Biochemistry 1993, 32, 6563-6575
6563
Conformational Differences between Bulged Pyrimidines (C-C) and Purines (A-A, 1-1) at the Branch Point of Three-Stranded DNA Junctionst Mark A. Rosent and Dinshaw J. Patel*.tJ Department of Biochemistry and Molecular Biophysics, College of Physicians and Surgeons, Columbia University, New York, New York 10032, and Cellular Biochemistry and Biophysics Program, Memorial Sloan- Kettering Cancer Center, 1275 York Avenue, New York, New York 10021 Received December 9, 1992; Revised Manuscript Received April 8, 1993
ABSTRACT: We have synthesized D N A oligomers that can combine to form three-way junctions containing six base pairs in each stem and two unpaired bases a t the branch point. Gel electrophoresis experiments indicate that the oligomers form stable complexes with equimolar stoichiometry. Using two- and threedimensional proton nuclear magnetic resonance spectroscopy, we have completed nonexchangeable proton chemical shift assignments for three junctions which differ only in the identity of the unpaired bases (C-C, A-A, or 1-1) at the branch point. Our results indicate that unpaired pyrimidines a t the branch point of junctions behave differently than do unpaired purines. In a junction with two unpaired cytidines, the 5‘ base loops out from the molecule to lie along the minor groove of the preceding duplex stem of the junction. The 3’ unpaired cytidine also demonstrates an unusual pattern of N O E connectivities with detected cross peaks to the subsequent base in the 3’ direction. Junctions with unpaired purines a t the branch point exhibit different behavior. Our data suggests that in these molecules the unpaired bases participate in stacking interactions among themselves and with the neighboring bases in the molecule. Despite these differences, the N O E patterns from each junction suggest the presence of a preferred, pair-wise stacking between two of the helices within the molecule. The structural differences between bulge-pyrimidine and bulgepurine junctions are discussed in light of the functional significance unpaired bases might have in the structure and dynamics of multistranded D N A junctions and, by extension, to junctions within cellular RNAs.
DNA junctions arise whenever three or more helices come together at a single point. The most frequently encountered DNA junction in biology is the four-stranded, or Holliday, junction (Holliday, 1964). However, simpler junctions comprising just three helices, as well as more complex junctions containing five or six helices, have been described in vitro (Ma et al., 1986; Wong et al., 1991). Holliday junctions are thought to occur within the cell during meiotic recombination (Meselson & Radding, 1975; OrrWeaver et al., 198 l), a process which allows the cell to shuffle genetic material among homologous chromosomes. However, three-stranded DNA junctions are also believed to play a role in certain recombination events (Broker & Doermann, 1975; Mingawa et al., 1983;Jensch & Kemper, 1986). Furthermore, as the simplest junctions, three-stranded junctions can serve as a model for the physical interactions present in Holliday junctions. When compared with Holliday junctions, three-stranded junctions appear to be more flexible in solution (Ma et al., 1986). There is disagreement in the literature whether the arms in a three-stranded junctions are found in a unstacked, “Y”conformation (Duckett & Lilley, 1990) or in an asymmetric conformation, possibly including base stacking across thebranchpoint (Guoet al., 1990;Luet al., 1991). However, it is likely that helical stacking in three-stranded junctions, if present, is less stable than that predicted to occur in Holliday junctions. This research was supported by NIH Grant GM34504 to D.P.M.R. supported by NIH Medical Scientist Training Program Training Grant 5-T32-GM07376. TheNMR spectrometerswere purchased from funds donated by the Robert Wood Johnson Trust toward setting up an NMR center in the Basic Medical Sciences at Columbia University. t Columbia University. Memorial Sloan-KetteringCancer Center. was
Chart I C2
G39
A3
T38
T4
A37
A34 G33 G32 A31 T30
X7
ys
T21 C22 C 2 3 T24 A25
G9
Czo
Cio
Gig
TII
A18
XIY
A12 Ti7
Cl3
GIG
G14
Cis
1
Stem II
J3CC J3AA J3II
c-c A-A 1-1
Interestingly, it has recently been shown (Leontis et al., 1991) that three-stranded junctions could be stabilized with the addition to one strand of unpaired, or ”bulged”, bases at the branch point. The addition of two or more unpaired bases allowed stable three-stranded junctions to form with just five base pairs in each arm. This finding allows one to construct smaller junctions more amenable to detailed structural analysis. In order to investigate the role of unpaired bases in directing helix-helix interactions within a three-way DNA junction, we have constructed three such junctions, each containing two unpaired bases on one strand at the branch point. The sequence and numbering scheme of the junctions are shown in Chart I. The junctions comprise 17 base pairs (six in two of the arms, five in the other). The three stems are indicated with Roman numerals. One junction, which we call “J3CC”, contains two unpaired cytosine bases at the branch point. The second, called “J3AA”, contains two adenine bases at this point, whereas “J3II” contains two unpaired inosines. In each
0006-2960/93/0432-6563$04.00/0 0 1993 American Chemical Society
6564 Biochemistry, Vol. 32, No. 26, 1993 of the molecules, two of the strands have been connected to form a hairpin with a four-base loop. We have studied these molecules by two- and threedimensional homonuclear N M R spectroscopy. Our aim was to describe the structural features of the three-way junction with regard to (1) distortions, if any, from the expected B-DNA structure in the double helical regions, (2) the presence or absence of Watson-Crick base pairs adjacent to the branch point, (3) the position and conformation of the unpaired bases, and (4) the overall tertiary structureof the molecules,including the presenceof any pair-wisestacking among the three helices. We present the results of this study in this paper and its accompanying counterpart. In this paper we demonstrate the formation of junction complexes from the component strands and present the sequential IH-NMR assignments of nonexchangeable protons. In the subsequent paper, we examine the exchangeable proton spectra of J3CC in order todetermine the status of the base pairs adjacent to the branch point. We then present a structural model for the J3CC junction that demonstrates the presence of a preferred pairwise helical stacking arrangement within the molecule.
MATERIALS AND METHODS DNA Synthesis and Purification. DNA oligomers were synthesized on solid supports (10-pmol scale) using standard phosphoramidite chemistry on an Applied Biosystems 39 1 automated DNA synthesizer. All reagents were supplied by Applied Biosystemsexcept 5-methylcytidine phosphoramidite, which was supplied by Pharmacia. Oligomers were cleaved from the column and deprotected by treatment with concentrated ammonium hydroxide for 16 h at 5 5 OC. The tritylcontaining oligomers were purified by reverse-phase HPLC, followed by detritylation in 80%acetic acid and repurification through reverse-phase HPLC. Organic cations were removed via gel filtration through Sephadex G-25, followed by passage through a Dowex cation-exchange column to form the Na+DNA salt. DNA Strand Quantification. Oligomer strands were quantified by UV absorbance at 260 nm in 89 mM Trisborate (pH 8.3) with 200 mM NaCl and 5 mM MgC12. Extinction coefficients were calculated with consideration of nearest-neighbor effects (Fasman, 1975). Hypochromicity effects were accounted for in the double-stranded hairpin stem region. No attempt was made to correct for the change in extinction coefficient due to the presence of the 5-methylcytidine residue. Gel Electrophoresis. Gels contained 20% acrylamide (20: 1 mono:bis) with 89 mM Tris-borate (pH 8.3), 200 mM NaC1, and either 5 mM MgC12 or 2 mM EDTA. Samples contained 5-10 pg of either the individual strands or one-to-one mixtures in 10 p L of gel buffer. They were heated to 80 “ C for 15 min and allowed to cool slowly to room temperature, following which dye was added, and the samples were loaded onto the gel. The gels were run overnight in a 4 OC cold room, without buffer recirculation, at a constant voltage of 100 V. After electrophoresis was complete, the gels were stained with 5 pg/L ethidium bromide in H2O for 30 min, followed by destaining for 30 min in distilled H20. DNA bands were visualized by transillumination of the gels with 300-nm light and photographed. Preparation of N M R Samples. Equimolar amounts of the two strands, as determined by gel electrophoresis, were mixed and lyophilized to dryness. The samples were next dissolved in 0.4 mL of 10 mM phosphate buffer (pH 6.2) containing 200 mM NaCl and 0.1 mM EDTA and were heated to 80 OC
Rosen and Pate1 for 15 min, followed by slow cooling to room temperature. The samples were then repeatedly lyophilized from 99.9% D20 and were finally dissolved in 0.4 mL of 99.96% DzO. For some experiments, 5 mM MgC12 was included through the addition of an aliquot of concentrated MgC12 in D20, followed by reannealing of the strands. Final DNA concentrations were 2-3 mM in each strand. N M R Spectroscopy. One-dimensional proton NMR spectra were recorded on a Bruker AM-400WB spectrometer. Spectra were acquired with presaturation of the residual HDO peak during the recovery period. The free induction decay, which contained 1024 complex data points, was apodized with a 90’ shifted sinebell and zero-filled to 4096 complex points prior to Fourier transformation. All spectra were referenced relative to an internal sodium 4,4-dimethyl-4-silapentane-lsulfonate (DSS) standard. Two-dimensional nuclear Overhauser effect spectroscopy (NOESY) experiments were acquired in the phase-sensitive mode (States et al., 1982) on a Bruker AM-500 spectrometer. The two-dimensional data sets were acquired with 1024 complex points in a 10 ppm spectral width in the t 2 dimension. Two different mixing times, 50 and 200 ms, were used in the NOESY experiments. Typically, 256 real and imaginary increments were acquired in the tl dimension. The twodimensional data sets were processed on a VAX/VMS 6310 computer using the software package FTNMR (D. Hare, Hare Research, Inc., Woodinville, WA). The data were subjected to Gaussian multiplication with 12-1 5 Hz of line narrowing in each dimension. Zero-filling in t l led to a matrix with 1024 complex points in each dimension. A three-dimensional homonuclear NOESY-NOESY experiment was run on a Bruker AMX-600 spectrometer in the phase-sensitive mode using time-proportional phase incrementation (Bodenhausen et al., 1980; Marion & Wuthrich, 1983) in both the t 2 and tl dimensions. The pulse scheme used was as reported by Boelens et al. (1989). We used 150 ms for the mixing time in each dimension, with no solvent irradiation during these periods. The matrix comprised 512 X 200 X 110 data points in an 8.0 ppm spectral width. The three-dimensional data set was processed on a Silicon Graphics IRIS using the FELIX software package (D. Hare, Hare Research, Inc., Woodinville, WA). The data was Gaussian multiplied with 12 Hz of line narrowing in each dimension, with zero-filling to a final matrix size of 512 X 256 X 256 complex points.
RESULTS Formation of Three-Way Junctions. We performed native polyacrylamide gel electrophoresis on mixtures of the different strands to determine whether stable junctions were forming under the experimental conditions. Figure 1 shows the results for a representative gel experiment. Lanes 1 and 2 contain the 14-base oligomers (residues 1-14 in Chart I) with either two cytidines or two adenines in the center. Both oligomers migrate quickly through the gel and stain lightly. The greater retardation of the adenine-containing oligomer may be due to transient formation of homodimers through mismatched base pairing. Lanes 3 and 4 contain bulge-loop duplexes, 12 base pairs in length, formed by one-to-one mixtures of either the cytidine or the adenine 14mer with the “complementary” dodecamer d(CGTAGCCGATGC). The hairpin oligomer alone (residues 15-40 in Chart I) is shownin lane 9. It migrates somewhat more rapidly than either of the two bulged duplexes, suggesting that this molecule does not dimerize under these conditions. We have noted that sequence variants of the
Biochemistry, Vol. 32, No. 26, 1993 6565
Branch Point Bulges in Three-Stranded DNA Junctions 1 2 3 4 5 6 7 0 9 1 0
FIGURE 1 : Gel electrophoresisexperiment showing formationof threeway junctions from component strands. Sample identities are as follow^: (1) d(GCATCG&GCTACG), (2) d(GCATCGEGCTACG), (3) d(GCATCGCCGCTACG) + d(CGTAGCCGATGC), (4) d(GCATCGAAGCTACG) + d(CGTAGCCGATGC), (5) d(GCATCGCCGTTACG) + d(CGTAGCTCCTA[TTTA]TAGGACGATGCT(6) d(GCATCGAAGCTACG) + d(CGTAGCTCCTA[TTTA]TAGGACGATGCT(7) d(GCATCGgGCTACG) d(CGTAGCATCCTA[TTTA]TAGGATCGATGC), (8) d(GCATCGAAGCTACG) + d(CGTAGCATCCTA[TTTA] TAGGATCGmGC) ,(9) d(CGTAGCTCCTA[TTTA]TAGGACGATGC), (10) d(CGTAGCATCCTA[TTTA]TAGGATCGATGC). Underlined residues represent unpaired bases (CC or AA) within the bulge-duplexor three-wayjunction complexes. The tetraloop residues from the hairpin strand are enclosed in brackets. Lanes 7,8,and 10 contain a variant of the hairpin strand with one additional base pair in the stem.
+
hairpin molecule with an additional GC base pair at the end of the hairpin stem run as a mixture of monomeric and dimeric species on gels (data not shown). Lanes 5 and 6 represent the three-way junctions, J3CC and J3AA, respectively. These two lanes each show a single,sharp band migrating more slowly than the individual components, confirming the formation of a stable junction in each case. We have compared the mobility of these junctions to that of a larger three-way junction without bulged bases, derived from three separate, unrelated oligomers, each 16 bases in length. This junction runs as a single, sharp band with lower mobility than that of J3CC or J3AA (data not shown), confirming the strand stoichiometry of the J3CC and J3AA junctions. Ionic Strength Effects on the Stability of the Three-Way Junctions. It has been reported that divalent cations play an important role in the stability and tertiary structure of threeand four-stranded DNA junctions (Cooper & Hagerman, 1989; Duckett et al., 1990; Leontis et al., 1991; Clegg et al., 1992). We have repeated the electrophoresis experiment shown in Figure 1 with 2 mM EDTA instead of 5 mM MgC12 in the gel buffer. Under these conditions, the junctions appear less stable and are characterized by higher mobility bands or streaks. This confirms the dependence of junction stability on ionic strength. One-Dimensional Proton Nuclear Magnetic Resonance of the Three-Way Junctions. Figure 2 shows the downfield and methyl regions of the one-dimensional proton N M R spectra in D20 for the three junctions, J3CC, J3AA, and J3II. DNA strand concentrations are approximately 0.4 mM. Buffer conditionsare 200 mM NaC1,2 mM MgC12,lO mM phosphate buffer (pH 6.5), and 0.1 mM EDTA. The resonance lines in the spectra are relatively sharp, implying that stable, oneto-one complexes are forming between the two strands in each case. Minor peaks visible in the spectra may reflect a slightly inequivalent stoichiometry of the two strands in the NMR samples. We noted that as the temperature of the sample was
8.5
8.0
7.5
7.0
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1.6
FIGURE 2: One-dimensional proton NMR spectra at 400 MHz of three-way junctions in D20 buffer (10 mM sodium phosphate, 200 mM NaCl, 2 mM MgC12, and 0.1 mM EDTA). DNA concentration is approximately 0.4 mM per strand. Base (7.0-8.5 ppm), H1’ (5.26.5 ppm), and methyl (1 A 2 . 2 ppm) regionsare shown. (A) Spectrum of J3CC at 25 “C. (B) Spectrum of J3AA at 30 “C. (C) Spectrum of J3II at 30 OC. The asterisks mark the unusual positions of the A29(H1’) and T27(CH3) resonances in each of the spectra. The triplet at 1.3 ppm represents a residual triethylammonium impurity in the samples.
raised above 35 OC, the minor peaks became more numerous and grew in intensity. By inspecting the N M R spectra of the individual strands (data not shown), we determined that these minor peaks originated from the component oligomers of the junctions. This “melting” effect was even more pronounced at lower NaCl concentrations. Thus, the stability of the junctions seemed to be sensitive to counterion concentration, as was found in gel electrophoresis experiments. Interestingly, the presence of Mg2+ cations had no additional stabilizing effect when the NaCl concentration was 200 mM or greater. In fact, the addition of MgC12 served only to broaden the proton resonance lines in the NMR spectra, indicatingtransient aggregation of the sample. We therefore elected to proceed with our N M R experiments without MgC12 in the sample buffer. The TTTA Loop Retains Its Unique Conformation in the Three-way Junctions. When joined to a stem of six base pairs, the DNA hairpin loop, 5’-TTTA-3’, posesses a unique conformation, resulting in several unusual proton chemical shifts (Blommers et al., 1991). We find these same chemical shift patterns in the spectra of the three-way junctions. The T27(CH3) resonance at 1.95 ppmand theA29(H1’) resonance at 6.40 ppm are two such features visible in the one-dimensional spectra of the junctions (asterisks, Figure 2). This result leads us to conclude that the unusual loop structure within the hairpin remains essentially unchanged when part of a larger threeway junction. NOESYSpectrum of J3CCin D20. In order to investigate the structure of the three-way junctions in detail, complete sequential assignments are necessary. These assignments are based on the right-handed nature of the DNA helices, which places proton pairs from neighboring residues within a short distance (
