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Analysis of Strand Slippage in DNA Polymerase Expansions of CAG/CTG Triplet Repeats Associated with Neurodegenerative Disease*

  • John Petruska
    Affiliations
    Department of Biological Sciences, Hedco Molecular Biology Laboratories, University of Southern California, Los Angeles, California 90089-1340
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  • Michael J. Hartenstine
    Affiliations
    Department of Biological Sciences, Hedco Molecular Biology Laboratories, University of Southern California, Los Angeles, California 90089-1340
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  • Myron F. Goodman
    Affiliations
    Department of Biological Sciences, Hedco Molecular Biology Laboratories, University of Southern California, Los Angeles, California 90089-1340
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  • Author Footnotes
    * This work was supported by National Institutes of Health Grants AG 11398 and GM 21422.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
      Lengthy expansions of trinucleotide repeats are found in DNA of patients suffering severe neurodegenerative age-related diseases. Using a synthetic self-priming DNA, containing CAG and CTG repeats implicated in Huntington's disease and several other neurological disorders, we measure the equilibrium distribution of hairpin folding and generate triplet repeat expansions by polymerase-catalyzed extensions of the hairpin folds. Expansions occur by slippage in steps of two CAG triplets when the self-priming sequence (CTG)16(CAG)4 is extended with proofreading-defective Klenow fragment (KF exo) fromEscherichia coli DNA polymerase I. Slippage by two triplets is 20 times more frequent than by one triplet, in accordance with our finding that hairpin loops with even numbers of triplets are 1–2 kcal/mol more stable than their odd-numbered counterparts. By measuring triplet repeat expansions as they evolve over time, individual rate constants for expansion from 4 to 18 CAG repeats are obtained. An empirical expression is derived from the data, enabling the prediction of slippage rates from the ratio of hairpin CTG/CTG interactions to CAG/CTG interactions. Slippage is initiated internally in the hairpin folds in preference to melting inward from the 3′ terminus. The same triplet expansions are obtained using proofreading-proficient KF exo+, provided 10–100-fold higher dNTP concentrations are present to counteract the effect of 3′-exonucleolytic proofreading.
      The human genome has an abundance of simple sequence repetitions that are unstable and tend to expand in large numbers in some genetic loci. A prime example is the CAG/CTG class of triplet repeats whose large expansions occur in genes associated with Huntington's disease and six other neurological disorders (
      • Ashley Jr., C.T.
      • Warren S.T.
      ). Such expansions represent a novel form of mutation whose cause is unknown. An attractive possibility under investigation is primer/template slippage during DNA replication or repair of tandemly repeated sequences (
      • Sinden R.R.
      • Wells R.D.
      ,
      • Wells R.D.
      ,
      • Richards R.I.
      • Sutherland G.R.
      ,
      • Schlotterer C.
      • Tautz D.
      ,
      • Behn-Krappa A.
      • Doerfler W.
      ,
      • Ji J.
      • Clegg N.J.
      • Peterson K.R.
      • Jackson A.L.
      • Laird C.D.
      • Loeb L.A.
      ,
      • Ohshima K.
      • Wells R.D.
      ).
      Strand slippage can occur when there is local strand separation in DNA regions containing tandem repetitions (
      • Levinson G.
      • Gutman G.A.
      ). For example, in a region of CAG/CTG repeats, local separation creates single-strand loops of CAGs and CTGs whose repetitive character may allow the two strands to be displaced (i.e. slipped) by an integral number of triplets. Such slippage in the presence of DNA polymerases can result in the addition of integral numbers of triplets to give triplet repeat expansions (
      • Sinden R.R.
      • Wells R.D.
      ,
      • Wells R.D.
      ,
      • Richards R.I.
      • Sutherland G.R.
      ,
      • Schlotterer C.
      • Tautz D.
      ,
      • Behn-Krappa A.
      • Doerfler W.
      ,
      • Ji J.
      • Clegg N.J.
      • Peterson K.R.
      • Jackson A.L.
      • Laird C.D.
      • Loeb L.A.
      ,
      • Ohshima K.
      • Wells R.D.
      ). Large loops that form stable hairpin structures are expected to increase the probability of slippage and expansion (
      • Ohshima K.
      • Wells R.D.
      ,
      • Darlow J.M.
      • Leach D.R.F.
      ,
      • Kang S.
      • Ohshima K.
      • Shimizu M.
      • Amirhaeri S.
      • Wells R.D.
      ,
      • Gacy A.M.
      • Goellner G.
      • Juranic N.
      • Macura S.
      • McMurray C.T.
      ,
      • Mitas M.
      • Yi A.
      • Dill J.
      • Kamp T.J.
      • Chambers E.J.
      • Haworth I.S.
      ,
      • Petruska J.
      • Arnheim N.
      • Goodman M.F.
      ).
      We (
      • Petruska J.
      • Arnheim N.
      • Goodman M.F.
      ) and others (
      • Darlow J.M.
      • Leach D.R.F.
      ,
      • Gacy A.M.
      • Goellner G.
      • Juranic N.
      • Macura S.
      • McMurray C.T.
      ,
      • Mitas M.
      • Yi A.
      • Dill J.
      • Kamp T.J.
      • Chambers E.J.
      • Haworth I.S.
      ,
      • Smith G.K.
      • Jie J.
      • Fox G.E.
      • Gao X.
      ) have shown that single strands of CTG repeats form stable hairpin structures as a result of base pairing between antiparallel CTGs, yielding G·C and C·G base pairs alternating with T·T mispairs. Similar hairpins also form in strands of CAG repeats but are not as stable (
      • Petruska J.
      • Arnheim N.
      • Goodman M.F.
      ,
      • Smith G.K.
      • Jie J.
      • Fox G.E.
      • Gao X.
      ,
      • Yu A.
      • Dill J.
      • Mitas M.
      ), probably because A·A mispairs are bulkier and more destabilizing than T·T (
      • Aboul-ela F.
      • Koh D.
      • Tinoco I.J.
      • Martin F.H.
      ). Our thermal denaturation studies of strand folding (
      • Petruska J.
      • Arnheim N.
      • Goodman M.F.
      ) indicated that hairpin stability increases more slowly than expected with increasing numbers of repeats. A plausible explanation is that strands with more repeating triplets have more degrees of freedom and tend to form several shorter hairpin loops instead of one long one. For example, a strand with 10 triplet repeats may fold into a single hairpin structure, whereas a longer strand with 30 repeats may form a more complex structure with two or three hairpin folds of similar size (
      • Petruska J.
      • Arnheim N.
      • Goodman M.F.
      ).
      Evidence of strand slippage in CAG/CTG repeat regions during DNA polymerization has been obtained using various gel-based primer extension assays (
      • Schlotterer C.
      • Tautz D.
      ,
      • Behn-Krappa A.
      • Doerfler W.
      ,
      • Ji J.
      • Clegg N.J.
      • Peterson K.R.
      • Jackson A.L.
      • Laird C.D.
      • Loeb L.A.
      ,
      • Ohshima K.
      • Wells R.D.
      ). Our assay differs from earlier ones mainly in the priming event. We engineered a DNA oligonucleotide of triplet repeats so that it is primed by intramolecular folding, rather than by intermolecular association. Being independent of concentration, the self-priming remains efficient at low DNA concentrations, unlike intermolecular priming that requires higher DNA concentrations. By using low concentrations, we avoid uncertainties arising from multimeric associations between DNA strands. Also, our starting primer 3′-end is fully complementary to template repeats, instead of partially complementary as in the only previous study of self-priming with triplet repeats (
      • Behn-Krappa A.
      • Doerfler W.
      ).
      In the present study we use a self-priming strand containing a large number of CTGs relative to CAGs, to measure the influence of CTG/CTG interactions on strand slippage in polymerase-catalyzed expansions of correctly paired (CAG/CTG) repetitions. This approach provides for the first time an opportunity to measure slippage and expansion rates as a function of the number of interacting triplets, for DNA polymerases with and without proofreading 3′-exonuclease activity.

      EXPERIMENTAL PROCEDURES

       Materials

       DNA Polymerases

      Escherichia coli DNA polymerase I Klenow fragment (KF)
      The abbreviations used are: KF, Klenow fragment;A, self-priming DNA sequence, 5′-(CTG)16(CAG)4-3′; An, initial hairpin fold of sequence A, having n CTG triplets at the 5′-end available as template for extending the primer 3′-end by n CAG triplets; An + (CAG)n, blunt-end hairpin formed by extendingAn by n CAG triplets with DNA polymerase.
      mutant, exo (D355A,E357A), devoid of proofreading 3′-exonuclease activity, was purified from overproducing strains (
      • Derbyshire V.
      • Freemont P.S.
      • Sanderson M.R.
      • Beese L.
      • Friedman J.M.
      • Joyce C.M.
      • Steitz T.A.
      ). Normal proofreading-proficient KF polymerase (exo+) was obtained commercially (U. S. Biochemical Corp./Amersham Corp.).

       Oligonucleotide Synthesis

      The self-priming DNA 60-mer, (CTG)16(CAG)4, and 30-mer (CTG)6(CAG)4 were synthesized by an Applied Biosystems DNA/RNA synthesizer, using β-cyanoethyl phosphoramidites, and purified by denaturing polyacrylamide gel electrophoresis. The 90-mer DNA marker (CTG)16(CAG)14 was purchased from Operon Technologies Inc. and obtained lyophilized after high pressure liquid chromatography purification. Purified DNA samples were dialyzed extensively against a low ionic strength buffer (5 mm NaH2PO4, 5 mmNa2HPO4, 1 mm Na4EDTA, pH 7.0) and stored at −70 °C.

       Methods

       Melting Analysis

      Thermal denaturation profiles were obtained for the 60- and 30-mer DNAs at the same strand concentration (1.9 μm) in low ionic strength buffer, by measuring UV absorbance A260 versus temperature, while raising temperature from 25 to 85 °C at a constant rate of 2 °C/min.

       End Labeling and Equilibration

      Samples of the self-priming 60-mer used in polymerase reactions were 5′-end-labeled with32P, using [γ-32P]ATP and T4 polynucleotide kinase (U. S. Biochemical Corp./Amersham Corp.) in kinase buffer (50 mm Tris-HCl, pH 7.6, 10 mmMgCl2, and 10 mm 2-mercaptoethanol). Labeled samples at a strand concentration of 100 nm were heat-denatured at 100 °C for 5 min and allowed to renature by cooling at room temperature. The results obtained in polymerase reactions showed no dependence on the rate of cooling, as expected for intramolecular folding. The labeled strands were stored at 4 °C to avoid any potential intermolecular associations promoted by freezing (
      • Petruska J.
      • Arnheim N.
      • Goodman M.F.
      ).

       Extension Reactions

      Radiolabeled DNA at 10 nmstrand concentration was incubated 5 min at 37 °C in reaction buffer (50 mm Tris-HCl, pH 7.5, 10 mmMgCl2, 1 mm dithiothreitol, and 0.05 mg/ml bovine serum albumin) to allow equilibration of DNA structure. Typically, 60 μl of DNA solution was then micropipeted into 15 μl of enzyme + dNTP solution (same buffer) held in a microcentrifuge tube at 37 °C, at which point running time (t) for reaction was started. Final enzyme concentration was approximately 60 nm in each case; the dNTPs used (N = C, A, G) were in equimolar amounts ranging from 0.1 to 10 μm. At times indicated (t = 0.5 min, etc.) a 5-μl aliquot of reaction mixture was removed and quenched with 25 μl of 20 mm EDTA + 20 m formamide.

       Denaturing Gel Electrophoresis

      Reaction products were separated into bands according to product size, by denaturing gel electrophoresis at 2000 V on 12% polyacrylamide (40 cm × 40 cm × 0.2 mm) with 16 m formamide as denaturant, in TBE buffer (89 mm Tris borate, pH 8.3, 2 mmNa2EDTA). Gels dried on paper were scanned with Molecular Dynamics Storm 860 PhosphorImager, and band intensities were integrated by FragmeNT Analysis software. Each band intensity was evaluated as percent of total integrated band intensities in the corresponding lane.

       Expansion Rate Analysis

      The 60-mer sequence (A) forms hairpin loops (An) with even and odd numbers of bases in the hairpin bend (Fig. 1). In the presence of DNA polymerase and dNTPs, the loops are rapidly extended (in seconds) to blunt-ended forms, An + (CAG)n, seen as gel band intensities In, where n is the number of CAG triplets added (Fig. 3). The band intensities change gradually (in minutes) because of slippage and further expansion. Since bands with n = 0, 2, 4, etc. are much more intense than those with n = 1, 3, 5, etc., even-numbered loops are evidently more stable then odd-numbered, as suggested earlier (
      • Darlow J.M.
      • Leach D.R.F.
      ). Thus slippage by two triplets and expansion by (CAG)2 is favored over slippage by one triplet and expansion by (CAG)1. To verify that added triplets have the sequence CAG, chain termination with dideoxy-NTPs is used. In separate reactions, a single dideoxy-NTP is added, at 100 × the corresponding dNTP concentration, to obtain termination atN = C, A, or G. In this way (data not shown) we confirm the positions of bands corresponding to addition of (CAG)n for both even and odd values of n.
      Figure thumbnail gr1
      Figure 1Possible hairpin folds of self-priming 60-mer DNA sequence (CTG)16(CAG)4. The even-numbered loops (A0,A2, etc.) and less stable odd-numbered loops (A1, A3, etc.) are extendable by even and odd numbers of CAG triplets, respectively. the solid dots indicate correct base pairs; the asterisk indicates the 5′-end radiolabeled with32P. The most stable loop is blunt-endedA0, having the most correct base pairs, namely 23. The loops indicated as much less stable have less than 4 CAGs correctly paired with complementary antiparallel CTGs. Upon reaction with DNA polymerase and dNTPs (N = C, A, G), the initial loops An are rapidly extended (in seconds) to blunt-end products, An + (CAG)n, seen as band intensities In (n = 0, 1, 2, etc.) by denaturing gel electrophoresis. With increasing reaction time, the changes in In indicate the rates of strand slippage in the blunt-ended hairpins and further expansion by addition of more CAG triplets.
      Figure thumbnail gr3
      Figure 3Band patterns on denaturing gel obtained by extending self-priming 60-mer with DNA polymerase KF exo. Extreme left and rightlanes contain the 90-mer DNA marker (M),32P(CTG)16(CAG)14. The second lane from the left shows the 60-mer32P(CTG)16(CAG)4 without polymerase. The other lanes show the 60-mer extended by the addition of CAG triplets with DNA polymerase + 0.2 μmdNTPs (N = C, A,G) at 37 °C, removing samples and quenching with EDTA at reaction times indicated.
      Assuming band intensities change successively by 2-triplet slippage and expansion, so that In converts toIn+2 with rate constantkn, we evaluated rate constantsk0 for I0I2, k2 for I2I4, etc. (Table I). The evaluation is made by applying the differential equations describing the corresponding rates of intensity change with timet, namely dI0/dt = −k0 I0, dI2/dt =k0 I0k2 I2, … , dIn/dt =kn−2 In−2kn In. SinceI0 decays exponentially with t, the k0 value is obtained directly from the slope of the straight line fitted by least squares, logI0 = log I0(t→ 0) − k0 t, whereI0(t → 0) is the initialI0 found by extrapolating to t = 0. Then, using k0 and the value of I0 at each time point, we obtaink2 by a least squares fit to the rates of change of I2. Thus, after determiningk0 directly, we are able to determinek2 relative to k0,k4 relative to k2, etc.
      Table IInitial amounts of hairpin folds in sequence A = (CTG)16(CAG)4 and corresponding slippage rate constants for 2-triplet expansions
      Hairpin foldBand intensityInitial amountSlippage expansionRate constant
      %min−1
      Even-numbered loops
      A0I0 = [A0]61I0I20.81 ± 0.02
      A2I2 = [A2 + (CAG)2]7I2I40.48 ± 0.02
      A4I4 = [A4 + (CAG)4]5I4I60.40 ± 0.03
      A6I6 = [A6 + (CAG)6]6I6I80.34 ± 0.02
      A8I8 = [A8 + (CAG)8]6I8I100.28 ± 0.02
      A10I10 = [A10 + (CAG)10]7I10I120.08 ± 0.03
      A12I12 = [A12 + (CAG)12]1.5I12I140.06 ± 0.03
      A14I14 = [A14 + (CAG)14]0.5I14I16ND
      Odd-numbered loops
      A1I1 = [A1 + (CAG)1]2I1I3(0.7 ± 0.1)
      Slippage rate constant k1 for I1 → I3, found along withk0′ = 0.04 ± 0.02 min−1 for I0 → I1 (slippage ofA0 by 1 triplet), by analyzing the changes in I1 versus t in accordance with dI1/dt =k0′I0 −k1I1.
      A3I3 = [A3 + (CAG)3]1I3I5ND
      A5I5 = [A5 + (CAG)5]0.5I5I7ND
      1-a Slippage rate constant k1 for I1I3, found along withk0′ = 0.04 ± 0.02 min−1 for I0I1 (slippage ofA0 by 1 triplet), by analyzing the changes in I1 versus t in accordance with dI1/dt =k0I0k1I1.
      For comparison, we also evaluated a rate constant (k0′) for slippage by 1 triplet (I0I1), converting the even-numbered loop A0 to the odd-numbered loop A1 (Fig. 1). The assumption is thatI0I1 occurs with rate constant k0′, whereasI1I3 occurs with rate constant k1 for 2-triplet slippage. Then, applying the corresponding differential equation, dI1/dt =k0I0k1 I1, we find thatk0′ (for 1-triplet slippage) is an order of magnitude less than k0 (for 2-triplet slippage), whereas k1 for 2-triplet slippage of an odd-numbered loop is consistent with k0 and k2 for 2-triplet slippage of even-numbered loops.

      RESULTS

      The self-priming sequence, A = (CTG)16(CAG)4, forms a series of hairpin loops (Fig. 1), to which integral numbers of CAG triplets can be added by polymerase-catalyzed reaction with appropriate dNTP substrates (N = C, A, G). In Fig. 1,A0 represents the most stable loop in the form of a blunt-ended hairpin; A1 corresponds to slippage of A0 by a single triplet;A2, slippage by two triplets; etc. Initially, there is an equilibrium distribution of loops reflecting their thermodynamic stability in the absence of polymerase. We verify that stable folds are formed by examining thermal denaturation profiles obtained by plotting UV absorbance (A260)versus temperature (Fig. 2).
      Figure thumbnail gr2
      Figure 2Comparison of thermal melting profiles for DNA 60-mer (CTG)16(CAG)4 and 30-mer (CTG)6(CAG)4. The heat denaturation curves and indicated melting temperatures in low ionic strength buffer, found by plotting UV absorbance A260 versus temperature, are shown at the same (1.9 μm) strand concentration.
      When DNA polymerase and dNTPs are added, the original loops are extended to blunt ends in a matter of seconds, so that the rates at which blunt-end hairpins rearrange by slippage and expand by triplet additions can be accurately measured over a period of minutes. To establish the equilibrium amounts of individual hairpins and their extension rates, we analyzed polymerase-catalyzed reaction products separated into discrete bands by electrophoresis on a denaturing, formamide-polyacrylamide gel. The products are first analyzed as a function of reaction time (t) at physiologically low (0.1 to 1 μm) dNTP concentration, using a proofreading-deficient DNA polymerase, Klenow fragment exo. The results shown (Fig. 3) are obtained with 0.2 μm each of the three dNTP substrates (N = C, A, G). This concentration appears high enough to obtain near-maximum velocity of extension with a minimum of artifacts such as terminal transferase activity, seen at higher [dNTP] as an increase in background “pause” bands (data not shown).

       Thermal Denaturation Profiles

      Upon heat denaturation (Fig. 2), the 60-mer (CTG)16(CAG)4 shows two sigmoidal transitions, with melting temperature Tm = 56 and 80 °C at low ionic strength. By comparison, the 30-mer (CTG)6(CAG)4, having 10 fewer CTG repeats, shows only the higher transition with Tm near 80 °C. The biphasic melting curve of the 60-mer is consistent with the kind of hairpin folding shown in Fig. 1, in which the 4 CAG triplets are correctly base-paired with 4 CTG repeats to give the more stable component melting at 80 °C, whereas the remaining 12 CTG triplets are more weakly paired to give the less stable component melting at 56 °C. The latter Tm value is close to that observed for hairpin folding of CTG triplets alone,e.g. (CTG)10 and (CTG)30, each withTm = 51 °C (
      • Petruska J.
      • Arnheim N.
      • Goodman M.F.
      ). However, the melting curve is unable to resolve the kinds of hairpin folds present. The various possible folds (Fig. 1) initially have the same strand length, but when extended with polymerase they acquire increasing lengths, which can be separated by electrophoresis on formamide-polyacrylamide gel.

       Initial Hairpin Loops

      As seen in Fig. 1, starting at the 5′-end marked by *, the 60-mer sequence A has a potential template of 16 CTG repeats followed by a primer of 4 CAG repeats. Upon hairpin folding, so that the CAGs align with CTGs in antiparallel fashion, the (CAG)4 primer can be correctly base-paired with any 4 successive antiparallel CTGs, shown as (GTC)4 in Fig. 1. The first loop, A0, is a blunt-ended structure with 3′- and 5′-ends juxtaposed. This is the most stable fold because it allows remaining CTGs to form the maximum number of base pairs with each other.
      The even-numbered loops A2,A4, … , A10belong in the same group as A0, because they each have the same kind of 4-base hairpin bend and an even number of overhanging triplets. The odd-numbered loops A1,A3, … , A11belong in a separate group, because they each have a 3-base bend and an odd number of overhanging triplets. The even-numbered loops are expected to be somewhat more stable than the odd-numbered (
      • Darlow J.M.
      • Leach D.R.F.
      ), because they each have one more base pair stabilizing the hairpin bend. For completeness, much less stable loops, A12 toA15, having fewer than 4 correctly paired CAG/CTG combinations, are also shown (Fig. 1).
      Being blunt-ended, A0 is not suitable for extension by DNA polymerases until it rearranges by strand slippage into one of the other possible forms with an overhanging 5′-template, (GTC)n* (Fig. 1). These forms, An(n = 1, 2, etc.), are easily extended to blunt-end products, An + (CAG)n, by the addition of (CAG)n opposite (GTC)n*. The products are resolved as a series of bands on a denaturing gel, with band intensityIn proportional to the amount of product, (An + (CAG)n). The 5′-end (*) is radiolabeled with 32P to obtain quantitative band intensity measurements with a PhosphorImager.

       Loop Resolution by Electrophoretic Analysis of Polymerase-catalyzed Extension Products

      Using proofreading-deficient DNA polymerase KF exo, we observed complete extension of initial loops within 0.5 min of reaction (Fig. 3). A 6-fold excess of polymerase is used to saturate all the self-primed loops originally present so that they can be completely extended to blunt ends in seconds, before appreciable primer/template slippage can occur. Also, we use a dNTP concentration low enough (0.2 μm) to avoid the formation of anomalous pause bands arising by base misinsertion or by blunt end addition of a single nucleotide (
      • Clark J.M.
      ).
      In Fig. 3 it is clear that the most intense bands correspond to additions of even numbers of CAG triplets. The bands formed by adding 0, 2, 4, etc. triplets are much more intense than those formed by adding 1, 3, etc. triplets. This striking result shows that even-numbered loops (Fig. 1) are significantly more abundant,i.e. more stable, than odd-numbered, in agreement with earlier observations (
      • Darlow J.M.
      • Leach D.R.F.
      ). The way the band intensitiesIn change with t, the time of reaction, indicates the rates at which the blunt-end loops, An+ (CAG)n, rearrange by slippage and expand by the addition of more CAGs.

       Measurement of Expansion Rates for Primer/Template Slippage in Polymerase KF exo Reactions

      By examining the band patterns in Fig. 3, we find that as the original 60-mer band (0 triplets added) decays with t, each subsequent even-numbered band, representing addition of 2, 4, etc. CAG triplets, intensifies to a maximum before decaying. This is better illustrated in Fig. 4, where the band intensityIn, expressed as percent of total intensity in each lane, is shown plotted versus t for the first three even-numbered bands, n = 0, 2, and 4. Since odd-numbered bands (additions of 1, 3, etc. triplets) remain faint at all times, we see that slippage by two triplets is strongly favored over slippage by one triplet.
      Figure thumbnail gr4
      Figure 4Plot of band intensities versusreaction time for the first three main bands representing extensions by 0, 2, and 4 CAG triplets. Band intensity I0indicates the amount of the original blunt-ended loopA0 (Fig. ), and I2 and I4 indicate the amounts of the extension products, A2 + (CAG)2 andA4 + (CAG)4, respectively. Each band intensity is expressed as percent of total integrated band intensities in the corresponding lane (Fig. ), measured by PhosphorImager.
      To describe how each band intensity In changes because of slippage by 2 triplets and subsequent expansion by (CAG)2, we use the kinetic scheme in Fig. 5. The rate constantskn with n = 0, 2, 4, etc. represent “macroscopic” rates for converting each even-numbered blunt-end loop (In) to the next (In+2) by 2-triplet slippage and expansion. For n = 0, the intensityI0 decreases exponentially with t(Fig. 4), and the exponential curve fitted by least squares yields the slippage rate constant, k0 = 0.81 min−1. By extrapolating to t = 0, we findI0(t → 0) = 61% as the initial amount of hairpin A0 (Table I). For n = 2, band intensity I2 rises rapidly until it reaches a maximum and then gradually declines (Fig. 4). By assumingk0 is the rate constant for convertingI0 to I2 by 2-triplet slippage, we obtain a good fit with the relationship, dI2/dt =k0 I0k2 I2, enabling us to determine k2 relative tok0. Also, by extrapolating to t= 0 we obtain I2(t → 0), the initial amount of hairpin A2 (Table I). Similarly, using dIn/dt =kn−2 In−2kn In, we are able to evaluatekn relative to kn−2and by extrapolation to estimate the initial amounts of the even-numbered hairpins with n = 4–14, i.e. A4 to A14. In the case ofA14, the initial amount (0.5%) is barely above the minimum level of detection, and the rate constant for slippage is not determined (ND, Table I).
      Figure thumbnail gr5
      Figure 5Kinetic scheme used to evaluate rate constants for slippage and expansion by two triplets. The rate constants kn (n = 0, 2, 4, etc.) indicate the rate at which band intensity In is converted to In2 as a result of 2-triplet slippage and polymerase-catalyzed expansion by 2 CAG triplets.
      Because odd-numbered loops give much less intense bands, comparable to faint pause bands in the background, we can only evaluate initial amounts for A1, A3, andA5 (Table I). In the case ofA1, there is sufficient intensity to estimate a rate constant k1 for 2-triplet slippage (I1I3), assumingA0 contributes to A1(I0I1) with rate constant k0′ for 1-triplet slippage. In this case, dI1/dt =k0′I0k1 I1, when applied to our data, yields k0′ = 0.04 ± 0.02 min−1 and k1 = 0.7 ± 0.1 min−1 (Table I, in brackets).

       Relationship between Slippage Rate and Number of Interacting Triplets

      In Table I (last column) we see that the slippage rate constant kn decreases with increasing number,n, of added CAGs. The rate constant is highest (0.8 min−1) for n = 0, the initial blunt-end hairpin A0 (Fig. 1), which has the highest number of CTG/CTG interactions relative to CAG/CTG interactions. As Fig. 1 shows, A0 has 4 strong CAG/CTG base pairing interactions and 5 weaker CTG/CTG interactions, so that (CTG/CTG)/(CAG/CTG) = 5/4. The longer blunt-end hairpins,An + (CAG)n, formed by adding nCAG triplets complementary to the template (GTC)n* (Fig. 1), gain n more CAG/CTG interactions while losing n/2 CTG/CTG interactions. Thus, while (CAG/CTG) = 4 + nincreases with n, both (CTG/CTG) = 5 − (n/2) and the ratio, (CTG/CTG)/(CAG/CTG) = (10 −n)/(8 + 2n), decrease with n.
      The kn value (on a logarithmic scale) shows a positive correlation with (CTG/CTG) = 5 − n/2 and a negative correlation with (CAG/CTG) = 4 + n (Fig. 6 A). In each case, the correlation is nonlinear for even values of n from 0 to 8. However, when log kn is plotted against the ratio, (CTG/CTG)/(CAG/CTG) = (10 − n)/(8 + 2n), the points fall close to a straight line (Fig. 6 B). Furthermore, k1 obtained for 2-triplet slippage between odd-numbered loops (Table I, in brackets) also falls on this line, at the point corresponding to n = 1 (Fig. 6 B, open square).
      Figure thumbnail gr6
      Figure 6Semi-log plot of rate constant values as a function of interacting triplets. A, plot of logkn (n = 0, 2, … , 10)versus the corresponding number of triplet interactions, (CAG/CTG) = 4 + n (upper scale) or CTG/CTG = 5 − (n/2) (lower scale). B, plot of log kn versus the relative number of CTG/CTG interactions to CAG/CTG interactions, i.e.(CTG/CTG)/(CAG/CTG) = (10 − n)/(8 + 2n). The straight line is a least squares fit tok0, k2, … ,k10 (solid squares) found for 2-triplet slippage in even-numbered loops; the open squareis the lone (k1) value found for 2-triplet slippage in odd-numbered loops (Table , brackets).
      The equation of the line fitted by least squares (Fig. 6 B) is log10 kn = 0.38x − 0.56, where x = (CTG/CTG)/(CAG/CTG). This equation can also be written as kn = αe βx, where α = 0.28 min−1 and β = 0.87. We can interpret α as the slippage rate constant for CAG/CTG interactions alone and e βx as the factor arising from the influence of (CTG/CTG) on the slippage of (CAG/CTG). This factor appears to hold as long as slippage by 2 triplets does not change the character of the hairpin bend made by CTGs.
      The even-numbered loops A0 toA10 (Fig. 1) have the same kind of 4-base bend made with two CTGs. However, in A12 the bend acquires a CAG in place of a CTG at the expense of a CAG/CTG base-pairing interaction, making it more difficult for A10A12 slippage to occur. As seen in Table I (last column), the slippage rate constant drops dramatically, from 0.28 min−1 for k8 (I8I10) to 0.08 min−1 for k10 (I10I12). This marked reduction in slippage rate is also evident in the accumulation of band intensityI10, corresponding to 10 CAG triplets added (Fig. 3). Subsequently, the slippage rate declines further to 0.06 min−1 for k12(I12I14) and to an even lower value for k14, undetermined (ND) in Table I.

       Effect of Proofreading Activity on Expansion Rate

      When KF exo is replaced by normal proofreading-proficient Klenow fragment, the 3′-exonuclease activity of proofreading markedly inhibits expansion at low dNTP concentrations (Fig. 7). At 0.2 μm dTNP, band intensities similar to those found initially with KF exoare observed in the 1st min of reaction, but expansion by slippage is greatly reduced because of 3′-end degradation with increasing time (Fig. 7). As dNTP concentration is increased to counteract the degradation, the band patterns for expansion by slippage are recovered for longer times. At 1.0 μm dNTP, the same patterns of band intensities as in Fig. 3 are observed for the first 10 min, but some decreases in long expansions (notably I14and I12) are evident at longer times. At 10 μm, no significant decreases are evident for at least 60 min. Thus, to obtain the same degree of expansion in the presence of proofreading as in its absence, apparently 10 to 100 times as much dNTP is required.
      Figure thumbnail gr7
      Figure 7Band patterns obtained by extension with proofreading-proficient DNA polymerase KF (exo+) at three different dNTP concentrations. Under incubation conditions like those used for proofreading-deficient KF exo (Fig. ), at low (0.2 μm) dNTP concentration, the self-priming sequence 32P(CTG)16(CAG)4 is extended to blunt-ended states in less than 1 min as before, but expansion by slippage is markedly reduced because of strand degradation by proofreading 3′-exonuclease activity. To counteract the degradation, much higher dNTP concentrations are needed. A 5-fold increase (1.0 μm) restores the pattern of expansion by slippage seen in Fig. for the first 10 min; another 10-fold increase (10 μm) is needed to maintain the pattern for the full 60 min.

      DISCUSSION

      Single DNA strands containing tandemly repeated CTG or CAG triplets form hairpin structures which, although not as stable as normal DNA duplexes, are nonetheless remarkably stable (
      • Darlow J.M.
      • Leach D.R.F.
      ,
      • Kang S.
      • Ohshima K.
      • Shimizu M.
      • Amirhaeri S.
      • Wells R.D.
      ,
      • Gacy A.M.
      • Goellner G.
      • Juranic N.
      • Macura S.
      • McMurray C.T.
      ,
      • Mitas M.
      • Yi A.
      • Dill J.
      • Kamp T.J.
      • Chambers E.J.
      • Haworth I.S.
      ,
      • Petruska J.
      • Arnheim N.
      • Goodman M.F.
      ,
      • Smith G.K.
      • Jie J.
      • Fox G.E.
      • Gao X.
      ,
      • Mitas M.
      ). To investigate the influence that such secondary structures have on strand slippage in polymerase-catalyzed repeat expansions, we have constructed a strand sequence, (CTG)16(CAG)4, forming a series of self-primed hairpins (Fig. 1) to which repeats of CAG triplets can be added by DNA polymerases. This sequence provides an opportunity to measure the influence of hairpin CTG/CTG interactions on primer/template slippage in repeating CAG/CTG regions. By using a DNA polymerase devoid of exonuclease activity (KF exo), we find that initial hairpin loops (formed at equilibrium) are fully extended to blunt-ended forms in a matter of seconds, so that the rates at which blunt-end loops rearrange by slippage and add more repeats can be measured over a period of minutes to about 1 h (Fig. 3). With this construct, we observe triplet repeat expansions in a series of discrete slippage steps for which rate constants can be evaluated.
      Of the various hairpins formed initially (Fig. 1), those with an even number of bases in the bend appear most stable since they are found in the largest amounts (Table I). The series of even-numbered loops fromA0 to A10 account for over 90% of all folded structures. As expected, the most stable loop is the blunt-ended structure A0, whose initial amount is 61% of total structures at 37 °C. The next most stable loops are initially present in amounts of 5–7%, these being the five even-numbered loops, A2 toA10. Surprisingly, each of the latter is as abundant or more abundant than all the odd-numbered loops collectively. The abundance of even-numbered loops does not decrease fromA2 to A10 as might be expected but instead shows a minimum at A4(5%). Possibly the longer overhanging templates, shown simply as (GTC)n* in Fig. 1, form a second hairpin fold providing some additional stability.
      The initial amounts of loops formed at equilibrium, in the absence of polymerase, can be used to calculate relative loop stabilities in terms of standard free energy, ΔG o = −RTlnKeq. From the ratio of equilibrium amounts, Keq = 7/61, we findA2 is less stable than A0by RTln(61/7) = 1.3 kcal/mol at T = 37 °C or 310 K. Similarly, from Keq = 2/61,A1 is found to be 2.1 kcal/mol less stable thanA0 and thus 0.8 kcal/mol less stable thanA2. Finding A1 about 1 kcal/mol less stable than A2 is consistent with our observation (Table I, footnote) that slippage by 1 triplet, converting A0 to A1, is an order of magnitude slower than the rate for 2-triplet slippage converting A0 to A2.
      As shown in Fig. 6 B, the rate of slippage by 2 triplets may be simply related to the ratio of CTG/CTG interactions to CAG/CTG interactions involved in slippage. The logarithm of the slippage rate constant, when plotted against the ratio (CTG/CTG)/(CAG/CTG) in the blunt-ended hairpin undergoing slippage, shows a linear relationship with a positive slope. The slope indicates that slippage rate increases exponentially with the ratio. The implication is that hairpin CTG/CTG interactions promote slippage by 2 triplets and thereby provide a favorable way of propagating waves of expansion in CAG/CTG repeat regions.
      In the expansion of the blunt-ended loops in our system, there are several reasons to think that slippage begins internally with CTG/CTG interactions rather than at the primer 3′ terminus with CAG/CTG interactions. We observe (Fig. 2) that the CTG/CTG region has a melting temperature 24 °C lower than the CAG/CTG region, so that local melting needed to initiate slippage is much more likely to occur in the CTG/CTG region. To initiate slippage by 2 triplets, three CAG/CTG interactions need to be disrupted at the primer 3′-end, but only one next to the CTG/CTG region where two weaker CTG/CTG interactions are more easily disrupted. Slippage by 2 triplets is favored over slippage by 1 triplet, in accordance with even-numbered loops of CTGs being more stable than odd-numbered (Table I). The rate constant for 2-triplet slippage increases with CTG/CTG interactions but decreases with CAG/CTG interactions (Fig. 6 A), so that the rate constant increases exponentially with the ratio of CTG/CTG to CAG/CTG interactions, as seen by the linear plot of log(rate constant) versus this ratio (Fig. 6 B). Furthermore, in the presence of 3′-exonucleolytic proofreading (Fig. 7), as long as there are CTG/CTG interactions present (n = 0–8) and slippage rates are 0.28 min−1 or higher (Table I), less than 1.0 μm dNTP concentrations are needed to restore normal slippage patterns, whereas 10 μm concentrations are needed when CTG/CTG interactions are absent (n = 12 and 14) and slippage rates fall to 0.08 min−1 or lower (Table I).
      Since our system is single-stranded and relatively short, it is not certain how many of the points above may apply to long double-stranded DNA molecules in vivo. Nevertheless, with these points in mind, we can propose a simple model (Fig. 8) indicating how triplet repeat expansion may occur when there is local strand separation in a region of triplet repeats. Such separation could arise, for example, by negative supercoiling (
      • Gellibolian R.
      • Bacolla A.
      • Wells R.D.
      ) of the kind needed to activate transcription or replication. Once separated in a region of triplet repeats, the strands may fold on themselves to form hairpin loops that are initially opposite each other but because of repetition are able to migrate apart by strand slippage and remain separated for longer periods. While apart, if a nick (single-strand cleavage) occurs opposite one of the loops, that loop can stretch and become linear so that a DNA polymerase, perhaps a proofreading-deficient polymerase involved in DNA repair, may fill in the gap to create an expansion on one strand. After the gap is filled and sealed by ligase, a nick opposite the other loop can lead to expansion on the other strand. According to this model, single-strand loops that become separated and nicked in the presence of polymerase may behave like the simple hairpin system we have studied.
      Figure thumbnail gr8
      Figure 8Proposed model of strand slippage and repeat expansion in vivo suggested by results of current experiments. The repeating arrows symbolize sequence repeats such as triplets. After strand separation and possible loop migration, a nick (cleavage by endonuclease) on one strand allows the loop on the opposite side to be stretched so that the nicked strand can be extended by DNA polymerase (possibly proofreading-deficient polymerase β in eukaryotes) and sealed by DNA ligase.
      Our simple system enables one to evaluate the contributions that individual single-strand interactions make to strand slippage in duplex repeat regions. By using the sequence (CTG)16(CAG)4, we have made an evaluation of CTG/CTG interactions contributing to slippage and expansion of CAG/CTG repeats. Similarly, by using the sequence (CAG)16(CTG)4, a corresponding evaluation can be made for CAG/CAG interactions. Since CAG/CAG interactions are known to be weaker, forming less stable hairpin folds (
      • Petruska J.
      • Arnheim N.
      • Goodman M.F.
      ,
      • Smith G.K.
      • Jie J.
      • Fox G.E.
      • Gao X.
      ,
      • Yu A.
      • Dill J.
      • Mitas M.
      ,
      • Mitas M.
      ), it is of interest to see how much difference this makes in the slippage rate constants. Previously it has been generally considered (
      • Sinden R.R.
      • Wells R.D.
      ,
      • Wells R.D.
      ,
      • Ohshima K.
      • Wells R.D.
      ,
      • Kang S.
      • Ohshima K.
      • Shimizu M.
      • Amirhaeri S.
      • Wells R.D.
      ,
      • Gacy A.M.
      • Goellner G.
      • Juranic N.
      • Macura S.
      • McMurray C.T.
      ,
      • Mitas M.
      ) that the greater the stability of single-strand folding, the greater the probability of strand slippage and expansion in repeat regions. However, it is also possible that a difference in folding stability on opposite strands may be important in determining strand slippage and expansion rates. This may be a reason why repeats of triplets such as GAA/TTC, having only purines on one side and pyrimidines on the other, also expand to give diseases such as Friedrich's ataxia (
      • Cossee M.
      • Schmitt M.
      • Campuzano V.
      • Reutenauer L.
      • Moutou C.
      • Mandel J.-L.
      • Koenig M.
      ).

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