Mismatch Repair in Human Nuclear Extracts

EFFECTS OF INTERNAL DNA-HAIRPIN STRUCTURES BETWEEN MISMATCHES AND EXCISION-INITIATION NICKS ON MISMATCH CORRECTION AND MISMATCH-PROVOKED EXCISION*

Huixian Wang and John B. Hays {ddagger}

From the Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, Oregon 97331-7301

Received for publication, March 20, 2003 , and in revised form, May 14, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
DNA mismatch repair (MMR) couples recognition of base mispairs by MSH2·MSH6 heterodimers to initiation, hundreds of nucleotides away, of nascent strand 3'-5' or 5'-3' excision through the mispair. Mismatch-recognition complexes have been hypothesized to move along DNA to excision-initiation signals, in eukaryotes, perhaps ends of nascent DNA, or to remain at mismatches and search through space for initiation signals. Subsequent MMR excision, whether simple processive digestion of the targeted strand or tracking of an excision complex, remains poorly understood. In human cell-free extracts, we analyzed correction of a mismatch in a 2.2-kilobase pair (kbp) circular plasmid containing a pre-existing excision-initiation nick for initiation, and measured MMR excision (in the absence of exogenous dNTPs) at specific locations. Excision specificities were ~100:1 for nicked versus continuous strands, 80:1 for mismatched versus homoduplex DNA, and 30:1 for shorter (0.3-kbp) versus longer (1.9-kbp) nick-mispair paths. To test models for recognition-excision coupling and excision progress, we inserted potential blockades, 20-bp hairpins, into nick-mispair paths, using a novel technique to first generate gapped plasmid. Continuous strand longer-path hairpins did not affect mismatch correction, but shorter-path hairpins reduced correction 4-fold, and both together eliminated it. Shorter-path hairpins had little effect on initiation of (3'-5') excision, measured 30–60 nucleotides 5' to the nick, but blocked subsequent progress of excision to the mismatch; longer-path hairpins blocked the (lower level) 5'-3' excision to the mismatch. Thus, (a) MMR excision protein(s) cannot move past DNA hairpins. Hairpins at both ends of substrate-derived 0.5-kbp DNA fragments did not prevent ATP-induced dissociation of mismatch-bound human MSH2·MSH6, so recognition complexes at mismatches might provoke excision at nicks beyond hairpins, or loosely sliding MSH2·MSH6 dimers might move to the nicks.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Evolutionarily conserved prokaryotic and eukaryotic mismatch-repair (MMR)1 systems promote genomic stability by correcting DNA replication errors, antagonizing homeologous recombination between diverged DNA sequences, and responding to a variety of DNA lesions (for reviews, see Refs. 13). The Escherichia coli pathway, fully reconstituted from purified proteins, provides a mechanistic paradigm. Here, MutS homodimers bind to DNA mismatches and MutH proteins recognize hemi-methylated d(GATC) sequences, transitory consequences of a brief post-replication delay in GATC-adenine methylation of nascent DNA, to thus provide the two requisite specificity elements for correction. Interaction of MutL homodimers with MutS-mismatch complexes activates DNA-strand nicking of nascent DNA at unmethylated d(GATC) sites. DNA helicase II (UvrD protein) loads at the nick if and only if MutS, a DNA mismatch, and MutL are present; one or more steps in this chain of events require ATP hydrolysis. UvrD presumably separates strands to facilitate excision of the nicked strand toward and beyond the mismatch, by one of several 3'-5' or 5'-3' ssDNA-specific exonucleases, depending on the nick-mismatch orientation. Gap filling by the replicative DNA polymerase III holoenzyme and DNA ligation follow.

In eukaryotes, mismatches are recognized by heterodimers with different specificities: base mismatches, and one or two looped-out extra nucleotides by MSH2·MSH6 and a range of extrahelical loopouts by MSH2·MSH3 in most eukaryotes (13), a subset of base-base mismatches by an additional MSH2·MSH7 in plants (4). The high similarity between MutS and its eukaryotic homologs suggests conservation of the biochemistry of MMR initiation. In contrast, although MutL-homolog MLH1·PMS2 (MLH1·PMS1 in yeast) heterodimers are required for mismatch correction, neither homologs of UvrD or MutH, nor of any ssDNA-specific exonucleases, have been implicated in eukaryotic MMR. This suggests less evolutionary conservation of post-recognition mechanisms. The absences of DNA-adenine methylation and, apparently, of MutH orthologs, indicate that different nascent-DNA features, perhaps leading-strand 3' ends and lagging-strand 3' or 5' ends, are used to strand-specifically initiate excision toward the mismatch. In mammalian cell extracts, mismatches provoke initiation of excision at pre-existing nicks or gaps in exogenous DNA substrates, with high efficiency and specificity (5, 6).

Despite the apparent divergence of mechanisms of MMR excision, numerous partial-reaction studies with purified proteins suggest general conservation of the initial steps involving MutS and MutL homologs. However, the mechanism by which mismatch-recognition is coupled to site-specific initiation of excision remains controversial. In one class of coupling models, mismatch-bound MutS/MSH dimers are postulated to bind ATP and then to move away along the DNA contour, searching for excision-initiation signals either by ATP hydrolysis-dependent translocation (7), or by ATP binding-dependent diffusional sliding, with hydrolysis occurring later (8). Or searching might be performed by more elaborate sliding complexes, to which other proteins have been recruited (25), Alternatively, MutS homolog proteins complexed with MutL homolog proteins and ATP are proposed to remain at or near mismatches, where they use DNA looping to search through space for excision initiation signals (9, 10).

Mechanisms of subsequent progressive excision are poorly understood, especially in eukaryotes. Most simply, an exonuclease might progressively digest one strand of dsDNA, as does the 5'-3' activity of eukaryotic exonuclease I, which has been firmly implicated in MMR (11, 12). No similar eukaryotic 3'-5' exonuclease has been linked to MMR, but the recent observation that aphidicolin inhibits 3'-5' excision points to exonucleases integral to the DNA replication polymerase {epsilon} and {delta} (13). Remarkably, recent reports show exonuclease I to be required for both 5'-3' and 3'-5' excision in MMR in cell-free extracts (11). Other mechanisms might involve DNA helicases that track along either strand and/or the DNA binding activities of MutL{alpha} (14).

Thus, internal DNA structures that can block protein movement might interfere with coupling of mismatch recognition to initiation of excision, and/or progress of excision toward the mismatch. We have used two recently described techniques (13, 15) to investigate both coupling of mismatch recognition to initiation of excision and the subsequent excision process. First, we have employed our new method for production of gapped circular plasmids (16) to create multiple gaps, into which we ligated not only mismatch-creating oligomers, but also other oligomers containing DNA hairpins, which might be expected to (a) block translocation of putative searching complexes and/or (b) impede progress of excision, in either case preventing mismatch correction. We also measured excision, near the nicks and elsewhere, to distinguish between case a, where no excision would be initiated, and case b, where it would be initiated but blocked before reaching the mismatch. We find a hairpin in the shorter nick-mismatch path to severely inhibit mismatch correction, but not initiation of excision; however, excision proves unable to continue past the hairpin.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids—Plasmid pUC19Y was previously derived (15) from pUC19 by removal of all recognition sequences for the sequence-specific nicking endonucleases N.BstNBI (5'-GAGTCNNNN{downarrow}) and N.AlwI (5'-GGATCNNNN{downarrow}). Further modification by standard techniques generated plasmids pUC19ASP, pUC19PPH, pUC19PA, and pUC19APH (Fig. 1, AD). Unprimed positions refer to the bla sense strand, and primed coordinates to the antisense strand; base pair 1/1' is the first C/G in the EarI endonuclease recognition site. Positions of incision sites for sequence-specific endonucleases refer to immediately preceding (5') nucleotides. All four new plasmids contain a site at position 23 for nicking endonuclease N.Bpu10I (5'-GC{downarrow}TNAGG), new N.AlwI nicking sites at positions 331 and 361, and a XhoI endonuclease site at position 339, overlapping a near site for endonuclease HindIII (Figs. 1 and 2). Each plasmid encodes one or more sites in the antisense strands, for nicking endonuclease N.BstNBI: unique sites at position 499' in pUC19ASP; four sites in pUC19PPH, at 160', 190', 469', and 499'; two in pUC19PA, 160' and 190'; and two in UC19APH, 469' and 499'.



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FIG. 1.
Substrate precursor plasmids. Plasmids shown were derived from pUC19Y (10) as described under "Experimental Procedures." Arrows indicate positions of cutting by site-specific nicking endonucleases used to introduce gaps (- -) or nicks (N.AlwI, N.BpuI01, and N.BstNBI) or dsDNA-specific endonuclease (HpaI) to select for gaps. Numbers in annuli indicate distances (nt) between endonuclease sites. Position 1/1' (inner bla-sense)/outer (bla-antisense)) corresponds to the first C/G of the EarI endonuclease site at position 2488 in pUC19.

 


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FIG. 2.
MMR substrates. Substrates containing indicated mismatches, hairpins, and nicks were constructed from plasmids pUC19ASP (A), pUC19PPH (B), pUC19PA (C), and pUC19APH (D), as described under "Experimental Procedures," and designated s19ASPgt, s19PPHgtdhp, s19PAgtshp, and s19APHgtlhp, respectively. The G/T mismatch is in a sequence such that correction in favor of G (excision of nicked strand) restores a site for endonuclease XhoI, and correction in favor of T (excision of continuous strand) restores a HindIII site. The site for AseI endonuclease, and the location of position 1/1', shown for substrate A, are the same in all substrates.

 

Construction of Substrates—After the four plasmids described above were digested with endonuclease N.AlwI, the 30-nt oligomers between nicks were removed by heating to 85 °C and reannealing to room temperature, over roughly 60 min, in the presence of a 50-fold excess of a synthetic oligomer complementary to the displaced 30-mer. Gapped plasmids were then purified by benzoylated-naphoylated-DEAE cellulose (BND-cellulose) chromatography, as described previously (16). We ligated oligomer XHMM30a, 5'-ACGTAAGCTTCGAGGTGAATAGGATCATCG, into the N.AlwI gaps to create T/G mismatches (T) whose correction to C/G or T/A would yield a site for XhoI (CTCGAG) or HindIII endonuclease (AAGCTT), respectively, and purified the resulting supercoiled substrates, correspondingly designated s19ASPgt, s19APHgt, s19PPHgt, and s19PAgt. We used endonuclease N.Bpu10I to singly nick s19ASPgt, generating substrate A (Fig. 2). We doubly nicked s19APHgt with endonuclease N.BstNBI and, as described above, created a new N.BstNBI gap in the potential longer nick-mispair path, into which we ligated oligomer HP1, designed to form a hairpin (underlined; 20-bp stem, (CC) loop): 5'-TCTATGCGACGTTAAGGCTGCTACCCTTCTGCCGT(CC)ACGGCAGAAGGGTAGCAGCCCATGGAGAGTCGCTC. The supercoiled product, s19APHgtlhp, was then singly site-specifically nicked with endonuclease N.Bpu10I to produce substrate D (Fig. 2). We similarly inserted hairpin-forming oligomer HP1 into a N.BstNBI gap in the potential shorter nick-mispair path, forming s19PAgtshp, after which N.Bpu10I nicking yielded substrate C (Fig. 2). To produce doubly gapped molecules from substrate s19PPHgt, we digested exhaustively with endonuclease N.BstNBI, removed the 30-nt oligomers by heating and reannealing as described above, and isolated putative doubly gapped (quadruply nicked) products by BND-cellulose chromatography. To remove any DNA containing one or no gaps, we digested with endonuclease HpaI, whose only sites are in the potential gap regions and that cannot cleave ssDNA, then treated with exonuclease V to destroy any linear dsDNA. We ligated oligomer HP1 into both N.BstNBI gaps and purified the covalently closed product, designated s19PPHgtdhp. Single nicking with endonuclease N.Bpu10I yielded substrate B (Fig. 2). To prepare the homoduplex control substrate s19A-SPhm from pUC19ASP, we proceeded as described for substrate A, except that the 30-nt oligomer inserted created a G/C base pair instead of a mismatch, then singly N.Bpu10I-nicked the product, s19ASPgc. To prepare the hairpin-containing homoduplex substrate s19PAgcshp, we ligated oligomer HP1 into a single N.BstNBI gap, producing plasmid pUC19PAgcshp. Although no nicked substrate (analogous to A, B, C, and D and HMA) was derived from pUC19PAgcshp, we designate it HMC in Fig. 6 and in the associated text, for convenience.



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FIG. 6.
Binding of hMutS{alpha} to mismatch- and hairpin-containing DNA fragments in the presence or absence of ATP. Isolation of the indicated 0.48-kbp ABS fragments from non-nicked substrates HMA, HMC, A, B, C, and D (see Fig. 2 and its legend) and radiolabeling of 3'-recessed ends by nucleotide incorporation, incubation of 6 fmol each with 0.25 pmol of purified hMutS{alpha} and 125 ng of nonspecific competitor DNA, with or without ATP at 200 µm as indicated, agarose gel electrophoresis and quantitative phosphorimaging were as described under "Experimental Procedures." Fractions (%) of mismatch-containing fragments shifted in the absence of ATP (lanes 1–5) were, respectively, 78 (A), 89 (B), 86 (C), 82 (D), and in the presence of ATP (lanes 6-10) were 9 (A), 10 (B), 10 (C), and 7 (D). In a separate experiment, binding of hMutS{alpha} to the ABS fragment from the single hairpin homoduplex HMC was similarly tested, in the absence or presence of ATP (lanes 11 and 12).

 

Verification of Hairpin Structures by Gel Electrophoresis—We used endonucleases AflIII and BglII to cut at the respective unique sites in substrates s19ASPgc (HMA), s19ASPgt (A), s19PPHgtdhp (B), s19PAgtshp (C), s19APHgtlhp (D), and s19PAgcshp (HMC) (for convenience, non-nicked substrates are given here the same designations as the mismatched (A, B, C, and D) or homoduplex (HMA, HMC) substrates generated from them by N.Bpu10I nicking, as described above under "Construction of Substrates"). Double cleavage with endonucleases AflIII and BglII at positions 511 and 22, respectively, generated two products: a larger ABL fragment (1.7 kbp), identical among all substrates; smaller ABS fragments (0.48 kbp) containing one (ABS-C, ABS-D, and ABS-HMC), two (ABS-B), or no (ABS-A and ABS-HMA) hairpins, as shown in Fig. 3 (left panel). Treatment with DNA polymerase I Klenow fragment (3' exo), in the presence of [{alpha}-32P]dCTP only, radiolabeled the 3' recessed ends generated by endonuclease AflIII, presumably with equal specific activities. After phenol extraction and ethanol precipitation, resuspended fragments were analyzed by electrophoresis through a 5% native polyacrylamide-gel (acrylamide:bisacrylamide at 37.5:1) and autoradiography (Fig. 3, right panel).



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FIG. 3.
Analysis of substrate structures. Substrates (letter designations when nicked shown in Fig. 2) s19ASPgt (A), s19PPHgtdhb (B), pUC19Pgtashp (C), and s19APHgtlhp (D), plus homoduplex substrate s19ASPhm (HM) were all digested with endonucleases AflIII and BglII, generating a 1.7-kbp fragment (ABL) and indicated 0.48-kbp fragments (ABS); radiolabeled nucleotides were incorporated into 3' recessed AflIII ends, and restriction digests were electrophoresed through 5% polyacrylamide and autoradiographed, as described under "Experimental Procedures." Schematic structures show ABS fragments released from the respective substrates, ABS-HM (lane 1), ABS-A (lane 2), ABS-B (lane 3), ABS-C (lane 4), and ABS-D (lane 5). Distances (bp) between fragment ends, hairpins, and mismatches are shown at the upper left.

 

Mismatch-Repair Reactions in HeLa Nuclear Extracts—Nuclear extracts were prepared from HeLaS3 cells (purchased from the National Cell Culture Center, Minneapolis, MN), as previously described (15). In brief, proteins were extracted with 0.15 M salt from nuclei released from cells broken by homogenization, then precipitated with ammonium sulfate. Standard MMR mixtures (15 µl) contained 100 ng of indicated substrates, 100 µg of nuclear extract, and 0.75 µg of bovine serum albumin, plus the following components at the indicated concentrations: 20 mM Tris-HCl, pH 7.6, 1.5 mM ATP, 1 mM glutathione, four dNTPs, each 0.1 mM, 5 mM MgCl2, 110 mM KCl. Mixtures were incubated at 37 °C for 12 min unless otherwise indicated. Reactions were terminated by the addition of 30 µl of stop solution (25 mM Na2EDTA, 0.67% sodium dodecyl sulfate, and 90 µg/ml proteinase K). After further incubation of mixtures at 37 °C for 15 min, DNA was extracted twice with an equal volume of phenol, and then precipitated with ethanol. DNA was resuspended in H2O and digested with 2 units of AseI endonuclease, plus 2 units of endonuclease HindIII or XhoI, as indicated, and 1 µg of RNase A, at 37 °C for 2 h in 15 µl of digestion buffer (10 mM Tris-HCl, pH 7.9, 50 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol). Digested products were separated by electrophoresis in 1% agarose gels made in 1x TAE buffer (40 mM Tris acetate, 2 mM EDTA). Correction of G/T mismatches to G/C restores a site for XhoI endonuclease, which thus cuts substrates already linearized by AseI endonuclease into 0.9- and 1.3-kbp fragments. DNA bands were visualized by staining with ethidium bromide, then imaged with UVP ImageStore 7500 and analyzed using ImageQuant software. Repair yield equals the ratio of the summed intensities of the 0.9- and 1.3-kbp fragments to the total of this sum plus the intensity of the 2.2-kbp band corresponding to singly cut (uncorrected) DNA.

Analysis of Excision Gaps—To freeze excision gaps generated during MMR, exogenous dNTPs were omitted from a standard reaction mixture containing nicked mismatched-DNA substrate or various control substrates. After incubation at 37 °C for 7 min, DNA was extracted and precipitated as described under "MMR Reactions in HeLa Nuclear Extracts," and digested at 37 °C for 2 h with 4 units of AhdI endonuclease plus 1 µg of RNase A in 15 µlof AhdI digestion buffer (20 nM Tris acetate, pH 7.9, 50 mM potassium acetate, 10 mM magnesium acetate, and 1 mM dithiothreitol). After addition of 0.5 pmol of a particular 32P-labeled oligomer probe (Fig. 5), the mixture was incubated at 85 °C for 5 min, then slowly cooled down to room temperature. Probes annealed to substrates were separated from free oligomers by electrophoresis in 1.2% agarose gels in 1x TAE buffer. After ethidium-stained DNA bands from agarose gels were measured quantitatively, as described under "Mismatch-Repair Reactions in HeLa Nuclear Extracts," gels were dried and the radioactivity in each sample measured by phosphorimaging. Dye-intensity measurements of bands from agarose gels were used to normalize radioactivity measurements for any variations in DNA recovery and loading.



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FIG. 5.
Analysis of mispair-provoked excision. Incubation of 500 ng of indicated substrates A, B, C, D (see Fig. 2) and HMA (s19ASPhm) for 7 min in 75-µl mixtures containing 500 µg of nuclear extract and all standard MMR components except dNTPs, extraction of DNA, resuspension in the same volumes and treatment with AhdI endonuclease, annealing of DNA in 15-µl aliquots with 0.5 pmol of the indicated radiolabeled probes, electrophoresis, and autoradiography or quantitative phosphorimaging, were as described under "Experimental Procedures." Probes are co-linear with upper (continuous; primed position numbers) or lower (nicked; unprimed position numbers) at the (left to right) positions indicated: probe 1 (5'-GATAGAGTCGCTAGACAGATAAAGCAAGTAG, 0.8 kbp from nick in longer nick-mispair path (position 1351–1381); probe 2 (5'-CCTTATTCCCGCTGTGCCTTTACAACTT), 0.045 kbp adjacent to nick in longer-path (2151–2178); probe 3 (5'-AGTCCCAATAACAGAGTACTCGCCTATGT), adjacent to nick in shorter nick-mispair path (52–80); probe 4 (5'-CGATGATCCTATTCACCTCGAAGCTTACGT), continuous (upper) strand at mispair position (323'-352'); probe 5 (5'-ACGTAAGCCTCGAGGTGAATAGGATCATCG), nicked (lower) strand at mispair position (323–352). A, representative autoradiograph for substrates and probes indicated. B, relative excision signal values determined by phosphorimaging, relative to value (100) for probe 2 with homoduplex control (HM), are shown at the respective probe positions, where they represent excision of the strand indicated. Data correspond to one of three highly reproducible experiments, among which relative signals differed by less than 10%. Positions along the upper strand are indicated by small printed numbers.

 

Purification of hMutS{alpha} and Analysis of Electrophoretic Mobility Shift—Human hMutS{alpha} protein heterodimers were prepared essentially as described (17). Briefly, the 30–65% ammonium sulfate fraction from HeLaS3 nuclear extracts was passed through a single-stranded DNA cellulose column and fractions were subsequently eluted with 1 mM ATP, then chromatographed on a 1-ml Pharmacia HR 5/5 Mono Q column. Purified hMutS{alpha} was made 1.0 mg/ml in bovine serum albumin and 10% (w/v) in sucrose, frozen in liquid nitrogen, and stored at –80 °C. SDS-polyacrylamide gel electrophoresis and Coomassie Blue staining showed the purified protein to be nearly homogeneous (>98% pure). For mobility shift analyses, 1 µg (~0.9 pmol) each of potential (not yet nicked) substrate DNA was treated with endonucleases AflIII and BglII and radiolabeled, essentially as described above under "Verification of Hairpin Structures by Gel Electrophoresis" (and again designated HMA, HMC, and A, B, C, D, for convenience), but with the following changes. Products were immediately separated by electrophoresis in a 1% agarose gel (without ethidium bromide); respective 0.48-kbp ABS fragments were cut out of the gels and recovered using a gel-extraction kit (Qiagen) as recommended by the manufacturer, after a narrow slice of each sample gel was stained with ethidium bromide to locate the fragments. Radiolabeling of 3'-recessed AflIII and BglII ends employed equal DNA inputs, measured by PicoGreen (Molecular Probes, Inc.) fluorescence, plus PolI Klenow (3' exo) and 0.2 µM [{alpha}-32]dCTP and [{alpha}-32P]dTTP, plus 16.7 µM dATP and dGTP, to ensure that little ssDNA remained at the ends; specific radioactivities were nearly identical among the five ABS fragments. DNA samples were extracted with phenol, precipitated, and resuspended in TE buffer. Equal amounts of ABS fragments (determined by radioactivities) were used for band-shift analyses. Binding reactions (25 µl) contained 10 mM Hepes-KOH, pH 7.5, 110 mM KCl, 1 mM dithiothreitol, 0.4% glycerol, 0.25 pmol of purified hMutS{alpha}, 6 fmol (2 ng) of 32P-labeled ABS fragments, and 125 ng of a 1-kbp DNA ladder (catalog number 15615-016, Invitrogen) as nonspecific competitor. Mixtures were either incubated on ice for 15 min without ATP; or ATP (200 µM) was added after 10 min and incubation continued on ice for 5 min. After addition of 5 µl of 50% sucrose, mixtures were immediately loaded onto a 5% polyacrylamide gel and electrophoresed at 6 V/cm in TBE buffer (89 mM Tris boric acid, 2 mM EDTA) with cooling. Protein-DNA complexes and free DNA were visualized by autoradiography after gel drying, and quantified by phosphorimaging.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Construction and Verification of Substrates—To test effects of internal DNA-hairpin structures on initiation and excision phases of mismatch correction, we constructed substrates A, B, C, and D in three stages (Figs. 1 and 2). First, we generated gaps at specific locations in circular substrates and ligated mismatch-creating oligomers into them. Second, we generated additional gaps, either on one side of the mismatch, or on the other side, or on both sides, and ligated hairpin-forming oligomers into the gaps. Finally, we specifically nicked the non-hairpin strand at an asymetrically located point, such that one hairpin site would be in a shorter nick-mismatch excision path and the other hairpin site in a longer-path. To incorporate hairpins into mismatch-repair substrates, we needed to anneal into gaps 72-nt oligomers that formed intramolecular stem-loop structures without significant intermolecular dimerization. When we tested three different 72-mers with different 42-nt hairpin structures and/or different flanking sequences, two of them mostly formed self-dimers under the annealing conditions tested, but the third (oligomer HP1) reproducibly formed a single structure with an electrophoretic mobility consistent with an intramolecular hairpin (data not shown). The respective sequences do not suggest any obvious explanation for this interesting phenomenon. The 20-bp stem is identical to one of several open-end cruciform or Y structures previously placed at the ends of mismatched linear DNA (10), and shown to prevent dissociation of bacterial MutS proteins from mismatched oligomers, even in the presence of ATP. We ligated oligomer HP1 into gapped circular plasmids and purified the supercoiled products, which were, as expected, resistant to endonuclease HpaI, the site for which is interrupted by the hairpin. The hairpin-containing structure remained mostly (>90%) supercoiled for at least 12 min at 37 °C in the HeLa nuclear extracts under standard mismatch-correction conditions, indicating that the hairpin was not cleaved or nicked.

We considered it essential to verify the structure and purity of the hairpin- and mismatch-containing structures, which had been prepared by a technically demanding multistep process. To that end, we cleaved the substrates with endonuclease AflIII at position 511, 20 bp right of the right-hand hairpin site, and with endonuclease BglII at position 22, 153 bp left of the left-hand hairpin site, generating the same 1.7-kbp ABL fragment from all substrates, plus 0.48-kbp ABS fragments containing the respective mismatch and hairpin features (Fig. 3, left panel). We then 32P-labeled the 3'-recessed AflIII ends and separated the fragments by electrophoresis in native 5% acrylamide gels (1:37.5 bisacrylamide:acrylamide), as shown in Fig. 3 (right panel). The ABS-HM and ABS-A fragments from the no-hairpin homoduplex and G/T substrates migrated the fastest (lanes HMA and A). The hairpin near the right-hand end of fragment ABS-D retarded it slightly (lane 5), whereas the more interior left-hand hairpin retarded fragment ABS-C more dramatically (lane 4); fragment ABS-B, containing both hairpins, was retarded the most (lane 3). No detectable amounts of other structures were identified, and the sharpness of the ABS-B, ABS-C, and ABS-D bands suggests that the hairpins are stable, i.e. do not migrate as a mixture of partially unwound molecules or other structures, even though one hairpin is only 20 bp from the open end.

Correction of Mismatches in Hairpin-containing Substrates—In all substrates, mismatch correction via removal of the continuous strand (restoration of HindIII sites) was negligible (Fig. 4, lanes 1, 3, 5, and 7). This confirms the nick requirement for initiation of MMR excision and the absence of adventitious nicks. We previously observed a plateau in mismatch correction after 10 min at 37 °C when the mispair-nick separation was 0.15 kbp (15). Here we measured correction yields (restoration of XhoI sites) after 12 min (Fig. 4, lanes 2, 4, 6, and 8). The 65% yield for the non-hairpin substrate A (Fig. 4, lane 2) remained constant up to 30 min (data not shown). The yield for the longer-path hairpin substrate D was not significantly lower (Fig. 4, lane 8). However, the shorter-path hairpin reduced the yield for substrate C to only 18% (Fig. 4, lane 6), and both hairpins virtually abolished repair of substrate B. Interestingly, extension of the incubation to 25 min increased the yield for substrate C to 27%; further incubation up to 40 min had no effect (data not shown).



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FIG. 4.
Correction of mismatches in hairpin-containing substrates. Incubation of 200 ng of substrates A, B, C, and D (see Fig. 2 and its legend) with 200 µg of HeLa nuclear extract for 12 min, digestion of half of recovered products each with endonuclease AseI plus endonuclease HindIII (lanes 1, 3, 5, and 7) or endonuclease XhoI (lanes 2, 4, 6, and 8), respectively, as indicated by (+), and electrophoretic analysis were as described under "Experimental Procedures." Fractions (%) of input substrate repaired (cleaved by indicated endonucleases) were calculated by quantitative imaging of bands. Data correspond to one representative experiment, from four (lanes 1–4) or five (lanes 5–8) with closely similar results (ranges were from 59 to 65% (lane 2), 15 to 18% (lane 4), and 59 to 64% (lane 6)).

 

Differential Effects of Hairpins on Initiation and Progress of MMR Excision—The levels of excision gaps generated at various positions during 7 min incubation in nuclear extracts at 37 °C were measured by binding of specific short radiolabeled ssDNA oligonucleotide probes (Fig. 5) to the non-excised strand. We previously showed that the fractions of substrates excised in the absence of exogenous dNTPs were equal to the fractions corrected in complete reactions (15). (Here up to 60% of input substrates were corrected (Fig. 4).) Excision signals shown in Fig. 5 are relative to an internal standard: nonspecific 5'-3' excision of the homoduplex control substrate, measured at a point just beyond the pre-existing nick, in what would be the longer-path direction in a mismatched substrate (probe number 1). As observed previously (15), this nonspecific 5'-3' excision of the homoduplex substrate, although substantial near the nick, was very low at a point 0.8 kbp farther along the longer-path (probe number 2), i.e. it was not processive.

In heteroduplex substrates, 3'-5' excision along the shorter nick-mispair path efficiently generated gaps in the nicked strand just downstream (30–60 bp) of the nick (probe number 3), not only in substrates A and D, which were efficiently corrected (Fig. 4), but also in poorly repaired substrate C and even negligibly-repaired substrate B. Thus, despite shorterpath hairpins in the latter two substrates, 3'-5' shorter-path excision toward the mismatch was initiated efficiently in all four cases. At the position of the mismatch, however, excision of the nicked strand (probe number 5) was high for the well repaired substrates A and D, but very low for the poorly repaired substrate C, and near background levels for substrate B; excision signals for the continuous strand at the mismatch position were at background level for all substrates (Fig. 5, probe number 4). Apparently, hairpins in the shorter nick-mispair path allowed initiation of excision, but not its progress. Interestingly, in mismatch substrates B and C, where hairpins blocked the shorter nick-mispair path, longer-path 5'-3' excision of the nicked strand measured at a site 0.8 kbp distal to the nick (probe number 1) was 5–6-fold higher than the homoduplex control value. Continuation of this mispair-provoked 5'-3' excision around the longer nick-mismatch contour in substrate C might account for the modest excision seen at the mismatch position (probe number 5) after 7 min, and for the significant but subnormal repair yields, 18% at 12 min (Fig. 4) and 27% at 25 min (not shown). Blockage of 5'-3' longer-path excision by the second hairpin in substrate B would account for the negligible excision (probe number 5) and correction (Fig. 4, lane 4) at the mismatch site.

The excision signals shown in Fig. 5 were all measured 7 min after initiation, roughly 1 min past the time at which excision typically reaches a plateau value in these extracts (13, 15), and thus most likely correspond to final yield values. We also assayed excision 2 min after initiation of reactions, in the linear initial phase of the time course (data not shown). For substrate D, whose shorter nick-mispair path was not blocked by a hairpin, the signal at 2 min for excision of nicked strand at the mispair position (probe number 5) was 34% of the signal measured just downstream of the nick (probe number 3). This corresponds to an excision progress rate of 4.7 nt/s, in good agreement with the 5.2 nt/s, measured previously (13). In contrast, signals at the mispair positions in substrate B and C were only 2% of the substrate D initial-excision signal (probe number 3), because of the excision progress blocking hairpins in their shorter nick-mispair paths. Notably, the initial-excision signals (probe number 3) for substrates B and C were 87 and 81% of that for substrate D, confirming that excision initiations at the nicks were at most slightly affected by hairpins between them and the mismatches. Thus, mismatch-provoked excision of the nicked strand, 3'-5' or 5'-3', appears to be blocked by hairpins in the continuous strand, suggesting that some excision components may track along this strand, but excision initiation is not affected. Comparison of probe number 5 versus number 4 signals (Fig. 4) for substrate A, after correction for background signals (substrate HMA), suggests that excision is over 100-fold specific for nicked versus continuous strands. Comparison of probe number 3 signals for substrates A versus HMA suggests that it is 80-fold specific for mismatched versus homoduplex DNA.

The substantial (non-processive) 5'-3' excision at the beginning of the longer nick-mispair path (probe number 2), seen for all mispair-containing substrates, is ascribed to enhanced initiation of 5'-3' excision at the gaps generated by concomitant mispair-provoked 3'-5' excision, as previously demonstrated (15).

Dissociation of hMutS{alpha} from Hairpin-bounded Mismatched DNA in the Presence of ATP—In the presence of ATP, both E. coli MutS and eukaryotic MutS{alpha} proteins rapidly dissociate from mismatch-containing linear DNA (8, 1820), but are retained when ends are blocked by streptavidin bound to biotinylated nucleotides (1921). Furthermore, terminal DNA-cruciform or hairpin-like open-end Y structures trap E. coli MutS protein on mismatched linear DNA in the presence of ATP (10). To determine whether or not putative ATP-dependent sliding of hMutS{alpha} might be similarly blocked by a hairpin structure, we analyzed binding of hMutS{alpha} to 0.48-kbp ABS fragments (cut out and purified from substrates with no, one, or two hairpins), in the absence or presence of ATP. In standard assays of electrophoretic mobility shift in the absence of ATP, hMutS{alpha} shifted almost all G/T-containing 0.48-kbp (ABS) fragments (cleaved from substrates A, B, C and D), as expected (Fig. 6, lanes 2–5); fractions of fragments shifted ranged from 78 to 89%. Very little of the 0.48-kb fragment from hairpin-free homoduplex substrate HMA was shifted (Fig. 6, lane 1). Complexes of hMutS{alpha} bound to mismatched (G/T) DNA containing zero, one, or two hairpin(s) migrated at nearly identical positions; the slight mobility differences closely corresponded to differences seen for the respective hMutS{alpha}-free DNA fragments (Fig. 3). Thus, the presence of hairpins did not dramatically decrease the mobility or yield of hMutS{alpha}-DNA complexes, or result in multiple shifted bands, as would have been the case if hMutS{alpha} bound to hairpins as well as to mispairs. To verify the absence of hMutS{alpha} binding to hairpins, we tested the ABS fragment derived from a homoduplex version of substrate C (HMC), which contains one hairpin. Binding was negligible, in the presence or absence of ATP (Fig. 6, lanes 11 and 12). Schofield et al. (10) similarly demonstrated lack of binding to cruciform structures by E. coli MutS protein. Surprisingly, binding to mismatch containing DNA fragments was severely reduced by the presence of ATP, to ~10% for all four G/T-containing fragments, notably including the two-hairpin ABS fragment (from substrate B) (Fig. 6, lanes 6–10). In contrast, E. coli MutS protein was previously reported to remain on 90-bp mismatch-containing oligomers, blocked at both ends by cruciforms or open-end Y structures in the presence of ATP (10). The binding conditions here were similar to those used previously, so the difference in results may reflect specific hMutS{alpha} and E. coli MutS properties.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We constructed circular DNA substrates, for measurement of mismatch repair in human nuclear extracts, in which hairpins (20-bp stem, 2-nt loop) were placed in the shorter-path between a G/T mispair and the pre-existing nick used to initiate MMR excision, or in the longer nick-mispair path around the substrate contour, or in both paths. Correction of the G/T mismatch was not significantly inhibited by the longer-path hairpin, but was substantially inhibited by the shorter-path hairpin and eliminated by the two hairpins together. However, efficiencies of initiation at the nick of 3'-5' MMR excision along the shorter-path (measured in the absence of exogenous dNTPs) were only slightly different among the four substrates. In the two shorter-path hairpin substrates in contrast, excision at the mismatch site beyond the hairpin was drastically reduced, consistent with the observed loss of mismatch correction. MMR excision measured at a point well around the longer-path from the nick was generally low but significant. Some longer-path excision even extended to the mismatch position, when measured in a substrate in which a hairpin blocked the shorter-path but not the longer-path, consistent with the low but appreciable mismatch correction in this case.

Inhibition of mismatch correction by a hairpin between the nick and the mispair, strong when the shorter-path was blocked and complete when both paths were blocked, indicates that progression of some critical MMR complex(es) along the DNA contour is (are) blocked. Excision at the mismatch position, although almost as high (81%) as that just downstream of the nick in the no-hairpin substrate, was reduced 14-fold by a shorter-path hairpin in the continuous strand, and a further 4-fold by an additional longer-path hairpin in the continuous strand. Thus, 3'-5' excision of the nicked strand may require concomitant tracking of one or more proteins along (5'-3') the continuous strand. If the proofreading exonuclease(s) of DNA polymerases {delta} and/or {epsilon} perform 3'-5' excision, as inhibition of this process by aphidicolin suggests (13), the polymerase(s) themselves and/or attached cofactors might be blocked by a continuous strand hairpin. Exonuclease I, which digests the 5'-3' strand in dsDNA (2224) is required for both 3'-5' and 5'-3' MMR excision and error correction (11, 12). A hairpin in the 5'-3' strand might block exonuclease I in either case, or interfere with tracking along the non-excised strand by a functional homolog of E. coli UvrD helicase, or of MutL{alpha}, which binds to ssDNA (14). These alternatives are not mutually exclusive.

Might hairpins also block mismatch-activated recognition complexes, including other proteins as well as hMutS{alpha}, that slide along DNA while searching for excision-initiation signals? At least two classes of MMR sliding-complex models have been proposed. In one class, ATP (perhaps its hydrolysis) is postulated to trigger sliding of MutS/MSH dimers away from mismatches along DNA contours (7, 8). In other models, mismatch containing DNA·MutS{alpha}·MutL{alpha} ternary complexes, relatively stable in the presence of ATP, which is in fact essential for their formation, without ATP hydrolysis being required (25), are thought to form, and then be released from the mismatch by proliferating cell nuclear antigen (PCNA). This release, which is more efficient when MgCl2 is present, might in these models generate a sliding PCNA·MutS{alpha}·MutL{alpha} complex, or both sliding MutS{alpha} and sliding PCNA·MutL{alpha} complexes, or these complexes further associated with other factors needed for MMR excision (25). In these cases, PCNA might play two roles even before DNA resynthesis: (i) tethering MutS{alpha} to homoduplex DNA via the known MutS{alpha}·PCNA interactions, but releasing it when a mismatch is encountered, thus enhancing the efficiency of mismatch recognition, as suggested by other work (26), and (ii) allowing more elaborate recognition complexes, perhaps involving as well MutL{alpha}, excision factors, and PCNA itself, to slide toward excision-initiation sites (25). We find here that purified hMutS{alpha} alone, apparently in contrast to bacterial MutS protein (9), is not retained on mismatched DNA blocked at both ends with hairpins in the presence of ATP. Thus, our experiments do not falsify any hypothesis in which ATP-dependent sliding of MutS{alpha} alone, either directly from a mismatch or after a cycle of MutL{alpha} recruitment and release by PCNA, searches for the excision-initiation signal, but do demand that the mode of any such sliding be loose enough to permit movement past the hairpin. Sliding of any more elaborate complex past a stiff hairpin extending about 70 Å from a 20-Å diameter helix would seem unlikely, virtually impossible if the complex included PCNA, whose central hole is 34 Å in diameter (27). Our data clearly do not falsify the hypothesis of Hsieh co-workers (9, 10), that mismatch-activated searching complexes, containing hMutS{alpha}, MutL{alpha}, and perhaps other proteins, might remain at or near the mismatch and search for the nick through space.

Our measurements of longer-path (5'-3') excision gaps at a point 0.8 kbp around the contour from the nick, presumably well beyond the range of nonspecific 5'-3' excision, provide a basis for estimation of shorter- versus longer-path preference in mispair-provoked excision. In mismatched-substrate A (no hairpins), the excision signal 0.8 kbp 5' to the nick was only 9 units, after correction for the nonspecific signal seen at this point in the homoduplex control substrate (HMA). The similarly corrected excision value 0.3 kbp 3' to the nick (at the mismatch position), was 248 units, of which 9 units or less could have been contributed by longer-path excision. Thus, shorter-path excision appeared 27-fold higher than longerpath; this may be a slight overestimate, if mispair-specific 5'-3' excision of 800 nt or so was not quite complete even after 7 min, or if some 5'-3' excision complexes had dissociated before reaching the 0.8-kbp point. In any event, this path specificity estimate is somewhat higher than the 10–20-fold estimated previously (15). Interestingly, the signal corresponding to low level mismatch-provoked longer-path (5'-3') excision at the 0.8-kbp points was increased to 23–30 units when a hairpin blocked the shorter-path, as if there were competition between excision in the two directions, and the hairpin made shorterpath excision slightly less competitive. This signal can easily account for the (corrected) excision signal of 15 units at the mismatch site in substrate C. The shorter- versus longer-path preference might be explained in several ways. Sliding MutS{alpha} might traverse 0.3 kbp much more rapidly than 1.9 kbp, with its orientation upon arrival at the nick dictating excision back along the path just traveled. On the other hand, if recognition complexes remaining at or near the mismatch directly contacted the excision-initiation via DNA looping, differences in properties of the longer versus shorter DNA contours between the two sites, intrinsic to the respective DNA segments or caused by proteins associated with them, might favor shorter-path excision.

In their study demonstrating (a) role(s) for human exonuclease I in both 3' and 5' mismatch repair, Genschel et al. (11) analyzed both 5'-3' and 3'-5' shorter-path excision by measuring loss of cutting by endonuclease NheII at a site closely adjacent to a G/T mispair, and longer-path excision by electrophoretically estimating the length of indirectly end-labeled strands, co-linear with the nicked strands of the substrate, that extended from a uniform end at an interior restriction site to a variable (gap-terminated) end for as much as 3.3 kbp (to the original nick site); shorter-path excision gaps were mapped as well. There are qualitative parallels between their observations and those reported here. However, the differences in specificities of excision signals for heteroduplex versus homoduplex DNA, typically 5–10-fold and occasionally higher for Genschel et al. (11) but 75–80-fold here (Fig. 5), and the reported relatively low ratios between G/T-provoked excision yields in MMR-proficient versus hMuts{alpha}-deficient extracts, typically 3–4-fold, suggest the presence of significant background activities, making quantitative comparisons difficult. Although their gap-mapping assay is not sensitive enough to measure the substantial 5'-3' longer-path excision of homoduplex substrates reported here and earlier, both studies show that this nonspecific excision is negligible a few hundred nucleotides farther along the longer (5'-3') path, i.e. is non-processive. In the case of G/T substrates, both groups see substantial longer-path excision, high just downstream of the nick, but low 0.8 kbp farther along in our case, detectable roughly 0.1 kbp downstream in their case.

Because we previously found that in nuclear extracts 5'-3' excision of a plasmid substrate was initiated at a pre-existing 30-nt gap roughly 2.5 times as efficiently as at a nick, we suggested that a gap generated by 3'-5' shorter-path excision toward a mismatch might concomitantly stimulate longer-path 5'-3' excision (15). Our observations here that a small but significant fraction of longer-path excision extends all the way around the longer-path, most likely accounting for the low but appreciable mismatch correction when the shorter-path (only) is blocked by a hairpin, suggest that some longer-path excision is in fact specifically mismatch-provoked. The relationship between this mismatch-provoked excision in nuclear extracts and the low but highly extended 5'-3' longer-path mismatch-dependent excision seen by Genschel et al. (11) using only purified human MutS{alpha} and exonuclease I is not clear. In 5' heteroduplex substrates both groups observed very little 3'-5' longer-path excision (11, 15). Whether any longer-path 3'-5' excision might extend far enough to accomplish significant mismatch correction when the shorter- (5'-3') path is blocked remains to be determined.

The experiments described here thus implicate a translocating complex in MMR excision, but do not rule out some searching through space or sliding/translocating models for mismatch-nick coupling. This work points toward further experiments, more stringent blocks, testing for putative multiprotein complexes that might be trapped, for example, and illustrates new techniques for constructing and testing complex mismatch-repair substrates.


    FOOTNOTES
 
* This work was supported by National Institutes of Health Grant ES09848 (to J. B. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Tel.: 541-737-1777; Fax: 541-737-0497; E-mail: haysj{at}bcc.orst.edu.

1 The abbreviations used are: MMR, mismatch repair; hMuts{alpha}, human MSH2·MSH6 protein heterodimers; hMutL{alpha}, human MLH1·PMS2 protein heterodimer; dsDNA, double-strand DNA; ssDNA, single-strand DNA; nt, nucleotides; kbp, one thousand DNA base pairs; PCNA, proliferating cell nuclear antigen. Back


    ACKNOWLEDGMENTS
 
We thank the National Cell Culture Center for HeLa cells and Stephen Hewitt for expert preparation of figures.



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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