High Affinity Cooperative DNA Binding by the Yeast Mlh1-Pms1 Heterodimer

doi:10.1006/jmbi.2001.4958 available online at http://www.idealibrary.com on J. Mol. Biol. (2001) 312, 637±647

MarkC.Hall1,HongWang2,DorothyA.Erie2andThomasA.Kunkel1*

1Laboratories of Molecular We demonstrate here that the Saccharomyces cerevisiae Mlh1-Pms1 hetero- Genetics and Structural dimer required for DNA mismatch repair and other cellular processes is Biology, NIEHS, RTP, NC a DNA binding protein. Binding was evaluated using a variety of single 27709, USA and double-stranded DNA molecules. Mlh1-Pms1 bound short substrates with low af®nity and showed a slight preference for single-stranded 2Department of Chemistry DNA. In contrast, Mlh1-Pms1 exhibited a much higher af®nity for long University of North Carolina DNA molecules, suggesting that binding is cooperative. High af®nity Chapel Hill, NC 27599, USA binding required a duplex DNA length greater than 241 base-pairs. The rate of association with DNA was rapid and dissociation of protein-DNA complexes following extensive dilution was very slow. However, in competition experiments, we observed a rapid active transfer of Mlh1-Pms1 from labeled to unlabeled DNA. Binding was non-sequence speci®c and highly sensitive to salt type and concentration, suggesting that Mlh1- Pms1 primarily interacts with the DNA backbone via ionic contacts. Cooperative binding was observed visually by atomic force microscopy as long, continuous tracts of Mlh1-Pms1 protein bound to duplex DNA. These images also showed that Mlh1-Pms1 simultaneously interacts with two different regions of duplex DNA. Taken together, the atomic force microscope images and DNA binding assays provide strong evidence that Mlh1-Pms1 binds duplex DNA with positive cooperativity and that there is more than one DNA binding site on the heterodimer. These DNA binding properties of Mlh1-Pms1 may be relevant to its partici- pation in DNA mismatch repair, recombination and cellular responses to DNA damage. Keywords: mismatch repair; Mlh1; Pms1; MutL homologs; DNA binding; *Corresponding author recombination

Introduction nearbyhemimethylated50-GATC-30sequence.7±9 The incision serves as an entry point for UvrD The DNA mismatch repair systems of prokar- (DNAhelicaseII)10todisplacetheerror-containing yotes and eukaryotes serve a vital function in DNA strand, which is degraded by one of several maintaining genomic stability by correcting repli- exonucleases. The resulting gap is ®lled in by DNA cation errors that escape polymerase proofreading polymerase III holoenzyme and sealed by DNA and by discouraging recombination between home- ligase.4 ologous DNA sequences (for recent reviews see Two of the E. coli mismatch repair proteins, Buermeyer et al.; Kolodner & Marsischky; Harfe & MutS and MutL, have homologs in eukaryotic mis- Jinks-Robertson).1±3TheEscherichiacoliDNAmis- match repair systems, suggesting that early steps match repair pathway is the best characterized and in the pathway are evolutionarily conserved. hasbeenfullyreconstitutedinvitro.4Thedimeric Homologs have not been found for many of the MutSproteininitiatesrepairbyrecognizinga downstream components of the E. coli pathway, DNAmismatch.5MutLbindstoaMutS-mismatch indicating that following mismatch recognition, complex6andlinksittotheMutH-catalyzed there may be substantial divergence in the repair incision of the newly replicated DNA strand at a mechanism. While much attention has been focused on studying the MutS homologs and their Abbreviations used: ss, single-stranded; ds, double- role in initiating mismatch repair through mis- stranded; AFM, atomic force microscopy. match recognition, many details of the role of the E-mail address of the corresponding author: MutL homologs are still unknown. In the past few [email protected] years, substantial progress has been made in char-

0022-2836/01/040637±11 $35.00/0 638 Mlh1-Pms1 DNA Binding acterizing the E. coli MutL protein. MutL interacts withthreeotherenzymesinthepathway,MutS,6 MutH8,9andUvrD,11anditstimulatestheactivity ofeachoftheseproteins.8,9,12,13Thus,MutLactsas a central coordinator of the serial reactions that occur during the correction of a DNA mismatch. MutLpossessesaweakATPaseactivity8thatis essentialformismatchrepair.14Inaddition,MutL was shown to be a DNA binding protein with af®- nityforbothssDNAanddsDNA.8,15±17Inmostof those studies, DNA binding appeared to be relatively weak. Possibly for this reason, DNA binding by MutL has not yet been investigated extensively. In fact, most current models for DNA mismatch repair do not invoke direct MutL interaction with the DNA substrate. It has become clear in recent years that eukaryotic mismatch repair proteins participate in a variety of other DNA transactions. These include transcription-coupled nucleotide excision repair, DNA damage surveillance, cell cycle checkpoint Figure 1. DNA binding by Mlh1-Pms1 is highly sensi- control and apoptosis, meiotic chromosome pairing tive to salt. Nitrocellulose ®lter binding assays were per- and crossing-over, and possibly somatic hyper- formed as described in Materials and Methods. Binding was measured as a function of Na concentration using mutation of immunoglobulin genes (reviewed by 3 1 5 mM[H]pGBT9 supercoiled DNA (nucleotide concen- Buermeyeretal. ).Ofparticularinterest,the tration) and 90 nM Mlh1-Pms1 (heterodimer concen- eukaryotic MutL homologs MLH1 and PMS2 are tration). Salt titrations were performed with NaCl in the the only mismatch repair proteins currently known presence (*) or absence (&) of 4 mM MgCl2 or with toplayacentralroleinmeiosis.1Micelacking sodium acetate in the presence of 4 mM magnesium MLH1 or PMS2 show defects in meiosis that acetate (~). All reactions were diluted and ®lters suggest a role in chromosome synapsis and meiotic washed with binding buffer containing the correspond- crossingover.18±20Similarly,disruptionofthe ing salt concentration. Data points represent the average yeast MLH1 gene results in defective meiotic of three individual experiments. Kaleidagraph (Synergy recombination.21Thewidespreadinvolvementof Software, Reading, PA) was used to apply a smooth these MutL homologs in DNA metabolic processes curve ®tting algorithm to the data sets to generate the continuous lines. raises two interesting questions. Do the heterodi- meric eukaryotic MutL homologs bind to DNA? Do all the biological roles of the eukaryotic MutL homologs involve a common set of biochemical properties or are certain properties (e.g. DNA bind- result from competition between Mg2 and the ing) specialized for individual applications? protein for DNA binding sites, as observed and Here, we demonstrate that the Saccharomyces cer- characterized previously for non-speci®c protein- evisiae Mlh1-Pms1 heterodimer does indeed bind to DNAinteractions.23ThebindingofMlh1-Pms1to DNA. We then report several novel features of this dsDNA was very sensitive to the concentration of DNA binding activity that have important impli- NaCl, as indicated by the steep slopes of the linear cations for the many biological functions of Mlh1- portionsoftheplotsinFigure1.Thesensitivityto Pms1. NaCl (i.e. the slope of the plots) was essentially

identical in the presence and absence of MgCl2. Results The stability of dsDNA-protein complexes was also measured as a function of increasing sodium Mlh1-Pms1 binds to DNA in a highly acetate concentration in the presence of 4 mM salt-sensitive manner magnesiumacetate(Figure1).Amuchgreatercon- The yeast Mlh1-Pms1 heterodimer was puri®ed centration of sodium acetate was required to desta- toapparenthomogeneityasdescribed.22Weused bilize the (Mlh1-Pms1)-DNA complex compared to a nitrocellulose ®lter binding assay to determine if NaCl. Acetate ions are expected to bind more Mlh1-Pms1 binds to 3H-labeled plasmid (pGBT9) weakly to proteins than Cl ions based on the Hof- DNA.Bindingwasobserved(Figure1),andwas meister series. Therefore, the requirement for a found to vary as a function of NaCl concentration, higher concentration of acetate to destabilize the protein-DNA complexes suggests that anion dis- both in the presence and absence of 4 mM MgCl2. placement from Mlh1-Pms1 is an important factor In the presence of MgCl2, the stability of the protein-DNA complex was greatly reduced compared in DNA binding. The sensitivity of Mlh1-Pms1 to the stability in the absence of MgCl2(Figure1). DNA binding to salt concentration, anion type, 2 The lower stability in the presence of MgCl2 may and the presence of Mg is consistent with a non- Mlh1-Pms1 DNA Binding 639 sequence speci®c interaction mediated primarily dilution resulted in a ®nal protein concentration of through ionic contacts with the sugar phosphate 3.7 nM, a concentration that yields very little bind- backbone. ing when added initially to the labeled DNA (see Figure3).Thus,thebindingobservedfourminutes after dilution cannot be explained by dissociation- Mlh1-Pms1 dissociates rapidly from labeled reassociation. Note also that the experiment with DNA only in the presence of unlabeled the153bplineardsDNAsubstrate(Figure2,inset) competitor DNA was performed at a low binding density (12 % of DNA molecules bound), demonstrating that indi- To investigate the stability of the (Mlh1-Pms1)- vidual protein-DNA interactions are stable during DNA complex, we measured DNA binding as a this time. A similar lack of detectable dissociation function of time after 50-fold dilution of the reac- was observed when M13mp2 ssDNA and short tionpriortoapplicationto®lters(Figure2).For ssDNA oligonucleotides were used (not shown). both circular plasmid DNA (pGBT9) and a 153 bp This slow rate of dissociation following dilution linear dsDNA, protein-DNA complexes did not demonstrates that ®lter binding is a valid approach dissociate for at least four minutes after dilution. for measuring Mlh1-Pms1 binding to DNA and For experiments using plasmid DNA, the 50-fold that differences observed with long versus short DNA substrates (see below) are not due to dissociation. In contrast to the slow dissociation from DNA following a 50-fold dilution, Mlh1-Pms1 dissociated very rapidly from [3H]pGBT9 in the pre- senceofanexcessofunlabeledpGBT9(Figure2). More than half of the initial complexes were com- pletely dissociated in less than ®ve seconds. As the concentration of competitor DNA was increased, the rate of dissociation increased (data not shown). This suggests that, despite its slow rate of passive dissociation from DNA, Mlh1-Pms1 can actively dissociate by a rapid transfer to another DNA strand. The 10 % residual binding after four minutes is observed even when competitor DNA is added prior to protein and therefore most likely does not represent a distinct set of long-lived complexes.

Apparent binding affinity is dramatically higher on long DNA substrates

8,16 Figure 2. Mlh1-Pms1 dissociates rapidly from labeled PreviousstudieswiththeE.coliMutLprotein DNA only in the presence of unlabeled competitor and circumstantial observations with Mlh1- DNA. Binding reactions were performed as described in Pms124,25suggestthatMutLhomologsbindto Materials and Methods using 5 mM[3H]pGBT9 super- short oligonucleotide DNA substrates with rela- coiled DNA (nucleotide concentration) and 183 nM tively low af®nity. To determine if Mlh1-Pms1 has Mlh1-Pms1 (heterodimer concentration). After a ten higher binding af®nity for speci®c DNA substrates minute incubation, 168-fold excess unlabeled pGBT9 or structures, ®lter binding assays were performed was added and the reaction incubated for the indicated with a variety of ss and dsDNA molecules amount of time (*) or reactions were diluted with a (Figure3).AllDNAmoleculesconsistingofrela- 50-fold excess of binding buffer and incubated for the indicated amount of time (&) prior to application to the tivelyshortss(Figure3(a))andds(Figure3(b)) ®lter. Results were normalized to the fraction of protein- DNA oligonucleotides were bound with low af®- bound DNA in the absence of competitor DNA (*), or nity. This collection included DNA substrates that without further incubation after dilution (&), which was mimic recombination intermediates, such as a Hol- set at 100 %. Data represent the average of three (&)or lidayjunction(Figure3(b)(!))andaY-junction four (*) independent trials. Inset A 250 nM 32P-labeled (not shown), as well as dsDNA oligonucleotides 153 bp linear dsDNA (nucleotide concentration) was with 50 or 30 ssDNA extensions (not shown). used in binding reactions with 548 nM Mlh1-Pms1 (het- In contrast to short DNA substrates, the appar- erodimer). After ten minutes, reactions were diluted ent binding af®nity of Mlh1-Pms1 for long ssDNA with 50-fold binding buffer and incubated for the indi- anddsDNAmoleculeswasmuchhigher(Figure3). cated amount of time prior to application to the ®lter. In this case, the actual fraction of protein-bound DNA mol- Both M13 ssDNA and duplex plasmid DNA ecules was plotted. Horizontal lines in the main graph (pGBT9) yielded high af®nity binding curves com- and inset are linear regressions generated by Kaleida- pared to the short substrates. Higher af®nity was graph. The smooth line connecting data points (*) was not due to differences in the DNA concentrations drawn by Kaleidagraph. used for long and short substrates because control 640 Mlh1-Pms1 DNA Binding

Figure 3. Binding of Mlh1-Pms1 to a variety of DNA substrates. Filter binding assays were performed as a function of protein concentration as described in Materials and Methods. (a) [3H]M13mp2 circular ssDNA (~) was 5 mM, [32P]poly(dA) (*) was 5 mM, and [32P]100mer ssDNA oligonucleotide (&) was 90 nM. (b) [3H]pGBT9 supercoiled DNA (*) and [3H]pGBT9 linear DNA (&) were 5 mM, [32P]153 bp dsDNA substrate (~) was 250 nM and the Holli- day junction substrate (!) was 100 nM. [3H]M13mp2 circular ssDNA (^, broken line) from (a) was replotted in (b) for direct comparison to the dsDNA substrates. All substrates are expressed as DNA nucleotide concentration. Data represent the average of three or four individual trials and smooth lines connecting data points were drawn with Kaleidagraph. experiments with several different substrates because M13 RFI dsDNA and pGBT9 yielded iden- demonstrated convincingly that binding was inde- tical binding curves (data not shown). It is also pendent of DNA concentration under our reaction worth noting that the af®nity of Mlh1-Pms1 for lin- conditions. One explanation for the higher binding ear pGBT9 was still much higher than the af®nity af®nity of Mlh1-Pms1 for long DNA substrates forshortdsDNAmolecules(Figure3(b)),andthat is positive cooperativity. Interestingly, single- there was no difference in af®nity for linear or cir- stranded poly(dA) of nearly the same length as the cular M13 ssDNA (data not shown). The impli- M13 ssDNA was not a high af®nity substrate for cations of these observations are discussed below. Mlh1-Pms1binding(Figure3).Similarlowaf®nity binding was observed using poly(dT) (not shown). These long synthetic homopolymeric substrates do Effect of linear dsDNA length on apparent not have the same potential to form secondary binding affinity structures as M13 ssDNA, implying that high af®- We measured the interaction of Mlh1-Pms1 with nity binding to M13 ssDNA re¯ects Mlh1-Pms1 linear dsDNA as a function of the DNA chain interactions with dsDNA regions. length to determine the length of dsDNA required toobtainhighaf®nitybinding(Figure4).Restric- A hierarchy of apparent binding affinities for tion fragments of pGBT9 were generated and 32 long DNA substrates labeled with P in identical fashion. A single concentration of Mlh1-Pms1 was used that results in Under identical reaction conditions, supercoiled less than half-maximal binding on the longest plasmid DNA was bound with signi®cantly and DNA substrates, and all DNA substrates were pre- reproducibly higher apparent af®nity than the sent at an identical nucleotide concentration. The same plasmid DNA linearized by a restriction results reveal a sharp increase in binding as the endonuclease(Figure3(b),compare*to&).This DNA length increased from 241 bp to 513 bp. As effect was not related to the superhelicity of the the length continued to increase, binding remained closed circular plasmid DNA, because a similar essentially constant. The amount of protein-DNA difference was observed between linear and nicked complex formed with the longer substrates present circular plasmid DNA (data not shown). Further- hereat250nM(30to40%inFigure4)issimilarto more, M13 ssDNA was bound with consistently the 30 % binding observed when a 20-fold higher higher apparent af®nity than the supercoiled plas- concentration of long, linear duplex is used mid DNA. This effect was unrelated to the differ- (Figure3).Thiscon®rmsthecontrolexperiments ence in length between M13 ssDNA and pGBT9 mentioned above and indicates that the higher Mlh1-Pms1 DNA Binding 641

absence of DNA ends is a signi®cant factor in the interaction between Mlh1-Pms1 and DNA.

Cooperative binding observed visually using atomic force microscopy Atomic force microscopy (AFM) was used to directly visualize Mlh1-Pms1 bound to dsDNA. A 1.8kbplasmid(pMH10;Figure5)andeithercircu- larorlinearM13dsDNA(Figure6)weredeposited onto mica in the presence and absence of Mlh1- Pms1 in the same buffer used for the ®lter binding assays.IntheabsenceofMlh1-Pms1(Figures5(a) and6(a)),theDNAmoleculesshowednohighfea- tures and appeared very similar to previously pub- lishedimagesofcirculardsDNA.29Inthepresence of Mlh1-Pms1, the appearance of the DNA molecules was drastically altered by the bound protein (Figure5(b)-(d)andFigure6(b)and(c)).Inspection of a large number of such images revealed that the Figure 4. Effect of dsDNA length on binding by interaction between Mlh1-Pms1 and DNA usually Mlh1-Pms1. Filter binding assays were performed as occurred as long, continuous tracts of protein coat- 32 described in Materials and Methods. All [ P]DNA sub- ingtheDNA(e.g.seeFigures5(c)and6(c)and strates were present in reactions at 250 nM (nucleotide (d)).Singlyboundproteinmoleculeswere concentration). The concentration of Mlh1-Pms1 in all reactions was 180 nM (heterodimer). Each data point observed at a much lower frequency than those represents the average of four independent experiments found in tracts. These observations indicate coop- and error bars are standard deviations. erative DNA binding by Mlh1-Pms1. In the majority of the images of continuous tracts of bound protein, two separate dsDNA regions appeared to be in contact with the protein tracts (Figures5(b)-(d)and6(b)-(d)).Whiletherewere DNA binding af®nity depends on chain length, not some long stretches of bound protein associated DNA concentration. Therefore, these results are withonlyonedsDNAregion(Figure6(c),blue again consistent with a cooperative binding mech- arrow), long, cooperatively bound tracts of protein anism for Mlh1-Pms1 in which a DNA chain were observed more often associated with two length of greater than 241 bp is required to achieve dsDNAregions(e.g.seeFigure6(c),redarrow). the cooperative effect. We also observed long tracts of Mlh1-Pms1 bound Figure4andthedissociationexperimentspre- simultaneously to two separate linear M13 dsDNA sentedinFigure2alsosuggestthattranslocation molecules(Figure6(d))althoughtheoccurrence or sliding of Mlh1-Pms1 off DNA ends was not was much less frequent than with the circular responsible for the difference in apparent af®nities dsDNA. This result is consistent with the lower between short and long DNA molecules, as shown af®nity of Mlh1-Pms1 for linear versus circular inFigure3.Ithasbeensuggestedthatthehuman dsDNA observed in ®lter binding assays (see Msh2-Msh6 heterodimer, a mismatched DNA rec- Figure3).TheAFMresultssuggestthattheMlh1- ognition protein, forms a topologically constrained Pms1 heterodimer contains more than one DNA ring around DNA capable of sliding or translocat- binding site and that binding of two duplex DNA strands by Mlh1-Pms1 promotes the formation of ing, and that requires a free DNA end to dis- long tracts of cooperatively bound protein (see Dis- sociate.26CrystalstructuresofbacterialMutS cussion). proteinsareconsistentwiththisidea.27,28Asimilar A statistical analysis of the heights of the DNA concept of DNA binding via formation of a topolo- and DNA-protein complexes observed in the AFM gically constrained ring around DNA also has been images was performed to con®rm that the struc- proposed for the E. coli MutL protein, based on the tures we interpret as two dsDNA regions coated crystal structure of an N-terminal 40 kDa by Mlh1-Pms1 do contain protein and are not 16 fragment. However,ifdissociationfromfreeends simply two interwound dsDNA regions. The wereresponsiblefortheobservationsinFigure3, height of the DNA alone versus the DNA-protein then we would not expect to see such a sharp tran- complexes provides a quantitative comparison of sition from low to high af®nity binding as a func- thedifferentstructures.30,31Intheabsenceofpro- tionofDNAlengthinFigure4,andwewould tein, the average height of dsDNA was expect to see more rapid dissociation of Mlh1- 0.44(Æ0.08)nm,consistentwithapreviousstudy.32 Pms1 from the short 153 bp dsDNA molecule com- At intersections where two dsDNA regions cross paredtocirculardsDNAlackingends(Figure2). overeachother(e.g.Figure6(a),arrow),theaver- Therefore, it is unlikely that the presence or age height was 0.67(Æ0.06) nm. In the presence of 642 Mlh1-Pms1 DNA Binding

Figure 5. Atomic force microscopy images of Mlh1-Pms1 binding to a 1.8 kb circular dsDNA. AFM images were obtained as described in Materials and Methods. (a) Representative top view of pMH10 DNA alone. The DNA concentration was 15 mM (nucleotide) and the scan size was 900 nm. The color bar (height) representing 0-3.0 nm above the mica surface applies to (a) and (b). (b) Representative top view of 15 mM pMH10 in the presence of 35 nM Mlh1-Pms1. The scan size was 900 nm. (c) and (d) Zoom surface plots of the boxed regions from (b). The plane of the mica was inclined 40 to show the topography of the surface.

Mlh1-Pms1, structures presumably containing pro- iments provide additional support that the high tein tracts associated with two dsDNA regions had af®nity binding of Mlh1-Pms1 involves multiple an average height of 1.42(Æ0.24) nm. This latter dsDNA binding sites in addition to cooperativity. value is too large to be accounted for by two inter- First, the heterodimer binds with higher af®nity to wound dsDNA regions and we conclude that they circular duplex DNA, whether it is supercoiled or are coated with Mlh1-Pms1. relaxed,thantolinearduplexDNA(Figure3).Cir- cular DNA molecules are more likely than linear molecules to contain two dsDNA regions adjacent Discussion to or crossing over each other, thus presenting This study provides the ®rst direct evidence that better opportunities for the binding of Mlh1-Pms1 a eukaryotic MutL protein complex binds to DNA. to both strands and consequent nucleation of a Nitrocellulose ®lter binding and AFM experiments tract of cooperatively bound protein. Second, high demonstrated that yeast Mlh1-Pms1 binds to both af®nity binding is only seen when the linear ssDNA and dsDNA in a non-sequence speci®c duplexisatleast513base-pairs(Figure4).Ifone manner. In ®lter binding assays, the af®nity of assumes that the geometry of the DNA binding Mlh1-Pms1 for short DNA molecules was low with sites on Mlh1-Pms1 precludes their occupying a slight preference for ssDNA over dsDNA. This adjacent sites on a DNA molecule, then a short, result is consistent with previous work on E. coli relatively in¯exible linear duplex might permit MutL.16Surprisingly,theaf®nityofMlh1-Pms1for interaction with only a single DNA binding site. long DNA molecules was much greater than for Only when the linear duplex DNA molecule is short DNA. The data indicate that this increase in long (i.e. 513 base-pairs), and therefore suf®ciently af®nity on long DNA molecules is a result of ¯exible to fold back on itself, will an opportunity strong positive cooperativity, a property not pre- exist for a second DNA binding site on Mlh1-Pms1 viously reported for any MMR protein. AFM to be engaged. Although separate DNA molecules images of protein tracts on dsDNA provide visual in solution would also be available for interaction con®rmation of cooperative binding. with the second binding site, intramolecular events In addition to demonstrating cooperative bind- should be more favorable. It is also possible that a ing, the AFM images also suggest that the Mlh1- minimum length of DNA (>241 bp) is required to Pms1 heterodimer contains more than one DNA establish a stable tract of cooperatively bound binding site. Moreover, because the majority of Mlh1-Pms1 and that this contributes to the DNA protein tracts observed by AFM were associated length dependence. Third, Mlh1-Pms1 binds with with two duplex DNA regions, the concomitant higher af®nity to long M13 ssDNA than to long occupation of two DNA binding sites on Mlh1- synthetic homopolymeric ssDNA. M13 ssDNA, but Pms1 may promote the cooperative binding. Sev- not homopolymeric ssDNA, forms a complex array eral observations from the ®lter binding exper- ofsecondarystructuresinsolution,29providing Mlh1-Pms1 DNA Binding 643

Figure 6. Atomic force microscopy images of Mlh1-Pms1 binding to M13mp2 dsDNA. AFM images were obtained as described in Materials and Methods. (a) Representative top view image of M13mp2 RFI DNA alone. The DNA concentration was 8.7 mM (nucleotide) and the scan size was 1500 nm. The color bar (height) representing 0-3.5 nm above the mica surface applies to (a), (b) and (c). The white arrow points to two dsDNA regions crossing over each other. (b) Representative top view image of 8.7 mM M13 RFI DNA in the presence of 35 nM Mlh1-Pms1. The scan size was 1500 nm. (c) Zoom view of the boxed region from (b). The light blue arrow indicates a tract of cooperatively bound Mlh1-Pms1 associated with a single dsDNA region. The red arrow indicates a tract of cooperatively bound Mlh1-Pms1 associated with two dsDNA regions of a single M13 molecule. (d) Surface plot showing two linear M13 dsDNA molecules held together by a tract of bound Mlh1-Pms1. The DNA concentration was 8.7 mM (nucleotide) and Mlh1-Pms1 was present at 35 nM. The image is an enlargement from an original scan size of 1800 nm. The plane of the mica was inclined 60 to show the topography of the surface. many opportunities for Mlh1-Pms1 to bind two within a central hole in the protein. Our results for non-adjacent duplex DNA regions. Furthermore, binding of Mlh1-Pms1 to long duplex DNA imply circular and linear ssDNA would adopt essentially that alternatives to this model exist for the eukary- the same con®guration in solution, providing an otic heterodimer. Nonetheless, the extensive hom- explanation for the observation that circular and ology and conservation of function implies that linear ssDNA are bound by Mlh1-Pms1 with simi- similarities may exist in certain DNA binding lar af®nity, unlike the situation with circular and properties of bacterial MutL and the eukaryotic linear dsDNA. Finally, two binding sites are con- MutL homologs. For example, Mlh1-Pms1 binding sistent with the observation that addition of to DNA is highly sensitive to salt concentration unlabeled competitor DNA to binding reactions andaniontype(Figure1),suggestinginteractions containing stable protein-DNA complexes leads to of positively charged amino acid side-chains with very rapid dissociation of protein from the labeled the DNA backbone. Consistent with this obser- DNA(Figure2).Giventheslowrateofpassivedis- vation, an Arg to Glu mutation has been shown to sociation of complexes after dilution, this rapid stronglydecreasebindingofMutLtoDNA.16That ``switching'' of protein from labeled to unlabeled arginine in MutL is not conserved in Mlh1 or Pms1 DNA most likely involves an intermediate in and the region of E. coli MutL predicted to bind which the protein interacts simultaneously with DNA16isnothighlyconservedamongMutL twoDNAmolecules.33 homologs at the primary sequence level. However, The concept of more than one DNA binding site modelbuildingbyBanetal.16suggeststhataposi- in the Mlh1-Pms1 heterodimer differs from a tive potential is conserved in the corresponding model proposed for DNA binding by homodimeric region of human MLH1-PMS2. We are currently E.coliMutL.16Basedonstructuralinformation,it attempting to map the location of DNA binding was suggested that MutL binds ssDNA in a sad- sites in Mlh1-Pms1 for functional analysis. Similar dle-shaped groove created by the dimerization of efforts to identify the sites responsible for coopera- MutL N-terminal domains. However, that groove tive protein-protein interactions also should be is too small to accommodate duplex DNA. It was informative regarding Mlh1-Pms1 functions. further predicted that the C-terminal residues of CurrentmodelsforDNAmismatchrepair2,3,34 intact MutL might contribute to formation of a have not invoked a direct interaction of MutL dimeric protein structure that encircles the DNA homologs with DNA. Our results indicate that molecule, thus implying a single DNA binding site these models may now need revision given the 644 Mlh1-Pms1 DNA Binding ability of Mlh1-Pms1 to bind with high af®nity to ile with spermatocytes exhibiting defective long dsDNA. There is already some evidence that chromosomesynapsis.18Maleandfemalemice DNA binding by MutL plays a role in mismatch de®cient in MLH1 are sterile and although repair in E. coli. The interaction of MutL with DNA chromosome synapsis is more or less normal, is important for its ability to stimulate DNA heli- meiosis is arrested prematurely due to decreased caseII,17thehelicaseinvolvedintheexcisionstep crossing-over.19,20Moreover,MLH1immunoloca- of the mismatch repair pathway. Furthermore, lizes to sites of crossing over in wild-type sperma- DNAenhancestheATPaseactivityofMutL,16 tocytes. In yeast, Mlh1 is also required for normal whichisrequiredformismatchrepair14andispro- crossingoverduringmeiosis.21Itiscurrently posed to trigger the transformation of MutL from unclear exactly how the MutL homologs act in aninitiationmodetoaprocessingmode.16 meiosis to facilitate proper chromosome synapsis The presence of multiple DNA binding sites on and crossing over. They could serve as part of the Mlh1-Pms1 may be important for communication physical linkage between homologous chromo- between the strand discrimination signal (e.g. poss- some pairs or at chiasmata. However, MLH1 and ibly a nick or the primer termini at a replication PMS2 may not even act together during meiosis, fork) and proteins bound at the mismatch, such as since the mice results show defects at different Msh2-Msh6. For example, in the presence of ATP, stages of meiosis depending on which gene is binding of E. coli MutS protein to a mismatch de®cient. Also, it appears in yeast that Pms1 resultsinformationofa-loopstructures12thatmay (homolog of mammalian PMS2) is not involved in be intermediates in the search for the strand dis- meioticcrossingover.21Understandingtheroleof crimination signal. E. coli MutL enhances the yield DNA binding by MutL homologs in recombination of a-loops and it is found in a complex with MutS promises to be an exciting area of future research. at the base of these structures where two dsDNA It will be interesting to determine if other regions are juxtaposed. Perhaps MutL stabilizes dimeric MutL complexes possess a similar high this putative repair intermediate not only by inter- af®nity cooperative DNA binding activity. This acting with MutS, but also by binding simul- may improve our understanding of the biological taneously to the two dsDNA regions that merge at function of this DNA binding property. For the base of the a-loop. Other possible functions for example, if E. coli MutL protein binds coopera- multiple DNA binding sites exist as well. Two tively, then cooperative DNA binding would likely Mlh1-Pms1 binding sites for dsDNA may be rel- be relevant for biological processes common to evant to repair on the leading and lagging strands prokaryotes and eukaryotes, such as mismatch during replication. repair, transcription-coupled nucleotide excision Current models of DNA mismatch repair also do repair or prevention of homeologous recombina- not accommodate the present evidence for the tion. In eukaryotes there are multiple dimeric cooperativity of DNA binding by Mlh1-Pms1 MutL homolog complexes such as Mlh1-Mlh2 and (Figures 3-6). In principle, cooperative interactions Mlh1-Mlh3 in yeast and MLH1-PMS1 and MLH1- with duplex DNA could facilitate communication MLH3 in mammals. The different roles of these between the strand discrimination signal and the multiple complexes are only beginning to be mismatch. Alternatively, physical interactions of understood. Studies of the DNA binding properties Mlh1 and Pms1 with other components of the mis- of these other heterodimers may also help provide match repair machinery may prevent cooperative further insight into their biological roles. assembly of Mlh1-Pms1 on DNA during mismatch repair. Cooperative interactions between Mlh1- Pms1 heterodimers, and DNA binding in general, Materials and Methods may be relevant to the other DNA transactions in Overproduction and purification of Mlh1-Pms1 which these MutL homologs participate. These processes include meiotic recombination, transcrip- The procedure for overproduction and puri®cation of tion-coupled excision repair of DNA adducts, and Mlh1-Pms1isdescribedindetailelsewhere.22Protein other cellular responses to agents that damage concentrations were determined by the method of DNA (for reviews on biological roles of mismatch Bradford35usingdyesolutionfromBio-Radandbovine repair proteins, see Buermeyer et al. and Harfe & serum albumin (BSA) as a standard. Jinks-Robertson1,3). The possible role of DNA binding by Mlh1-Pms1 Materials in genetic recombination is of particular interest. Recombination involves homologous pairing of Strain Sé103 (relA1 thyA144 rpsL254(Strr) metB1 two DNA molecules over relatively long stretches. deoC4)36wasobtainedfromtheE.coliGeneticStockCen- 0 The cooperative, multiple-site DNA binding ter (Yale University, New Haven, CT). Strain Sé103 F activity of Mlh1-Pms1 could be relevant here, was created by mating Sé103 with XL1-Blue (recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F0 proAB although the two duplexes with which Mlh1-Pms1 lacIqZÁM15 Tn10 (Tetr)])(Stratagene) and selecting trans- simultaneouslyinteracts(Figures5and6)neednot conjugants on LB agar plates containing 25 mg/ml strep- be homologous. It is clear from studies in mice that tomycin and 10 mg/ml tetracycline. Media for growth of the MLH1 and PMS2 proteins are required for nor- Sé103 F0 contained M9 minimal media salts, 12 mM mal meiosis. Male mice de®cient in PMS2 are ster- glucose, 1 mM MgSO4, 2.7 g/l salt-free casamino acids, Mlh1-Pms1 DNA Binding 645

20 mM thymidine, and 25 mg/ml streptomycin. Sé103 activities (cpm/mg) were: 84 bp, 2.2 Â 107; 153 bp, carries thyA and deoC mutations that allow growth in 1.5 Â 107; 241 bp, 6.6 Â 106; 513 bp, 4.1 Â 106; 1054 bp, media containing a very low thymidine concentration. 1.9 Â 106; 1801 bp, 1.5 Â 106; 3482 bp, 9.6 Â 105; 5339 bp, This maximizes speci®c activity when labeling DNA 7.1 Â 105. 32P-labeled Holliday junction and Y-junction in vivo with [3H]thymidine. Phage T4 polynucleotide substrates with arms of 27 bp were donated by Dr kinase, calf intestinal phosphatase, T4 DNA ligase, and Anna Fabisiewicz and Dr Leroy Worth (NIEHS). all restriction enzymes were from New England Biolabs To construct the 1.8 kb plasmid pMH10, pET3c (Nova- and were used under recommended conditions. DNase I gen) was digested with BsrBI. The resulting 1.8 kb frag- (ampli®cation grade) was from Life Technologies. ment containing the origin of replication and the gene 6-[3H]thymidine and [g-32P]ATP were from Amersham encoding b-lactamase was gel-puri®ed, religated with T4 Pharmacia Biotech. Poly(dA) was purchased from Sigma DNA ligase and recovered in E. coli XL1-Blue. and the 100mer ssDNA oligonucleotide was synthesized by Life Technologies. Nitrocellulose filter binding Preparation of DNA substrates Nitrocellulose ®lters (25 mm, 0.45 mm, HAWP, Milli- pore Corporation) were soaked in 0.4 M KOH for 20 To prepare [3H]M13mp2 phage DNA, a 10 ml over- 0 minutes, rinsed thoroughly with deionized distilled night culture of Sé103 F was grown at 37 C in minimal water and soaked in binding buffer (25 mM Tris-HCl media described above. The overnight culture was (pH 8.0), 10 % glycerol, 25 mM NaCl, 4 mM MgCl , diluted in 500 ml minimal media and grown for one 2 9 1 mM DTT) for at least one hour prior to use. Unless hour at 37 C prior to addition of 5 Â 10 pfu of puri®ed otherwise stated, binding reactions (20 ml) were per- M13mp2 phage and 5 mCi [3H]thymidine. Growth was formed in binding buffer supplemented with 100 mg/ml continued for another nine hours at 37 C. Phage par- BSA. These binding conditions were selected based on ticles were harvested and phage DNA puri®ed as 37 theresultspresentedinFigure1.Itshouldbenotedthat described. Thespeci®cactivitywas150,000cpm/mg. binding reactions were performed in the presence of To prepare [3H]pGBT9 plasmid DNA, pGBT9 was trans- MgCl2 for consistency with atomic force microscopy formed into Sé103 and a single transformant was grown experiments (see below), which require magnesium for at 37 C up to one liter in minimal media supplemented deposition of DNA on the negatively charged mica sur- with 150 mg/ml ampicillin and 4 mCi of [3H]thymidine. face. DNA substrate and Mlh1-Pms1 were included at Growth was continued overnight to saturation at 37 C, the indicated concentrations. All binding reactions were cells were harvested by centrifugation and plasmid DNA incubated at room temperature (23-25 C) for either ®ve puri®ed using a QIAgen mega prep kit followed by a or ten minutes. It was empirically determined that bind- CsCl/EtBr gradient. The speci®c activity was 92,000 3 ing was completed in less than 30 seconds and varying cpm/mg. Nicked circular [ H]pGBT9 was generated by the length of incubation did not in¯uence results. Fol- treating 9 mg of supercoiled DNA with 0.01 unit of lowing incubation, binding reactions were diluted with DNase I for ®ve minutes at room temperature. The reac- 1 ml of binding buffer and passed through nitrocellulose tion was quenched by adding EDTA to 2.5 mM and 3 ®lters at a ¯ow rate of approximately 2 ml/minute using incubating at 65 C for 20 minutes. Linear [ H]pGBT9 a Millipore 1225 sampling vacuum manifold. Filters was generated by digestion of approximately 25 mg were rinsed once with 750 ml of binding buffer, dried supercoiled DNA with 20 units PstI restriction endonu- under a heat lamp and quantitated with a Beckman LS clease at 37 C overnight followed by puri®cation from a 7800 liquid scintillation counter and EcoLume scintil- 0.8 % (w/v) agarose gel using the QIAquick gel extrac- lation ¯uid (ICN). Background counts were measured in tion kit (QIAgen). mock reactions lacking protein and although they varied 0 Poly(dA) and the 100mer oligonucleotide were 5 end- somewhat depending on the DNA substrate, were typi- 32 labeled with P by reaction with T4 polynucleotide cally less than 1 % of the total radioactivity. kinase and [g-32P]ATP at 37 C for one hour. The 100mer oligonucleotide was separated from unincorporated nucleotide using a QIAquick nucleotide removal kit Atomic force microscopy (QIAgen). Poly(dA) was separated from unincorporated nucleotide on an STE-equilibrated Bio-Gel A-15 m col- Binding reaction mixtures (20 ml) contained 35 nM umn. The speci®c activities (cpm/mg) were 1.4 Â 109 and Mlh1-Pms1 and either 15 mM pMH10 or 8.7 mM 4.8 Â 107 for the 100mer and poly(dA), respectively. The M13mp2 dsDNA (nucleotide concentration) in binding series of variable length linear duplex DNA molecules buffer. Reactions were incubated at room temperature was generated by digestion of 68 mg of unlabeled pGBT9 for one minute, then deposited on freshly cleaved ruby plasmid DNA with ten units of BsrBI, ®ve units FspI, ten mica (Spruce Pine Mica Company, Spruce Pine, NC). units HincII, or ten units AccI overnight at 37 C. Restric- Following an additional one minute incubation, excess tion reaction products were separated on a 1 % (w/v) liquid was blotted with Whatman ®lter paper and the TAE-agarose gel run in the presence of 0.5 mg/ml EtBr mica was rinsed thoroughly with Nanopure water and and the 3482 bp, 1801 bp and 241 bp BsrBI fragments, dried under a nitrogen gas ¯ow. All images were the 1054 bp and 513 bp FspI fragments, the 5339 bp and obtained with a Nanoscope III (Digital Instruments, 153 bp HincII fragments and the 84 bp AccI fragment Santa Barbara, CA) operated in tapping mode in air. were puri®ed using the QIAquick gel extraction kit Non-contact/tapping mode Nanosensor PointprobeR sili- (QIAgen). Each fragment was treated with calf intestinal con cantilevers (Molecular Imaging Corporation) with phosphatase at 37 C for one hour, 50 end-labeled with force constants from 37.0 to 50.0 N/m were used for all T4 polynucleotide kinase and [g-32P]ATP at 37 C for one imaging. The typical resonant frequency of these tips hour and separated from unincorporated nucleotide was approximately 180 kHz. The images were collected using the QIAquick nucleotide removal kit. The concen- at 2.5-3.0 kHz scan speed and 512 Â 512 resolution. Sec- tration of each substrate was measured by absorption at tion analysis was performed using the Nanoscope soft- 260 nm and adjusted to 10 mM DNA nucleotide. Speci®c ware (version 4.42r4). 646 Mlh1-Pms1 DNA Binding

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Edited by M. Belfort Supplementary Material for this paper is available on IDEAL