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Base Stacking and Even/Odd Behavior of Hairpin Loops in DNA Triplet Repeat Slippage and Expansion with DNA Polymerase

2000; Elsevier BV; Volume: 275; Issue: 24 Linguagem: Inglês

10.1074/jbc.275.24.18382

1083-351X

Michael J. Hartenstine, Myron F. Goodman, John Petruska,

Genomics and Chromatin Dynamics

Repetitions of CAG or CTG triplets in DNA can form intrastrand hairpin loops with combinations of normal and mismatched base pairs that easily rearrange. Such loops may promote primer-template slippage in DNA replication or repair to give triplet-repeat expansions like those associated with neurodegenerative diseases. Using self-priming sequences (e.g.(CAG)16(CTG)4), we resolve all hairpin loops formed and measure their slippage and expansion rates with DNA polymerase at 37 °C. Comparing CAG/CTG loop structures with GAC/GTC structures, having similar hydrogen bonding but different base stacking, we find that CAG, CTG, and GTC triplets predominantly form even-membered loops that slip in steps of two triplets, whereas GAC triplets favor odd-numbered loops. Slippage rates decline as hairpin stability increases, supporting the idea that slippage initiates more easily in less stable regions. Loop stabilities (in low salt) increase in the order GTC < CAG < GAC < CTG, while slippage rates decrease in the order GTC > CAG ≈ GAC > CTG. Loops of GTC compared with CTG melt 9 °C lower and slip 6-fold faster. We interpret results in terms of base stacking, by relating melting temperature to standard enthalpy changes for doublets of base pairs and mispairs, considering enthalpy-entropy compensation. Repetitions of CAG or CTG triplets in DNA can form intrastrand hairpin loops with combinations of normal and mismatched base pairs that easily rearrange. Such loops may promote primer-template slippage in DNA replication or repair to give triplet-repeat expansions like those associated with neurodegenerative diseases. Using self-priming sequences (e.g.(CAG)16(CTG)4), we resolve all hairpin loops formed and measure their slippage and expansion rates with DNA polymerase at 37 °C. Comparing CAG/CTG loop structures with GAC/GTC structures, having similar hydrogen bonding but different base stacking, we find that CAG, CTG, and GTC triplets predominantly form even-membered loops that slip in steps of two triplets, whereas GAC triplets favor odd-numbered loops. Slippage rates decline as hairpin stability increases, supporting the idea that slippage initiates more easily in less stable regions. Loop stabilities (in low salt) increase in the order GTC < CAG < GAC < CTG, while slippage rates decrease in the order GTC > CAG ≈ GAC > CTG. Loops of GTC compared with CTG melt 9 °C lower and slip 6-fold faster. We interpret results in terms of base stacking, by relating melting temperature to standard enthalpy changes for doublets of base pairs and mispairs, considering enthalpy-entropy compensation. primer-template DNA duplex loop domain of template triplet interactions base pair(s) Repetitive DNA sequences such as tandemly repeated triplets of bases are abundant and highly polymorphic in the human genome, probably because of strand slippage promoted by repetition in DNA replication, repair, or recombination (1.Levinson G. Gutman G.A. Mol. Biol. Evol. 1987; 4: 203-221PubMed Google Scholar, 2.Schlotterer C. Tautz D. Nucleic Acids Res. 1992; 20: 211-215Crossref PubMed Scopus (817) Google Scholar, 3.Wells R.D. J. Biol. Chem. 1996; 271: 2875-2878Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 4.Petruska J. Hartenstine M.J. Goodman M.F. J. Biol. Chem. 1998; 273: 5204-5210Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 5.$$$$$$ ref data missingGoogle Scholar, 6.Jakupciak J.P. Wells R.D. J. Biol. Chem. 1999; 274: 23468-23479Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Occasionally, a repetitive region within a human gene expands sufficiently to cause inherited human disease (7.Sutherland G.R. Richards R.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3636-3641Crossref PubMed Scopus (301) Google Scholar). At least 12 neurological disorders are associated with triplet repeat expansions within human genes (5.$$$$$$ ref data missingGoogle Scholar). Eight such disorders arise by the expansion of CAG repetitions in gene regions encoding glutamine repeats in protein. In each case, involving a different gene (Huntingon's disease, spinobulbar muscular atrophy, dentatorubral-pallidoluysian atrophy, and spinocerebellar ataxias 1, 2, 3, 6, and 7), the number of CAG repeats in tandem is expanded from a normal range of 5–30 to a disease-causing range of 40–100 (5.$$$$$$ ref data missingGoogle Scholar). The resultant polyglutamine expansion in protein may cause disease by forming insoluble nuclear protein aggregates (8.Davies S.W. Turmaine M. Cozens B.A. DiFiglia M. Sharp A.H. Ross C.A. Scherzinger E. Wanker E.E. Magiarini L. Bates G.P. Cell. 1997; 90: 537-548Abstract Full Text Full Text PDF PubMed Scopus (1912) Google Scholar, 9.Skinner P.J. Koshy B.T. Cummings C.J. Klement I.A. Helin K. Servadio A. Zoghbi H.Y. Orr H.T. Nature. 1997; 389: 971-974Crossref PubMed Scopus (499) Google Scholar, 10.Matilla A. Koshy B.T. Cummings C.J. Isobe T. Orr H.T. Zoghbi H.Y. Nature. 1997; 389: 974-978Crossref PubMed Scopus (231) Google Scholar), possibly with cross-linking by transglutaminase (11.Kahlem P. Terre C. Green H. Dijan P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14580-14585Crossref PubMed Scopus (180) Google Scholar, 12.Cooper A.J.L. Sheu K.-F.R. Burke J.R. Onodera O. Strittmatter W.J. Roses A.D. Blass J.P. J. Neurochem. 1997; 69: 431-434Crossref PubMed Scopus (89) Google Scholar), an enzyme abundant in the brain (13.Gilad G.M. Varon L.E. J. Neurochem. 1985; 45: 1522-1526Crossref PubMed Scopus (48) Google Scholar, 14.Ohashi H. Itoh Y. Birckbichler P.J. Takeuchi Y. J. Biochem. (Tokyo). 1995; 118: 1271-1278Crossref PubMed Scopus (9) Google Scholar), enabling glutamine reaction with lysine to form a peptide-like cross-link between polypeptides. Another four disorders involve other triplet repetitions expanded in noncoding regions of genes. Myotonic dystrophy, for example, is associated with repeating CTG triplets expanded in an untranslated 3′-terminal gene region, from a normal range of 5–40 repeats to a disease-causing range of 50–3000 (5.$$$$$$ ref data missingGoogle Scholar). Expansions of this magnitude are also observed in CGG repeats and CCG repeats, found in 5′-untranslated regions of genes associated with fragile X syndromes A and E, respectively (5.$$$$$$ ref data missingGoogle Scholar). Similarly large increases are observed for GAA repetitions in an intron of a gene associated with Friedreich's ataxia (5.$$$$$$ ref data missingGoogle Scholar). In each case, the noncoding region involved is transcribed into RNA but not translated into protein. The increased number of repetitions in RNA may cause disease by interfering with RNA transcription or processing within cell nuclei (15.Krahe R. Ashizawa T. Abbruzzese C. Roeder E. Carango P. Giacanelli M. Funanage V.L. Siciliano M.J. Genomics. 1995; 28: 1-14Crossref PubMed Scopus (129) Google Scholar, 16.Davis B.M. McCurrach M.E. Taneia K.L. Singer R.H. Housman D.E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7388-7393Crossref PubMed Scopus (381) Google Scholar, 17.Verkerk A.M.J.H. de Graff E. De Boulle K. Eichler E.E. Konacki D.S. Reyniers E. Manca A. Poustka A. Willems P.J. Nelson D.L. Oostra B.A. Hum. Mol. Genet. 1993; 2: 399-404Crossref PubMed Scopus (86) Google Scholar, 18.Duval-Valentin G. Thuong N.T. Helene C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 504-508Crossref PubMed Scopus (257) Google Scholar). In DNA undergoing replication or repair, repeating triplets may enable one DNA strand to slip relative to the other so that the number of triplet repeats can be expanded by DNA polymerase (2.Schlotterer C. Tautz D. Nucleic Acids Res. 1992; 20: 211-215Crossref PubMed Scopus (817) Google Scholar, 4.Petruska J. Hartenstine M.J. Goodman M.F. J. Biol. Chem. 1998; 273: 5204-5210Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 19.Behn-Krappa A. Doerfler W. Hum. Mutat. 1994; 3: 19-24Crossref PubMed Scopus (29) Google Scholar). Also, because CNG triplets associated with disease can form intrastrand hairpin folds with secondary structure (20.Gacy A.M. Goellner G. Juranic N. Macura S. McMurray C.T. Cell. 1995; 81: 533-540Abstract Full Text PDF PubMed Scopus (519) Google Scholar, 21.Mitas M., Yu, A. Dill J. Kamp T.J. Chambers E.J. Haworth I.S. Nucleic Acids Res. 1995; 23: 1050-1059Crossref PubMed Scopus (134) Google Scholar, 22.Petruska J. Arnheim N. Goodman M.F. Nucleic Acids Res. 1996; 24: 1992-1998Crossref PubMed Scopus (121) Google Scholar), it is of interest to determine how such folding affects slippage and expansion with polymerase. Recently, we developed a convenient in vitroassay, using self-priming repeat sequences, to measure rates of slippage and expansion in relation to hairpin structure (4.Petruska J. Hartenstine M.J. Goodman M.F. J. Biol. Chem. 1998; 273: 5204-5210Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). In an earlier study of the major (CAG/CTG) class of disease-associated triplets (4.Petruska J. Hartenstine M.J. Goodman M.F. J. Biol. Chem. 1998; 273: 5204-5210Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), we examined DNA polymerase extension products of the self-priming sequence, (CTG)16(CAG)4, which forms a well defined series of hairpin loops. Using high resolution gel electrophoresis to analyze product lengths, we found that the CTG repeats form loops that are predominantly even-membered (i.e. have even rather than odd numbers of bases in the hairpin bend). The products of polymerase extension were observed to slip and expand in steps of two triplets at rates of ∼1 step/min at 37 °C. In the present study, a similar analysis is made for the complementary case, (CAG)16(CTG)4, and “sister” cases obtained by replacing CAG/CTG with GAC/GTC. Since these cases exhibit the same hydrogen bonding with different base stacking, they reveal the influence of base stacking on the even-odd character of hairpin folds and slippage rates leading to repeat expansions with polymerase. Like the self-priming DNA 60-mer, 5′-(CTG)16(CAG)4-3′ previously studied (4.Petruska J. Hartenstine M.J. Goodman M.F. J. Biol. Chem. 1998; 273: 5204-5210Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), the complement (CAG)16(CTG)4 and sisters, (GTC)16(GAC)4 and (GAC)16(GTC)4, were each synthesized using β-cyanoethyl phosphoramidites in an Applied Biosystems DNA/RNA synthesizer and purified by electrophoresis on denaturing 12% polyacrylamide gel. Each DNA 60-mer extracted from gel was dialyzed extensively against the same 0.02 m Na+phosphate buffer used previously (4.Petruska J. Hartenstine M.J. Goodman M.F. J. Biol. Chem. 1998; 273: 5204-5210Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) (5 mmNaH2PO4, 5 mmNa2HPO4, 1 mm Na4EDTA, pH 7.0) and stored at −70 °C. The KFexo−polymerase used to achieve rapid primer 3′ extension on template, anEscherichia coli DNA polymerase I Klenow fragment mutant (D355A,E357A) devoid of 3′ → 5′ as well as 5′ → 3′ exonuclease activity, was purified from overproducing strains (23.Derbyshire V. Freemont P.S. Sanderson M.R. Beese L. Friedman J.M. Joyce C.M. Steitz T.A. Science. 1988; 240: 199-201Crossref PubMed Scopus (301) Google Scholar). The dNTP substrates used in extension experiments were purchased from Amersham Pharmacia Biotech, along with ddNTPs employed for sequence analysis of extended products. Thermal denaturation profiles were obtained for each DNA 60-mer at the same strand concentration (2 μm) in 0.02 m Na+ phosphate buffer (4.Petruska J. Hartenstine M.J. Goodman M.F. J. Biol. Chem. 1998; 273: 5204-5210Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), by measuring UV absorbance (A 260)versus temperature, from 20 to 85 °C at 2 °C/min. For use in extension reactions, DNA 60-mers were 5′-labeled with 32P, using [γ-32P]ATP and T4 polynucleotide kinase (U.S. Biochemical Corp./Amersham Pharmacia Biotech) in kinase buffer (50 mm Tris-HCl, pH. 7.6, 10 mm MgCl2, and 10 mm2-mercaptoethanol). As before (4.Petruska J. Hartenstine M.J. Goodman M.F. J. Biol. Chem. 1998; 273: 5204-5210Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), labeled samples at 100 nm strand concentration were unfolded by heating at 100 °C for 5 min and refolded by slow cooling to room temperature and then stored at 4 °C to avoid intermolecular associations promoted by freezing (22.Petruska J. Arnheim N. Goodman M.F. Nucleic Acids Res. 1996; 24: 1992-1998Crossref PubMed Scopus (121) Google Scholar). Radiolabeled DNA samples at 10 nm strand concentration, in polymerase reaction buffer (50 mm Tris-HCl, pH 7.5, 10 mm MgCl2), were incubated at 37 °C for 5 min to allow equilibration. A 120-μl aliquot was then micropipetted into a 0.5-ml polypropylene microcentrifuge tube containing 30 μl of polymerase plus dNTPs in reaction buffer at 37 °C, at which point (within 3 s), running time (t) for reaction was started. The concentrations of DNA polymerase KFexo− and each dNTP in the reaction mixture were 60 and 400 nm, respectively. After short intervals of reaction time (t = 15 s, 30 s, etc.), a 5-μl aliquot of reaction mixture was removed and added (within 2 s) to 10 μl of 20 m formamide solution containing 20 mm EDTA to quench the reaction. Extension products of radiolabeled DNA were separated into bands of increasing chain length by electrophoresis at 2000 V on 12% polyacrylamide slab gel (40 cm × 40 cm × 0.2 mm) containing 16 m formamide as denaturant, in TBE buffer (90 mm Tris borate, pH 8.3, 2 mm Na2EDTA). Gels were dried on paper and scanned by a Molecular Dynamics Storm 860 PhosphorImager. FragmeNT Analysis software (Molecular Dynamics) was used to integrate the intensity of each radioactive band in a gel lane, expressed as a percentage of total integrated band intensities in the lane. In each of the four cases studied here, the initial 60-mer sequence (A) forms a series of self-primed hairpin loops, A n (n= 0, 1, 2, etc.), where n is the number of overhanging template triplets at the 5′-end, as illustrated (Fig. 1,a–d), with an asterisk indicating the 5′-end labeled with 32P. The loops are rapidly extended to their corresponding blunt-end products by adding DNA polymerase and sufficient amounts (0.4 μm each) of the three dNTPs needed to reach near maximum velocity of primer extension on template, with minimal background “pause” banding caused by misinsertion or terminal transferase activity (4.Petruska J. Hartenstine M.J. Goodman M.F. J. Biol. Chem. 1998; 273: 5204-5210Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). In 15 s of reaction time (t) with polymerase, the primer 3′-end ofA n is extended by n primer triplets to form the corresponding blunt-end product, A n plusn triplets, measured as a discrete gel band intensity (I n) by denaturing gel electrophoresis and PhosphorImager analysis. Each I n (n= 0, 1, 2, etc.) changes as reaction time t is increased, because each blunt-end product undergoes slippage and further extension with polymerase. By analyzing I n versus t and extrapolating back to t = 0, we evaluate I n0, indicating the approximate amount of loop A n present initially, before polymerase was added.Figure 1Possible hairpin loops formed by self-priming sequences of CAG/CTG and GAC/GTC triplet repeats. A, self-priming DNA sequence having 16 repeats of a template triplet followed by four repeats of complementary primer triplet;A n, hairpin fold of sequence A, havingn overhanging template triplets on which DNA polymerase can rapidly add n complementary primer triplets to the primer 3′-end. a, loops A n (n = 0, 1, 2, etc.) with hydrogen-bonded base pairs for sequence 5′-(CTG)16(CAG)4-3′; b, corresponding loops for “sister” sequence of same base composition, (GTC)16(GAC)4; c and d, corresponding loops for respective “complementary” cases, (CAG)16(CTG)4 and (GAC)16(GTC)4. The hairpin bends in even-numbered loops (n = 0, 2, … 14) are made with an even number of unpaired bases, minimally four as shown; those in odd-numbered loops (n = 1, 3, … 15) are made with an odd number of unpaired bases (at least three). The hydrogen-bonded, Watson-Crick base pairs are indicated bydots in a horizontal series; three dotsbetween triplets in parentheses indicate stably paired primer-template triplets in the p/t duplex domain; two dotsbetween triplets in parentheses indicate less stably paired template triplets in the t/t loop domain. The asterisk indicates the 5′-end of template labeled with 32P. Note that loopsA n have n template triplets available for extending primer 3′-end on template with DNA polymerase (e.g. polymerase KFexo−). In the presence of three dNTPs (n = C, G, and A or T), polymerase KFexo− rapidly extends A n byn primer triplets to form product blunt-end hairpins. The products in each case, A n plus (CAG)n(a), A n plus (GAC)n(b), A n plus (CTG)n(c), or A n plus (GTC)n(d), are observed as a series of band intensitiesI n (n = 0, 1, 2, etc.) by denaturing gel electrophoresis (Figs. 2 b and 3 b). The changes in I n with increasing reaction time are measured to determine rates at which successive blunt-end product hairpins undergo slippage resulting in further polymerase-catalyzed expansion. Extrapolation of I n to zero reaction time yields I n0, the initial amount ofA n formed by each sequence.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The loops are classified as even-numbered or odd-numbered, depending on whether an even or odd number of bases is enclosed within the loop. The even-numbered loops (n = 0, 2, … 10) have the same kind of hairpin bend made with an even number of unpaired bases, minimally four as shown (Fig. 1, a–d). The odd-numbered loops (n = 1, 3, … 11) have another kind of bend made with an odd number of unpaired bases (minimally 3). The other loops shown (n = 12–15) have one or more primer triplets in the bend region where there is less opportunity for correct base pairing; these are much less stable loops, indicating how bend regions change as primer triplets enter the bend region in extreme cases of slippage. The initial blunt-ended loop, A 0, which cannot be extended unless slippage occurs, is seen as band intensityI 0 remaining after 15 s of reaction. AsA 0 undergoes slippage to an extendable form (A 1, A 2, etc.),I 0 declines in a simple manner, suggesting a first order differential equation, dI 0/dt = −k 0 I 0, or in terms of measured differences (Δ) as follows, ΔI0/Δt=−k0I0Equation 1 where k 0 is the slippage rate constant and I 0 is the average intensity value measured in a short time interval, Δt, of 15 or 30 s. For Δt between reaction times t x andt y, the intensity change is ΔI 0 =I 0(t y) −I 0(t x), and the correspondingI 0 on the right side of Equation 1 is the average value, I o = 0.5(I 0(t x) +I 0(t y)). In our experiments, I 0 decays to a low residual “background” intensity (I 0b) that remains nearly constant, indicating that a small fraction ( 2. In each case, starting with the general form of Equation 5, ΔIn/(InΔt)=kn−2,n(In−2/In)+kn−1,n(In−1/In)−knEquation 6 we apply approximations (a)k n− 2,n ≫k n− 1,n, (b)k n− 2,n ≈k n− 1,n, and (c) k n− 2,n≪ k n− 1,n to determine which gives the best k n estimate by linear least squares fit. In all cases where approximation a applies ton = 2 (i.e. k 02 ≫k 12 in Equation 5), we find that the corresponding general approximation (k n− 2,n ≫k n− 1,n) also applies to Equation 6 for evaluating other even-numbered k nvalues (n = 4, 6, etc.). By using thek n values measured with corresponding simplified (linear) versions of Equations 5 and 6, we extrapolateI n back from t = 15 s tot = 0, to estimate I n0, indicating the initial amount of loop A n present before extension with polymerase. Each of the self-priming sequences examined here, like (CTG)16(CAG)4 previously studied (4.Petruska J. Hartenstine M.J. Goodman M.F. J. Biol. Chem. 1998; 273: 5204-5210Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), forms a series of hairpin loops, A n (n = 0, 1, 2, etc.), with stable p/t1duplex enclosing a less stable t/t loop domain (Fig.1, a–d). The subscriptn in A n indicates that there aren overhanging template triplets on which DNA polymerase can extend the primer 3′-end rapidly (in seconds) to yield blunt-end product (A n + n triplets) proportional to the amount of A n present. With increasing reaction time, each blunt-end product undergoes slippage, allowing more triplets to be added by DNA polymerase, as shown in Figs.2 and3.Figure 3Comparison of (GAC)16(GTC)4 and (GTC)16(GAC)4 melting transitions and corresponding band patterns showing triplets added with increasing time of DNA polymerase reaction. a, melting curves obtained by plotting UV absorbance A 260 versustemperature at the same (2 μm) strand concentration in low salt buffer, showing that (GAC)16(GTC)4 has higher first T m than (GTC)16(GAC)4 (54 versus 47 °C) but the same second T m (78 °C). b, patterns of bands on denaturing gel, obtained by reaction with DNA polymerase KFexo− at 37 °C, using 0.4 μm each of three dNTPs (N = C, G, and T or A) required for correct extension of primer triplets on template triplets. The outer lanes marked ddCshow bands corresponding to 1, 2, etc. primer triplets added, found by including ddCTP in the reaction mixture to cause termination with dideoxycytidine.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In each case (Fig. 1, a–d), the even- and odd-numbered loops A n up to n = 11 have all four primer triplets correctly hydrogen-bonded and stacked in Watson-Crick bp (indicated by dots) with four antiparallel, template triplets located n triplets from the 5′-32P-end (*). The p/t duplex in A n (n = 0, 1, … 11) encloses a t/t loop domain containing 12 − ntemplate triplets. The t/t domain has the hairpin bend made with an even or odd number of unpaired bases and also has mispaired bases held between correct bp formed between opposing (antiparallel) template triplets. Opposing triplets of type CTG or GTC form mispairs of type T opposite T (T/T) held between correct G/C and C/G bp (dots), as shown (Fig. 1, a andb) for sequences (CTG)16(CAG)4 and (GTC)16(GAC)4, respectively. On the other hand, opposing triplets of type CAG or GAC form mispairs of A opposite A (A/A), as shown (Fig. 1, c andd) for (CAG)16(CTG)4 and (GAC)16(GTC)4, respectively. Loops A 12 to A 15(Fig. 1, a–d), having fewer than four of the primer triplets correctly bound to template triplets, are much less stable and are hardly apparent initially. These loops are included to show how bends change in extreme cases of slippage after more then 11 triplets are added by DNA polymerase in longer reaction times (Figs.2 b and 3 b). To measure the relative stability of loop structures formed in absence of polymerase, thermal denaturation curves were obtained for each 60-mer sequence in low salt buffer (0.02m Na+), by plotting UV absorbance (A 260) versus temperature (Figs.2 a and 3 a). In each case, as found previously for (CTG)16(CAG)4 (4.Petruska J. Hartenstine M.J. Goodman M.F. J. Biol. Chem. 1998; 273: 5204-5210Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), two sigmoidal transitions are evident, the first with melting temperature (T m) below 60 °C and the second with T m near 80 °C. The first transition indicates the melting of the t/t loop domain containing the base pairs and mispairs of template triplet interactions. As anticipated from previous work (22.Petruska J. Arnheim N. Goodman M.F. Nucleic Acids Res. 1996; 24: 1992-1998Crossref PubMed Scopus (121) Google Scholar) and seen by comparing Figs. 2 a and 3 a, this domain is most stable for CTG triplets (T m = 56 °C) followed by GAC (54 °C), CAG (52 °C), and last GTC (47 °C). The second transition indicates the dissociation of stable p/t duplex, which melts at nearly the same temperature (T m ≈ 79 °C) in all four cases. If slippage in the stable p/t duplex is promoted by slippage in the less stable t/t domain, as we suggested previously (4.Petruska J. Hartenstine M.J. Goodman M.F. J. Biol. Chem. 1998; 273: 5204-5210Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), then (GTC)16(GAC)4, having the lowestT m for the first transition (47 °C, Fig.3 a), should also have the highest rate of slippage and expansion with polymerase. This is evidently the case, as seen by comparing gel band patterns (Figs. 2 b and 3 b) obtained by primer extension with DNA polymerase KFexo− at 37 °C, for reaction times ranging from 0.5 to 16 min. The addition of DNA polymerase KFexo− and appropriate (0.4 μm) dNTP substrates results in rapid extension of loops A n to their blunt-end products, A n + ntriplets, resolved as band intensities I n by electrophoresis and 32P PhosphorImager analysis (Figs.2 b and 3 b). After stopping the reaction at various times and resolving product bands in gel lanes, we evaluate band intensities I 0, I 1,I 2, etc. by integration in each lane, to measure the relative amounts of products A 0,A 1 + 1 triplet, A 2 + 2 triplets, etc. as a function of time. The I n values obtained at times of 0.25 and 0.5 min, before products rearrange significantly by slippage, are used for extrapolation back to 0 time, to obtain I n0, indicating the initial amounts of A n present when polymerase was added. In the case of (CTG)16(CAG)4, whose band patterns are shown in Fig. 2 b (left), we see that the loops are predominantly of the even-numbered type, as previously reported (4.Petruska J. Hartenstine M.J. Goodman M.F. J. Biol. Chem. 1998; 273: 5204-5210Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The complementary case, (CAG)16(CTG)4, shown in Fig. 2 b(right), also forms mainly even-numbered loops, as does (GTC)16(GAC)4, shown in Fig. 3 b(left) After 0.5 min of reaction time, the most intense bands correspond to even numbers of triplets added (0, 2, … 10). As reaction time is increased, the band intensities gradually change as blunt-end products rearrange by slippage and are expanded further, mainly in steps of two triplets. By examining band intensity changes with reaction time (Fig.2 b), we see that expansion by slippage is several times faster for (CAG)16(CTG)4 than for (CTG)16(CAG)4. A faster slippage rate is in keeping with the observation (Fig. 2 a) that the first transition has a 4 °C lower T m value, 52 °C, compared with 56 °C for (CTG)16(CAG)4, The second transition in both cases is the same (T m = 79 °C). The “sister” sequence (GTC)16(GAC)4, which has the same base composition as (CTG)16(CAG)4, has a much lower first transition (T m = 47 °C), with no significant change in the second transition (T m = 78 °C), as seen in Fig. 3 a. The first T m is 9 °C below that for (CTG)16(CAG)4, and the rate of expansion by slippage is also much faster as found by comparing the gel band patterns in Figs. 3 b (left) and 2b(left) Nevertheless, (GTC)16(CAG)4still forms even-numbered loops preferentially and slips in steps of two triplets, as do (CTG)16(CAG)4 and (CAG)16(CTG)4. In contrast, (GAC)16(GTC)4, which has the same base composition as (CAG)16(CTG)4, tends to form odd-numbered loops. Unlike the other three cases, the most intense bands in this case (Fig. 3 b, right) correspond to odd numbers of triplets added (1, 3, 5, etc.). The changes in band intensity with time indicate that expansion by slippage is much slower than observed for (GTC)16