high quality (Fig. 1C). The full- nant energy transfer (smFRET) (19, 20) confirm that MSH2/ length MSH2/MSH3 was expressed and copurified as a hetero- MSH6 and Escherichia coli (MutS) preferentially bind single base dimer, and each subunit, when resolved by PAGE, migrated as a mismatches or two base pair bulges. MSH2/MSH3 can recognize single band according to the expected molecular mass (Fig. 1C). some base-base mismatches (21), but has a higher apparent affi- To further test the conformational integrity of the protein, we nity and specificity for small DNA loops composed of 2–13 bases visualized full-length MSH2/MSH3 using small angle X-ray scat- (12–14, 22–24). Thus, defects in repair mediated by MSH2/MSH3 tering (SAXS) (34, 35)(Fig. 1D). Interestingly, the high-resolu- are poised to be a major source of insertion-deletion mutations. tion SAXS structure revealed that the N-terminal portion of the The mechanism by which MSH2/MSH3 discriminates between MSH3 subunit formed an unfolded “panhandle” structure, which repair-competent and repair-resistant loops (24–26), however extended beyond the heterodimeric interface of MSH2/MSH3. remains enigmatic. A small ðCAÞ4 loop of DNA can be faithfully The handle undergoes a visible broadening and conformational repaired by MSH2/MSH3 both in vitro (24, 26) and in vivo (25, change upon binding the ðCAÞ4 loop. But otherwise, the DNA- 27, 28). In contrast, hydrogen bonded CAG hairpin loops are not bound heterodimeric portion of human MSH2/MSH3 was similar excised, and confer genomic instability through insertion and in conformation to that of the human MSH2/MSH6 bound to amplification of CAG repetitive tracts (29–31). Although ðCAÞ4 template containing a G-T mispaired base (16). Using these well loops and CAG hairpins both harbor three-way junctions, MSH2/ MSH3 interacts with them distinctly (24, 26). Why one template Author contributions: W.H.L., J.M., I.R., and C.T.M. designed research; W.H.L., J.E.C., J.M., is repaired better than the other is not known, but the conse- G.L.H., and Y.L. performed research; W.H.L., J.E.C., J.M., G.L.H., I.R., and C.T.M. analyzed quence is remarkable: Inefficient repair of CAG loops results in data; and W.H.L., J.M., I.R., and C.T.M. wrote the paper. mutations that underlie more than 20 hereditary neurodegenera- The authors declare no conflict of interest. tive or neuromuscular diseases (30–33). *This Direct Submission article had a prearranged editor. Here, we address the underlying basis for discriminating repair- Freely available online through the PNAS open access option. competent and repair-resistant DNA loops by MSH2/MSH3. We 1W.H.L. and J.E.C. contributed equally to this work. find that MSH2/MSH3 binds with similar affinity to a repair- 2 ’ ð Þ Present address: Department of Surgery, St. Jude Children s Research Hospital, 262 Danny competent CA 4 loop and to repair-resistant CAG hairpins. How- Thomas Place, MS 332, Memphis, TN 38105. ever, the three-way hairpin junction adopts a conformational state 3To whom correspondence may be addressed. E-mail: [email protected] or irasnik@ that traps nucleotide-bound MSH2/MSH3, and inhibits its disso- physics.emory.edu. ciation from the hairpin. The biochemical and smFRET results See Author Summary on page 17247. imply that repair-resistant CAG hairpins provide a unique but This article contains supporting information online at www.pnas.org/lookup/suppl/ nonproductive binding site for nucleotide-bound MSH2/MSH3, doi:10.1073/pnas.1105461108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1105461108 PNAS ∣ October 18, 2011 ∣ vol. 108 ∣ no. 42 ∣ E837–E844 Downloaded by guest on September 30, 2021 nucleotide, the apparent affinity of MSH2/MSH3 for both the ðCAÞ4 loop and hairpin templates was in the low nanomolar range (Table 2), and was in good agreement with previous mea- surements (24, 26, 31). The presence of magnesium decreased the affinity of ATP-bound MSH2/MSH3 to any template by about 10-fold, but, in general, DNA binding of ADP- or ATP-bound MSH2/MSH3 did not distinguish repair-competent ðCAÞ4 loop from the repair-resistant ðCAGÞ7 hairpin or ðCAGÞ13 hairpin.

MSH2/MSH3 Stabilizes a High FRET State When Bound to the Repair- ð Þ ð Þ Competent CA 4 Loop. The CA 4 loop differs structurally from the ðCAGÞ13 DNA in that the latter forms a hairpin comprising G-C hydrogen bonded base pairs and A/A mispaired bases every third nucleotide in the stem (24, 26). To test for conformation differences between the two templates, we measured the protein- induced DNA conformational dynamics using smFRET. We pre- pared DNA substrates, which were identical to those used in the Fig. 1. Conformational Integrity of Mismatch Recognition Complexes and biochemical DNA-binding experiments, except that the bottom DNA templates. (A) Schematic structure of the three-way junction DNA tem- strand of 18 nucleotides was labeled with Cy3 (on the 5′ end, ð Þ plates. The CA 4 loop and CAG hairpins are centrally located in the upper green ball) and Cy5 (on the 3′ end, red ball) (Fig. 2 A, C, E). Thus, unlabeled strand. The duplex base comprising eighteen base pairs, which for each template used in the smFRET experiments, the local ′ ′ were labeled at the 5 end with Cy3 and at the 5 end with Cy5 for smFRET environment of the fluorophores was identical. For both tem- experiments, or with a 5′ fluorescein for the FA experiments. (B) The purified ′ templates resolved on native polyacrylamide gels and visualized by ethidium plates, the top strand at the 3 end contains a poly-dT extension bromide staining. SS is the 18nuc single strand DNA that is the complemen- and a biotin tag for immobilization on streptavidin coated cover tary strand for the looped templates, DS is the 18 bp homoduplex DNA, and slips for observation (Fig. 2 A, C, E, blue ball). The extension the heteroduplex looped substrates are as labeled. (C) Resolution of purified was designed to prevent potential interaction of the fluorophores human MSH2/MSH3 (middle lane) and MSH2/MSH6 (right lane) proteins by with the streptavidin surface. The smFRET was used to probe SDS-PAGE. The size markers (right) indicate the molecular weights. (D) SAXS proximity between the Cy3 and Cy5 tags and the conformational structure of MSH2/MSH3 protein alone (top, left) or in the presence of the dynamics of the DNA. ð Þ CA 4 loop (top, right) overlaid on the crystal structure of human MSH2/MSH6 E 2∕ 3 ð Þ We determined the FRET efficiency ( FRET) values for hun- bound to a G-T mispaired base (11). View of the MSH MSH - CA 4 loop dreds of individual molecules. In the absence of protein, the dis- complex from front (bottom, right) and side (bottom, left). E ð Þ tribution of FRET for CA 4 substrates was a single narrow peak at E ∼ 0.31 (Fig. 2A, DNA only; the peak at E ¼ 0 characterized materials, we tested whether there were biochem- FRET FRET represents substrates with an inactive acceptor). In addition to ical or conformational differences, which segregated with the re- E the FRET population distribution, we followed the dynamics of pair-competent or repair-deficient nature of looped templates. each individual FRET pair by plotting time traces of donor (Cy3) and acceptor (Cy5) emission. However, there were no observable MSH2/MSH3 Binds Nucleotides with High Affinity at both Repair-Com- transitions within our time resolution. To determine that the tran- petent and Repair-Resistant Templates. We observed little differ- sitions were fast but not absent, we measured the recovery rate of ence in nucleotide affinity when MSH2/MSH3 was prebound to ð Þ ð Þ the acceptor dye intensity from the transitions to nonfluorescent repair-resistant CAG 7 hairpin and CAG 13 templates, or to a states in the presence of 2-mercaptoethanol. We compared re- ðCAÞ4 loop, which is a good substrate for MSH2/MSH3 in vitro sults for the ðCAÞ4 loop relative to the homoduplex DNA (with (22, 24, 26) and in vivo (27, 28). As measured by UV-cross-linking an identical local environment of the fluorescent dyes). The recov- (Fig. S1), the affinity of ADP or ATP to either subunit of DNA- ery of the intensity for ðCAÞ4 loop was an order of magnitude bound MSH2/MSH3 was substantially weaker when MSH2/ faster then the homoduplex DNA (Fig. S2). The recovery is facili- MSH3 was bound to DNA (Table 1). However, the reduction in tated by close proximity (2–3 nm or closer) of the donor and nucleotide affinity for MSH2/MSH3 did not display striking dif- acceptor. Thus, the ends of the CA4 loop substrate came into close ferences among ðCAGÞ7, ðCAGÞ13, and the ðCAÞ4 loop templates proximity confirming that there were conformational fluctuations, (Table 1). even though they were not observable within our time resolution. Addition of MSH2/MSH3 to the ðCAÞ4 loop template, in the ð Þ E MSH2/MSH3 Binds with Similar Affinity to the Repair-Competent CA 4 absence of nucleotides, led to the appearance of a new FRET “ ” E ∼ 0 4 A Loop and to the Repair-Resistant CAG Hairpins. To test whether nu- peak (bound state, high FRET )at FRET . (Fig. 2 , cleotide binding to MSH2/MSH3 altered its association with þMSH2∕MSH3). The population in the high FRET state in- DNA, we labeled each DNA template with fluorescein at the creased with protein until the entire population had shifted to ′ E ∼ 0 4 A þ 2∕ 3 5 -end of the bottom strand, and measured the DNA-binding FRET . (Fig. 2 , MSH MSH ). Consistent with high affinity by fluorescence anisotropy (FA). In the absence of bound affinity binding (Table 2), the high FRET state saturated at a

Table 1. Nucleotide binding affinities of MSH2/MSH3 subunits determined by cross linking in the absence or presence of DNA templates, KD in nM Subunit Ligand No DNA CA4 CAG7 CAG13 MSH2 ADPð−Mg2þÞ 210 ± 90 250 ± 90 190 ± 50 530 ± 120 ADPðþMg2þÞ 150 ± 60 150 ± 120 250 ± 50 440 ± 130 ATPð−Mg2þÞ 730 ± 240 5,300 ± 700 4,800 ± 500 4,300 ± 800 MSH3 ADPð−Mg2þÞ nq nq nq nq ADPðþMg2þÞ 160 ± 90 7,200 ± 1,000 4,000 ± 1,100 3,700 ± 540 ATPð−Mg2þÞ 550 ± 190 53,800 ± 15,100 11,400 ± 4,000 59,200 ± 15,100 Nq: not quantifiable—lack of signal.

E838 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1105461108 Lang et al. Downloaded by guest on September 30, 2021 Table 2. DNA-binding affinities of wild-type MSH2/MSH3 smFRET population was observable when MSH2/MSH3 was PNAS PLUS determined by Fluorescence Anisotropy in the presence and added to homoduplex DNA, even at very high protein concentra- absence nucleotides, KD in nM tion (Fig. 2 C and D). Both MutS and MSH2/MSH6 bend G-T Template CA4 CAG7 CAG13 mismatched DNA at the site of the mismatch (15, 16, 18). Thus, we purified MSH2/MSH6 and added it to a comparably labeled − ( Mg) 2.5 ± 0.5 3.8 ± 0.6 6.4 ± 0.7 G-T base mismatch template. Indeed, for an MSH2/MSH6 com- (+Mg) 2.6 ± 0.7 5.1 ± 0.6 5.3 ± 0.9 − plex, we observed a high FRET shift, (Fig. 2E), which was similar ADP( Mg) 1.6 ± 0.06 1.6 ± 0.06 2.8 ± 0.04 ð Þ ADP(+Mg) 41.9 ± 5.3 41.6 ± 6.2 27.7 ± 2.4 in magnitude to that induced by MSH2/MSH3 on the CA 4 loop ATP(−Mg) 3.0 ± 0.05 4.2 ± 0.04 6.6 ± 0.05 (Fig. 2C). The single molecule traces indicated that the high ATP(+Mg) 39.7 ± 4.3 70.9 ± 11.6 43.0 ± 5.3 FRET state was stable (black horizontal line, Fig. 2F). MSH2/ MSH6 does not bind to the ðCAÞ4 loop (26, 31), and no high smFRET population was observable when MSH2/MSH6 was A þ 2∕ protein concentration in the nanomolar range (Fig. 2 , MSH added to that template, even at very high protein concentration. 3 MSH ). Thus, MSH2/MSH3 formed a stable complex with the Thus, MSH2/MSH6 and MSH2/MSH3 complexes displayed simi- ð Þ repair-competent CA 4 loop template in which the two ends of lar transitions when bound to their preferred repair-competent the heteroduplex loop were positioned more closely, suggestive of templates with an estimated bending angle of 40 to 45° (Fig. S4). bending. To monitor the conformational dynamics of the transitions, we Nucleotide Binding Increases the Dissociation of MSH2/MSH3 from the followed individual Cy3 (green) and Cy5 (red) emission traces for ð Þ 2∕ 3 ð Þ B CA 4 Loop Under Hydrolytic Conditions. MSH2/MSH6 and MSH2/ the MSH MSH - CA 4 loop complex (Fig. 2 , the calculated MSH3 couple DNA binding and ATP hydrolysis to initiate down- FRET efficiency curves are displayed in blue). The observation stream repair (11–13). Thus, we tested the effects of ATP binding time was limited typically to less than 60 s by photodestruction and hydrolysis on the conformational dynamics of the MSH2/ of the acceptor (indicated by black arrows, Fig. 2B). The single MSH3-bound ðCAÞ substrate. ATP was added to a complex con- molecule traces did not vary significantly (Fig. 2B). A few traces 4 taining MSH2/MSH3-bound ðCAÞ loop DNA in the presence captured conformational transitions (Fig. 2B, blue trace) consis- 4 (þMg) or absence (−Mg) of magnesium, and distribution of tent with detection of a protein-binding event (Fig. 2B, blue trace). However, there were no observable dynamics within smFRET efficiencies was measured under both hydrolyzing and B nonhydrolyzing conditions (Fig. 3). Induction of the high FRET our time resolution (Fig. 2 , black line), and the lifetime of BIOCHEMISTRY the average transition was longer than our maximum observation state by DNA-bound MSH2/MSH3 was independent of both time (Fig. S2). Similar results were obtained when MSH2/MSH3 bound to a comparably labeled A2 bulge, which is also a template for MSH2/MSH3-dependent repair (Fig. S3). We evaluated additional control DNA templates. MSH2/ MSH3 has weak affinity and rapidly dissociates from homoduplex DNA (24, 26, 31). Consistent with these properties, no high

Fig. 3. ATP increases dissociation of MSH2/MSH3 from a ðCAÞ4 loop sub- strate. FRET efficiency histograms for MSH2/MSH3 binding to the ðCAÞ4 substrate at different protein concentrations in the presence of 100 μMATP (A) without MgCl2 and (B) with 5 mM MgCl2. The MSH2/MSH3 concentration is indicated. (C) The dynamics of the ðCAÞ4 substrate upon MSH2/MSH3 binding in (B). The time traces of representative donor fluorescence (green, Cy3) and acceptor fluorescence (red, Cy5). The black line indicates the time of Fig. 2. Binding of MSH2/MSH3 and MSH2/MSH6 to their preferred repair the ðCAÞ4 loop in the high FRET state. Time of acceptor photobleaching is substrates increases FRET efficiency. The dynamics of different substrate mo- indicated by black arrow. (upper) Blue traces are the corresponding FRET lecules in the presence of or in the absence of added MSH2/MSH3. (A) smFRET efficiencies. (D) Time traces like the one shown in (C) analyzed in a two state efficiencies for MSH2/MSH3 binding to the ðCAÞ4 loop substrate without system using a Hidden Markov Model (36) to determine the average transi- (top) or with (bottom) MSH2/MSH3. The schematic of the labeled substrates: tion rates from initial to high and high to initial FRET states. The transition green ball is Cy3 label; red ball is Cy5 label (bottom); blue ball is biotin label. rates from the initial to high FRET states depends on protein concentration The protein concentration is indicated. (B) The time traces of representative (gray balls), while the transition rates from high to initial FRET state were donor fluorescence (green, Cy3) and acceptor fluorescence (red, Cy5). The independent of the protein concentration (black balls). (E) Model for confor- black line indicates the time of the ðCAÞ4 loop in the high FRET state. Time mational dynamics observed for MSH2/MSH3 binding to ðCAÞ4 loop sub- of acceptor photobleaching is indicated by black arrow. (upper) Blue traces strate: MSH2/MSH3 binds to the a high FRET state of the ðCAÞ4 loop, while are the corresponding FRET efficiencies. (C, D) Same as (A, B) for homoduplex addition of ATP or ADP under hydrolytic conditions increases dissociation of substrate. (E, F) Same as (A, B) for binding of MSH2/MSH6 to a G/T mis- MSH2/MSH3 from the ðCAÞ4 loop. The high FRET state is indicated as a bent matched substrate. structure.

Lang et al. PNAS ∣ October 18, 2011 ∣ vol. 108 ∣ no. 42 ∣ E839 Downloaded by guest on September 30, 2021 magnesium (Fig. S5) and nucleotide binding (Fig. 2A and Fig. 3 A The ðCAGÞ13 hairpin DNA (Fig. 4A) displayed a relatively E ∼ 0 3 and B). ATP(þMg) binding weakened the affinity of MSH2/ broad distribution of FRET efficiency, around FRET . B MSH3 for the ðCAÞ4 DNA (Table 2), and more nucleotide-bound (Fig. 4 , DNA only). Remarkably, binding of MSH2/MSH3 to the MSH2/MSH3 was required to saturate the high FRET shift ðCAGÞ13 hairpin resulted in two new FRET populations (Fig. 4B, (Fig. 3B) relative to the absence of nucleotide (Fig. S5). However, þMSH2∕MSH3), one of which was similar to that observed from ð Þ ATP binding, under hydrolytic conditions, resulted in a striking the CA 4 loop. The high and a low FRET distributions of the ð Þ E ∼ 0 43 E ∼ 0 20 alteration in the dynamics of MSH2/MSH3 binding (compare CAG 13 hairpin, around FRET . and FRET . , respec- Fig. 2B and Fig. 3C). Multiple transitions between high FRET tively, had the same dependence on MSH2/MSH3 concentration (Fig. 4B, þMSH2∕MSH3). Each FRET state was stable, with states and low FRET states were obvious in the single molecule an average lifetime longer than 30 s (Fig. 4 C and D). Thus, in traces, and the lifetime of nucleotide-bound MSH2/MSH3 on the ð Þ ð Þ ð Þ B C contrast to CA 4 loops, the MSH2/MSH3-bound CAG 13 hair- CA 4 loop dropped from minutes (Fig. 2 ) to seconds (Fig. 3 ). pin adopted two conformational populations in which and the þ Similar results were obtained when ADP( Mg) was the added majority of the ends (65%) had moved apart. nucleotide (Fig. S6). Thus, MSH2/MSH3 binding was sufficient Surprisingly, the high FRET state (35% of ends) largely disap- to stabilize the high FRETstate, and binding of ATPor ADP under peared when ðCAGÞ13-bound MSH2/MSH3 was occupied with hydrolytic conditions increased dissociation of MSH2/MSH3 from nucleotide (Fig. 4E, þMSH2∕MSH3). Under hydrolytic condi- ð Þ the CA 4 loop. tions, addition of ATP to ðCAGÞ13-bound MSH2/MSH3 shifted To determine the binding and dissociation kinetics, we applied the equilibrium populations towards the low FRET state (com- a hidden Markov model (36) to hundreds of time traces for sev- pare Fig. 4 B and E). Analysis of the single molecule traces in- eral MSH2/MSH3 concentrations to generate robust measures of dicated that ATP(þMg) occupancy of MSH2/MSH3 significantly the average transition times (Fig. 3D). We found that the transi- shortened the average lifetime for the high FRET state to around tion rate to the high FRET state increased with protein concen- 5–10 s (Fig. 4G, horizontal black line). Under the same condi- tration, but the transition rate back to the initial FRET state was tions, the low FRET state was stable, and dissociation was rarely F independent of protein concentration. Collectively, these findings observed (Fig. 4 ). Under hydrolytic conditions, the FRET þ þ indicated that the shift to the high FRET state depended on efficiencies for ATP( Mg) were similar to those of ADP( Mg) ð Þ (Fig. S7). Thus, the repair-deficient ðCAGÞ13 template differed MSH2/MSH3 binding to the CA 4 loop, while the reverse tran- ð Þ sition rate arose from MSH2/MSH3 dissociation (Fig. 3E). from the repair-competent CA 4 loop: nucleotide occupancy of MSH2/MSH3 promoted its dissociation from the high FRET ð Þ state and the nucleotide-bound MSH2/MSH3 was, instead, Binding of MSH2/MSH3 to the Repair-Deficient CAG 13 Hairpin Results in the Appearance of a Unique Conformational Population. trapped in the low FRET state. The differential dynamics between the repair-resistant Both the ðCAGÞ13 hairpin and the ðCAÞ4 loops bind well to ðCAGÞ hairpin and the repair-competent ðCAÞ loop templates MSH2/MSH3 (Table 2), but only the ðCAÞ loop is accurately ex- 13 4 4 were striking. The shift to the low FRET state could not be ex- cised and repaired in vitro (24, 26) and in vivo (27, 28). Therefore, ð Þ plained by differential affinity of nucleotide-bound MSH2/ we tested whether the conformational dynamics of the CAG 13 MSH3: the biochemical data indicated that nucleotide binding hairpin might be relevant to its repair-deficient nature. and DNA-binding affinity of MSH2/MSH3 to the ðCAGÞ13 hair- pin and the ðCAÞ4 loop were similar (Table 1). The differences in MSH2/MSH3-induced DNA conformational dynamics could also not be explained by oligomerization of MSH2/MSH3 on the DNA templates. We have previously reported that the stoichio- metry of MSH2/MSH3 on both the ðCAÞ4 loop and the ðCAGÞ13 hairpin is one heterodimer per DNA molecule as measured by sedimentation equilibrium analysis (31). Thus, models in which the low and high FRET states were stabilized by two or more MSH2/MSH3 heterodimers were unlikely. However, two models seemed plausible. The low and high FRET states could arise if two distinct nucleotide-bound forms of MSH2/MSH3 were able to bind to the ðCAGÞ13 hairpin, and induce distinct conformations. Alternatively, the low and high FRET states might arise if ðCAGÞ13 template itself formed two major DNA conformations that were able to bind MSH2/MSH3. In either case, the ratio of MSH2/MSH3 and ðCAGÞ13 template would be 1∶1. We considered both possibilities.

ð Þ The High and Low FRET States of the CAG 13 Hairpin Loop Do Not Fig. 4. Repair-resistant ðCAGÞ13 template traps nucleotide-bound MSH2/ MSH3 in the low FRET state. (A) A fold back loop of CAG DNA forms Arise from Binding of Distinct Nucleotide-Bound Forms of MSH2/ with A/A mispaired base every three nucleotides. Green ball is Cy3 label; Red MSH3. Different from MSH2/MSH6, the MSH2 and MSH3 sub- ball is Cy5 label (bottom). (B) FRET efficiency for MSH2/MSH3 binding to units of MSH2/MSH3 bind nucleotides stochastically (26), and the ðCAGÞ13 hairpin in the absence of nucleotides. The FRET efficiency histo- efficient hydrolysis results in formation of ADP-MSH2/MSH3- grams indicate that MSH2/MSH3 binding induces a high FRET state and a empty and empty-MSH2/MSH3-ADP in solution. Only ADP- low FRET state relative to the substrate alone. Individual time traces of MSH2/MSH3-empty stably binds to the ðCAÞ4 loop DNA (26). the low (C) and high (D) FRET states. The time traces of representative donor However, it was possible that the altered conformation of the fluorescence (green, Cy3) and acceptor fluorescence (red, Cy5). Black bars ðCAGÞ13 hairpin template permitted binding of both ADP-bound indicate the binding event, and the arrows indicate photo bleaching of forms of MSH2/MSH3 (Fig. 5A). In such a model, binding of the the acceptor dye. (E) Addition of ATP to (B) strongly reduces the relative ð Þ abundance of the high FRET state compared to the low FRET state. (F) The two distinct ADP-bound forms of MSH2/MSH3 to the CAG 13 individual time traces indicate that the low FRET state remains stable and templates would result in the high and low FRET states. (G) the time in the high FRET state is shorter in the presence of ATP relative To test this hypothesis, we created mutants of MSH2/MSH3 to its absence (D). in which only one of the Walker motifs was competent to bind

E840 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1105461108 Lang et al. Downloaded by guest on September 30, 2021 Table 3. Nucleotide binding affinities of wild-type and mutant PNAS PLUS MSH2/MSH3 proteins determined by Fluorescence Anisotropy, KD in nM Ligand WT Sgl2 Sgl3 Dbl ADP(−Mg) 217 ±12 1,124 ± 164 354 ± 19 >1,300 ADP(+Mg) 53.9 ± 3.5 60.8 ± 5.3 110.3 ± 4.1 > 1,200 ATP(−Mg) 409 ± 42 373 ± 39 415 ± 65 > 2,700 ATP(+Mg) 70.2 ± 5.1 37.9 ± 2.9 212.7 ± 2.0 > 3,000

sotropy, both sgl2 and sgl3 bound ADP with equivalent affinity as WT MSH2/MSH3 (Table 3). Thus, mutation in one site did not influence the nucleotide affinity in the other. Consequently, each mutant MSH2/MSH3 heterodimer was able to form a single nucleotide-bound complex, which varied only in the nucleotide- bound subunit. We next tested how well the mutant MSH2/MSH3 proteins could bind to DNA, by measuring FA of labeled DNA substrates (Table 4). With the exception of dbl, wt and mutant MSH2/MSH3 had good affinity for the ðCAGÞ13 hairpin in the presence of nu- cleotide (Table 4). We measured nucleotide affinity for the DNA- bound wt and MSH2/MSH3 mutants using fluorescently labeled ATP and ADP (Fig. 5 E and F). ATP bound well to the MSH2 or MSH3 subunit of wt, sgl2, or sgl3 as free heterodimers (Fig. 5E, open symbols), but none of these ATP-bound complexes adopted a stable ATP-bound state on the ðCAGÞ13 hairpin DNA (Fig. 5E, closed symbols). ADPðþMgÞ retained high affinity for ðCAGÞ13- ð Þ bound wt and sgl3, but had little affinity for the CAG 13-bound BIOCHEMISTRY sgl2 mutant (Fig. 5F, solid inverted triangles). Thus, nucleotide- bound MSH2/MSH3 associated to the ðCAGÞ13 hairpin only

Fig. 5. A ðCAGÞ13 hairpin binds only one nucleotide-bound MSH2/MSH3 when nucleotide occupied the MSH2 subunit and the MSH3 sub- complex, but displays both high and low FRET states. (A) Schematic diagram unit was empty (sgl3), yet both high and low FRET states were of two possible MSH2∕MSH3-ðCAGÞ13 hairpin complexes with nucleotide observed (Fig. 5G). These experiments argued against a model bound in either the MSH2 or MSH3 subunit. (B) Sequences of the mutant where the high and low FRET states arose from binding of MSH2 and MSH3 subunits aligned with the canonical Walker A box sequence two distinct nucleotide-bound MSH2/MSH3 complexes to the motif of the wt MSH2/MSH3. The conserved Lysine residue has been replaced ðCAGÞ13 hairpin. with a methionine in the mutant proteins to destroy the ATP binding pocket. (C) Resolution of the wild-type and mutant MSH2/MSH3 on denaturing gels. The Repair-Resistant ðCAGÞ Template Traps MSH2/MSH3 in the Low Wt, wild-type MSH2/MSH3 subunits, sgl2, Walker A box mutations in the 13 MSH2 subunit only, sgl3, Walker A box in the MSH3 subunit only, dbl, Walker FRET State. The experimental results raised the possibility that the 32 ð Þ A motif mutations in both subunits. (D) Binding of ½α- P-ATP to wild-type CAG 13 template adopted more than one DNA conformation and mutant MSH2/MSH3 proteins analyzed by UV-cross-linking followed by for binding of ADP-MSH2/MSH3-empty. Indeed, in the absence resolution on denaturing gel. Only intact nucleotide binding sites bind ATP efficiently. (E) Fluorescence anisotropy measurements of Bodipy-labeled ATP Table 4. DNA-binding affinities of mutant MSH2/MSH3 proteins binding to both wild-type and mutant MSH2∕MSH3-ðCAGÞ13 hairpin com- determined by Fluorescence Anisotropy, KD in nM plexes. (F) Mutation of the Walker (A) box in the MSH2 subunit only inhibits binding of Bodipy labeled ADP to a MSH2∕MSH3-ðCAGÞ13 hairpin complex. (G) Association of nucleotide-bound wild-type and mutant MSH2/MSH3 to Template CA4 CAG13 the ðCAGÞ13 templates result in high and low FRET states. ATP is retained poorly in the MSH3 subunit when bound to DNA (18). Thus, sgl3 and wild Mutant Sgl2 − type in the presence of nucleotides are similar, and sgl2 in the presence of ( Mg) 6.3 ± 1.1 4.8 ± 0.6 nucleotides is the same as wt without bound nucleotides. ATP is 100 μM. (+Mg) 3.7 ± 0.5 2.8 ± 0.4 ADP(−Mg) 4.1 ± 0.6 2.8 ± 0.04 nucleotides (Fig. 5B). We changed the critical lysine of the Walk- ADP(+Mg) 12.1 ± 1.2 20.1 ± 4.6 ATP(−Mg) 13.5 ± 2.9 3.9 ± 0.7 er A sites (GGKST/S) to a methionine in one, the other, or both ATP(+Mg) 45.9 ± 4.5 50.0 ± 3.4 of the ATP binding sites. These mutants are referred to as sgl2 (mutation in MSH2 only), sgl3 (mutation in MSH3 only), or dbl Mutant Sgl3 (both subunits mutated) (Fig. 5B), depending on the site(s) of the (−Mg) 4.1 ± 0.7 2.3 ± 0.4 amino acid change. The amino acid substitutions had no effect on (+Mg) 3.2 ± 0.4 2.4 ± 0.4 the expression of the protein relative to the WT protein, and each ADP(−Mg) 3.0 ± 0.5 3.8 ± 1.0 C ADP(+Mg) 2.3 ± 0.4 3.4 ± 0.5 subunit was expressed at stoichiometric levels (Fig. 5 ). Thus, we − purified each mutant MSH2/MSH3 heterodimeric complex and ATP( Mg) 14.4 ± 1.0 5.1 ± 0.7 ATP(+Mg) 43.2 ± 4.4 59.0 ± 6.4 characterized its behavior with respect to DNA and nucleotide binding. Double Mutant The mutant MSH2/MSH3 proteins had the expected nucleo- (−Mg) 16.0 ± 2.2 8.3 ± 1.3 tide binding properties. As judged by X-linking, neither ½α-32P- (+Mg) 28.0 ± 2.5 33.5 ± 12.9 ADP nor ½α-32P-ATPðþMgÞ bound to the MSH2/MSH3 dbl ADP(−Mg) 15.7 ± 1.9 2.8 ± 0.04 (Fig. 5D, lanes 4), while sgl2 and sgl3 bound nucleotides only in ADP(+Mg) 118.2 ± 16.1 >5,600 − their intact site (Fig. 5D, lanes 2 and 3) and wt MSH2/MSH3 ATP( Mg) 18.4 ± 5.6 No binding ATP(+Mg) No binding No binding bound both sites equally (Fig. 5D, lanes 1), as measured by ani-

Lang et al. PNAS ∣ October 18, 2011 ∣ vol. 108 ∣ no. 42 ∣ E841 Downloaded by guest on September 30, 2021 of protein, by increasing MgCl2 concentrations, we could resolve E ∼ 0 24 the broad FRET efficiency peak at FRET . into two closely E ∼ 0 24 E ∼ 0 21 spaced DNA populations around FRET . and FRET . (Fig. 6A). The single molecule traces indicated that these two DNA populations were rapidly interconverting (Fig. 6 B and C). The ðCAGÞ13 DNA can be characterized as a three-way DNA junction with two homoduplex and one heteroduplex arm (the ðCAGÞ13 stem). Perfectly paired three-way DNA junctions form a single stable conformation (37). Thus, we hypothesized that the unpaired A-A mispaired bases in the stem of a ðCAGÞ7 or ðCAGÞ13 loops might allow rearrangement of the junction into two major conformational populations of the DNA. If the ðCAGÞ13 DNA intrinsically adopted high and low FRET states, then MSH2/MSH3 might preferentially bind to one. To test this idea, we stabilized the junction by converting the two A-A pairs closest to the junction of the ðCAGÞ13 hairpin tem- plate into A-T pairs (AT-ðCAGÞ9) (Fig. 6D). Introduction of the two A-T pairs at the base of the junction “locked” it into a single narrow distribution, which did not show two populations upon Fig. 7. The junction of the AT-ðCAGÞ9 hairpin adopts only one stable three- increasing MgCl2 (Fig. 6E). Moreover, AT-ðCAGÞ9 adopted a F way junction, from which MSH2/MSH3 does not dissociate. (A) The FRET single stable state as shown in the single molecule traces (Fig. 6 efficiencies for binding of MSH2/MSH3 to the AT-ðCAGÞ substrate in the G ð Þ 9 and ). We next added MSH2/MSH3 to the AT- CAG 9,DNA, presence of (þMgCl2) results in a single low-FRET population. (B) The FRET and tested whether MSH2/MSH3 would promote the high and efficiency histograms for AT-ðCAGÞ9 in the presence of ATP under hydrolytic low FRET conformations. Remarkably, binding of MSH2/MSH3 conditions. The affinity of nucleotide-bound MSH2/MSH3 for the AT-ðCAGÞ9 þ to the AT-ðCAGÞ9, resulted in only a low FRET conformational substrate ( MgCl2) is reduced, but binding results in the same low FRET population (Fig. 7A). Adding ATPand MSH2/MSH3, under hydro- state as observed in the absence of nucleotides. (C) The individual time traces of the AT-ðCAGÞ9 substrate alone (top), AT-ðCAGÞ9 bound to MSH2/MSH3 lyzing conditions, lowered the affinity of MSH2/MSH3 to the ð Þ ð Þ B (middle), and AT- CAG 9 bound to MSH2/MSH3 at the indicated concentra- AT- CAG 9 hairpin (and reduced the shift) (Fig. 7 )butdidnot þ μ ð Þ tions in the presence of ATP ( Mg) (100 M) (bottom). All traces are similar alter the overall conformation of the CAG 13 hairpins. Further- and display no dynamics. (C) The time traces of representative donor fluor- more, MSH2-MSH3 binding did not increase the dynamics of escence (green, Cy3) and acceptor fluorescence (red, Cy5), and (D) the blue AT-ðCAGÞ9; few conformation transitions were observed even traces are the corresponding FRET efficiencies. (E) Proposed model for con- when MSH2/MSH3 was in the nucleotide-bound state (Fig. 7 C formational regulation of loop repair by MSH2/MSH3 at three-way DNA junc- and D). Thus, MSH2/MSH3 bound stably to the AT-ðCAGÞ9 tions. The conformational flexibility of the substrate determines the possible ð Þ junction and did not dissociate readily from the low FRETconfor- binding modes of MSH2/MSH3. (F) Binding of MSH2/MSH3 to a CA 4 loop binds, bends DNA. Upon downstream nucleotide hydrolysis and exchange, mation. When bound to MSH2/MSH3, the AT-ðCAGÞ9 hairpin MSH2/MSH3 adopts a doubly bound form which is verify and to leave the lesion to signal downstream repair by the MMR machinery. (G) The straigh- tened ðCAGÞ13 hairpin junction traps nucleotide-bound MSH2/MSH3 in a nonproductive complex, which cannot leave the lesion to initiate efficient repair by the MMR pathway. Successful mismatch repair couples DNA binding and ATP hydrolysis. Trapping does not allow processing of ATP in the MSH3 subunit, and prevent ADP/ATP exchange needed to leave the site. Circles with 2 and 3 represent the MSH2/MSH3 heterodimer. Red ball are ADP and blue balls are ATP.

adopted a single stable low FRETstate, which was not observed for the ðCAÞ4 loop template under any condition tested. Discussion How insertion and deletion mutations arise in the genome and why some loops are repaired better than others are unknown. Here, we show that MSH2/MSH3 discriminates between a repair- competent and a repair-resistant loop by sensing the confor- mational dynamics of their three-way junctions. We propose that the conformational properties of the substrate junction govern whether a loop is removed or becomes precursor for mutation. Fig. 6. The ðCAGÞ loop intrinsically adopts more than one conformational 13 We find the repair-competent ðCAÞ4 substrate is intrinsically a state in the absence of protein. (A) The FRET efficiency histograms of the ð Þ flexible hinge with dynamics that are faster than our time resolu- CAG 13 substrate in the absence of protein. Increasing MgCl2 concentration E resolves the presence of two FRET states. (B) Few transitions are observed tion. MSH2/MSH3 binds and stabilizes the bent state (Fig. 7 , within the time resolution for concentrations below 5 mM MgCl2, but be- bending), presumably to verify the lesion. Upon nucleotide bind- come more apparent at 20 mM MgCl2.(C) Blue trace is the corresponding ing, the enzyme undergoes a series of rapid nucleotide-dependent FRET efficiency. (D) A schematic of the sequence changes at the junction steps and eventually dissociates to signal downstream repair ð Þ of the AT CAG 9 template. The two A-A mismatches closest to the junction (Fig. 7E, sliding). Indeed, the smFRET results imply that the ð Þ in the CAG 13 substrate have been replaced by A-T pairs. (E) Conformation of substrate dynamics induced by nucleotide-bound MSH2/MSH3 AT-ðCAGÞ9 substrate at different MgCl2 concentrations. In the absence of at the ðCAÞ4 loop have a nonexponential dwell-time distribution protein, the substrate exists in a single conformation. Addition of MgCl2 in- creases the shift towards higher FRET values, (F, G) but the individual time consistent with the presence of more than one kinetic step traces are not dynamic. (F) The time traces of representative donor fluores- (Fig. S8). Rapid association and dissociation poises the protein cence (green, Cy3) and acceptor fluorescence (red, Cy5), and (G) the blue complex to verify and move away from the lesion and to initiate traces are the corresponding efficiencies. interactions necessary for downstream signaling.

E842 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1105461108 Lang et al. Downloaded by guest on September 30, 2021 The repair-resistant ðCAGÞ13 junction intrinsically adopts dis- our biochemical measurements that the G674A Walker A site PNAS PLUS crete conformational states as indicated by the two-state-FRET mutant in the MSH2 subunit binds ATP poorly, if at all, in the distribution (most noticeable at high MgCl2 concentrations) context of MSH2/MSH3 (Fig. S9). Thus, the G674A Walker A (Figs. 4 and 6). Unliganded MSH2/MSH3 recognizes both of site mutation does not block hydrolysis per se, but failure to bind these conformational states with similar affinity and further ATP in the MSH2 subunit prevents formation of ADP-bound separates them. MSH2/MSH3 can convert some of the hairpin MSH2/MSH3, the major lesion-binding form (26). junctions into a repair-competent bent state. However, upon While ATP hydrolysis is reduced in MSH2/MSH3-bound CAG k K nucleotide binding, MSH2/MSH3 dissociates from the bent state hairpin (31, 40), the apparent nucleotide affinity and the cat∕ M and, instead is trapped by a junction configuration from which for ATP hydrolysis are similar for MSH2/MSH3 when bound to a F it cannot dissociate (Fig. 7 , trapped). The nucleotide-bound repair-competent ðCAÞ4 loop and the repair-resistant ðCAGÞ13 protein becomes “stuck” on the lesion, and likely cannot carry out hairpin (31, 40). Thus, a second issue is the extent to which the the steps leading to ADP/ATP exchange, which is critical for dis- recognition properties of MSH2/MSH3 differ between these two sociation and downstream repair. These findings imply that the types of loops. smFRETresolves discrete populations, and our data repair-resistant CAG hairpins provide a unique but nonproduc- provide definitive evidence that MSH2/MSH3 captures a distinct tive binding site for nucleotide-bound MSH2/MSH3, which fails conformation of the ðCAGÞ13 hairpin, which significantly length- to effectively couple DNA binding with ATP hydrolysis. ens the lifetime of bound protein relative to repair-competent ð Þ The AT- CAG 9, junction differs by only two nucleotides ðCAÞ4 loop. Because MSH2/MSH3 binds with equal apparent relative to the ðCAGÞ13 hairpin, but only one intrinsic conforma- ð Þ k K affinity to the CA 4 and the CAG hairpin templates, the cat∕ M tion is available for MSH2/MSH3 binding. Similar to the repair- is expected to be similar, but the altered recognition properties resistant ðCAGÞ13 hairpin loop, MSH2/MSH3 cannot convert the ð Þ of MSH2/MSH3 on the low FRET population cannot be resolved AT- CAG 9 into a bent state, rather, the template exists in a in bulk measurements (40). The time scale of the changes requires single junction conformation, which captures MSH2/MSH3. ð Þ sensitive, high-resolution techniques to observe them. Because The residence of MSH2/MSH3 on the AT- CAG 9, is long lived, DNA binding inhibits ATP hydrolysis for MSH2/MSH6 (41, 42) whether or not the protein complex is bound with nucleotides C D and MSH2/MSH3 (31, 40), the longer lifetime of the MSH2/ (Fig. 7 and ). Thus, dynamics of the junction is an active MSH3 on the repair-resistant template implies a reduction of ATP participant in directing loop conformation. We envision that con- binding and/or hydrolytic activity in the straightened conformation formational regulation of small loop repair occurs at the level of (31). Collectively, our proposed model provides a basis for how the junction dynamics. an intact MMR complex can become inefficient when bound to BIOCHEMISTRY This mechanism has strong mechanistic implications for a particular types of loops. The junction dynamics are poised to be second class of mismatch repair deficits. Mutations in the MMR a pivot point for coupling DNA loop binding and ATP hydrolysis (Mismatch Repair) machinery lead to an increase in spontaneous mutation rate, which is typically referred to as a mutator pheno- by an intact MSH2/MSH3 to outcomes of mutation or repair. – type (1 3). For example, about 15% of patients with hereditary Methods nonpolyposis have widespread genome instabil- Detailed methods are provided as SI Methods. ity, characterized by single base changes or changes in copy num- ber at repetitive tracts (1–3). The mutational spectrum in this Protein Purification. His-tagged human MSH2/MSH3 and MSH2/MSH3 was class of MMR deficits reflects the inability of the mutated MMR overexpressed in SF9 insect cells using a pFastBac dual expression system machinery to correct postreplicative errors throughout the gen- (GIBCO-BRL) and purified as described previously (24, 26). ome (1–3). Our data provide a plausible mechanism for a second class of MMR defects in which the lesion itself prevents its Nucleotides and Oligonucleotides. Oligonucleotides used in the binding processing by the normal repair machinery (32). Defective repair studies were obtained from Operon or IDT, SI Methods. Fluorescently labeled arises when the repair-resistant loops trap the MMR proteins oligos were labeled at the 5′ end with fluoresceine for single label experi- during recognition of the lesion and they remain uncorrected. ments. Nucleotides of the highest grade were purchased from Sigma. ½α32P-ATP was purchased from Perkin Elmer, and ½α32P-ADP was derived The resulting insertion and deletion mutations, in this case, will 32 “ ” by incubation of ½α P-ATP with hexokinase. All preparations of nucleo- be site-specific in that they are limited to particular locations tides used contained less than 1% contamination of other nucleotides. where the repair-resistant lesions reside. The properties of trinu- cleotide expansion characterize this type of mutation. UV-Cross-Linking, DNA, Nucleotide Binding, Fluorescence Anisotropy. Experi- We do not as yet know whether the unusual junction dynamics ments were performed as described previously (26). provides a general mechanism underlying all “class two” insertion/ deletion mutations or whether the unusual dynamics are restricted SAXS. Data were collected at the SIBYLS (beamline 12.3.1), and analyzed with to only some junctions. However, our results provide, at the struc- the automated pipeline described previously (34). The Atsas program suite tural level, a glimpse into why some loops are recognized differ- (35) and GASBOR (43) were used to extract shapes from SAXS scattering ently by MSH2/MSH3 and imply that the junction dynamics is at curves (see SI Methods for more details). least one component in a complex process that leads to mutation. Integration of our biochemical and smFRET data clarifies two Single Molecule FRET. The single molecule FRET experiments were performed key issues bearing on the expansion mutation. First, the role of on a prism-type total internal reflection microscope which features 532 nm MSH2/MSH3 ATP hydrolysis activity in causing expansion has excitation from a Nd:YAG laser (50 mW, CrystaLaser), as previously described (44, 45). been unclear. A G674A Walker A site mutation in the MSH2 subunit suppresses CTG (Cytosine-thymine-guanine) expansion ACKNOWLEDGMENTS. This work was supported by the Mayo Foundation, in mice (38), and prevents GAA (Guanine-adenine-adenine) de- the National Institutes of Health Grants NS40738 (C.T.M.), GM066359 letion in yeast (39), implying that ATP hydrolysis in the MSH2 (C.T.M.), NS062384 (to C.T.M.), and CA092584 (C.T.M.), NS060115 (to C.T.M.) subunit is a requisite step in expansion. However, we observe in and National Science Foundation PHY-0748642 (I.R.).

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E844 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1105461108 Lang et al. Downloaded by guest on September 30, 2021