Chem. Res. Toxicol. 1994, 7, 319-328
319
lH NMR Characterization of a Duplex Oligodeoxynucleotide Containing Propanodeoxyguanosine Opposite a Two-Base Deletion in the (CpG)3 Frameshift Hotspot of Salmonella typhimurium hisD3052 James G. Moe,tv* G. Ramachandra Reddy,s Lawrence J. Marnett,s and Michael P. Stone’vt Center in Molecular Toxicology, Department of Chemistry, and A. B. Hancock, Jr., Memorial Laboratory for Cancer Research, Department of Biochemistry, Vanderbilt University, Nashville, Tennessee 37235 Received November 19, 1993”
The exocyclic lesion l,W-propano-2’-deoxyguanosine (PdG) was incorporated into 5’-
d[ATCGC(PdG)CGGCATG]-3’, derived from the hisD3052 gene of Salmonella typhimurium. The modified oligodeoxynucleotide was annealed with the complementary strand 5’-d[CATGCCGCGAT] -3’ which contained a CpG deletion. The resulting duplex 5’-d[ATCGC(PdG)CGGCATG]-3’-5’-d[CATGCCGCGAT] -3’ required PdG and one adjacent cytosine to be unpaired. Four thymine imino lH NMR resonances were observed a t temperatures below 25 “C, which demonstrated formation of a stable duplex with a two-base bulge. PdG was accommodated within the DNA helix, whereas the 3’-neighbor cytosine was poorly stacked and appeared to be extrahelical. The sequential nuclear Overhauser enhancement connectivities between aromatic and H1’ protons along the modified strand were interrupted between PdG and the 3’aeighboring unpaired cytosine. On the complementary strand no interruptions were observed. An NOE was observed between the PdG methylene protons H8,b and the imino proton of the 5’-neighbor base pair. Weaker NOESwere observed between the PdG H8,bprotons and the imino proton from guanine two nucleotides removed in the 3’-direction, and to the amino proton of cytosine located in the complementary strand two nucleotides removed in the 3’-direction. Chemical shift perturbations were also observed for the latter cytosine as compared to the corresponding cytosine in the unmodified fully complementary duplex. These observations provided evidence for a poorly stacked or an extrahelical conformation of this unpaired cytosine. The amino proton resonances of the 3’-neighbor cytosine were not observed, presumably due to increased exchange with solvent. The methylene protons from PdG were shifted upfield relative to the monomer PdG, probably as a result of aromatic ring current shielding, consistent with an intrahelical location. Multimeric derivative oligonucleotides containing the PdG bulge migrated anomalously on nondenaturing polyacrylamide gels, consistent with a structure in which the unpaired nucleotides induced a bend in the DNA.
Introduction Frameshift mutations arise when bases are inserted or deleted in the reading frame of DNA. The frequency of frameshifts is dependent upon sequence; these mutations are often associated with iterated bases, palindromes, and tandem repeats. Several mechanisms including slipped mispairing, insertion-slippage, and formation of quasipalindromes have been proposed to explain this sequence dependence (1-5). The hisD3052 mutation, located within the histidinol dehydrogenase gene from Salmonella t y phimurium, resulted from deletion of a cytosine by ICR191 (6-8). It is reverted by additions and deletions which restore the reading frame but do not necessarily reverse the forward mutation (9). A CG1deletion in the reiterated sequence (CG)4 represents one of the most common reversion events (10,11). Malondialdehyde (MDA),2a mutagen produced endogenously in humans during lipid Center in Molecular Toxicology and Department of Chemistry. $Present address: Gene-Trak Systems, Inc., 31 New York Ave., Framingham, MA 01701. 5 Center in Molecular Toxicology and A. B. Hancock, Jr., Memorial Laboratory for Cancer Research, Department of Biochemistry. e Abstract published in Aduance ACS Abstracts, March 15, 1994. f
peroxidation and prostaglandin biosynthesis, reverts hisD3052 (12-16). Although MDA exists as its tautomer 0-hydroxyacroleinin aqueous solutions, structure-activity studies (17) suggest that both carbonyl equivalents must react to generate a premutagenic lesion that induces 1 The oligonucleotides discussed in this paper do not have terminal phosphate groups-we abbreviate the nomenclature for oligonucleotides by leaving out the phosphodiester linkage. A, C, G, T, and X refer to mononucleotide units; Xis the exocyclic 1,Wpropanoguanosine nucleotide PdG. A right superscript refers to numerical position in the oligonucleotide sequence starting from the 5’-terminus of chain A and proceeding to the 3’-terminus of chain A and then from the 5’-terminus of chain B to the 3’-terminus of chain B. So as to keep the numbering scheme in register with previous studies on d(CGCXCGGCATG).d(CATGCCGCGCG),the nucleotides have been numbered beginningwith A-2, T-l, C1, etc. C2, C5, C6, C8, Cl’, C2’, C2”, etc., represent specific etc., represent the protons carbon nuclei. H2, H5, H6, H8,Hl’, H2’, H2”, attached to these carbons. 2 Abbreviations: AAF, N-acetyl-2-aminofluorene; AF, 2-aminofluorene; NOE, nuclear Overhauser enhancement; NOESY,two-dimensionalNOE nuclear Overhauser enhancement experiment; ppm, parta per million; MDA, maMlG, 3-(~-~-ribofuranosyl)pyrimido[1,2-aIpurin-10(3H)-one; DMT, dimethoxylondialdehyde;PdG, 1,iP-propano-2’-deoxyguauosine; trityl; PdG-2BD, d(ATCGC(PdG)CGGCATG)d(CATGCCGCGAT);DSS, sodium 4,4-dimethyl-4-silapentanesulfonate; PNK, T4 polynucleotide kinase; TBE, Tris-borate-EDTA buffer; TPPI, time-proportional phase increment; TOCSY, totalhomonuclear correlated spectroscopy;lD, onedimensional; 2D, two-dimensional.
0893-228~/94/2707-0319$04.50/0 0 1994 American Chemical Society
320 Chem. Res. Toricol., Vol. 7, No. 3, 1994
Moe et al.
Chart 1. Oligodeoxynucleotides Containing Iterative CG Base-Step Repeats Derived from the Frameshift-Prone Sequences from the hisD3052 Gene of S. typhimurium
v 1
2
3
4
5
6
7
6
9
1011
5’-C G C G C G G C A T G - 3 3 - G C G C G C C G T A C-5’ 22 21 20 19 18 17 16 15 14 13 12
PdG-28Q -2-1 1
2
3
4
5
6
7
6
9
1011
5’-A T C G C X C G G C A T G-3’ 3 - T A G C G * . * * - * * * * * C C G T A C -5’ 22 21 20 19 1s
X
17 15 15 14 13 12
= PdG Adduct
frameshifts. This is consistent with formation of the pyrimidopurinone adduct designated MlG, formed from addition of 1 equiv of MDA to deoxyguanosine, which is the major DNA adduct (18-21 ):
0 MDA
bR
p-hydroxyacrolein M,G adduct
Structural studies of M1G adducts in oligodeoxynucleotides have not been possible because the adduct is not stable during automated DNA synthesis. The exocyclic adduct lJ\n-propan0-2’-deoxyguanosine(PdG) is a structural analog of M1G which is readily incorporated into oligodeoxynucleotides (22) (Chart 1) and is amenable to high-field NMR studies. Patel and co-workers, using NMR spectroscopy, observed PdG(syn).A(anti) pairing at pH 5.8, simultaneous partial intercalation of the complementary PdG and A bases at pH 8.9, and PdG(syn).G(anti) pairing which was pH-independent (23,241. The solution structures of the pH 5.8 and pH 8.9 PdGSA adducts were refined using molecular dynamics calculations which incorporated nuclear Overhauser enhancement (NOE)distance restraints (25, 26). The Patel group also established that the exocyclic ring of PdG was inserted into the DNA duplex when positioned opposite an apurinic site (27). Thermodynamic measurements, obtained from UV melting assays by Breslauer and co-workers, revealed that PdG reduced the thermal stability, transition enthalpy, and transition free energy of the duplex when positioned opposite cytosine or adenine (28). Adduct-directed mutagenesis assays performed in vitro using a noniterated repeat sequence as the template revealed base-pair substitutions to be the predominant mutation induced by PdG. However, a measurable quantity of deletions were also observed (5). When PdG was incorporated into the (CG)4repeat from the hisD3052 sequence contained within a recombinant M13 phage (M13MB102) and transformed into Escherichia coli, twobase deletions of CG were observed (29). We previously demonstrated that placing PdG within a (CpG)3 repeat
resulted in structural perturbation of a single dinucleotide repeat unit (30). High-field lH NMR analysis of that modified oligodeoxynucleotide localized the structural perturbation to the modified base pair and the neighboring base pair in the 3’-direction. The results suggested potential mechanisms involving adduct-induced slippage of two base pairs, whereby PdG could induce twonucleotide deletions in the CG iterated repeat sequence. We have constructed a PdG-modified oligodeoxynucleotide (designated PdG-2BD) to model the putative strand slippage intermediate which would precede a twobase-pair deletion in the hisD3052 sequence (Chart 1). The present work utilizes high-field lH NMR and electrophoretic mobility assays to examine the consequences of introducing a dinucleotide deletion opposite PdG located within this CG repeat sequence. lH NMR studies indicate that a single bulge conformation exists, consistent with PdG and the 3’-neighbor cytosine being unpaired. The PdG is intrahelical and stacked, whereas the unpaired cytosine is poorly stacked and appears to be extrahelical. Oligodeoxynucleotides containing this PdG bulge migrate anomalously on nondenaturing polyacrylamide gels, an observation consistent with the introduction of a bend or kink in the DNA helix.
Materials and Methods Oligodeoxynucleotide Synthesis. The unmodified oligodeoxynucleotides were synthesized by the Midland Certified Reagent Co. (Midland, TX) and purified by anion-exchange chromatography. PdG was synthesized using the methodology of Marinelli and co-workers (22). The 5’-(dimethoxytrityl) (DMT) derivative of PdG was synthesized and purified as previously described (31) and incorporated into oligodeoxynucleotidesby the Midland Certified Reagent Co. The extinction coefficients of the oligodeoxynucleotideswere calculated on the basis of nearest-neighbor analysis as 1.22 X 105M-1 cm-1 for the modified strand [5’-d(ATCGCXCGGCATG)-3’], and 1.01X 106 M-l cm-l for the complementarystrand [5’-d(CATGCCGCGAT)3’1 (32).Equimolar amounts of each strand were combined,and the resulting duplex was dialyzed (lo00MWCO, Spectrum)with 1exchangeagainst 0.6 M NaCl and 3 exchangesagainst deionized H20. The sample was lyophilized. Spectroscopy. UV melting assays were performed using a CARY 2390 spectrophotometer (Varian Associates, Palo Alto, CA) interfaced with a Neslab (Newington, NH) cryobath and temperature gradient prog-rammer. Oligodeoxynucleotides(strand concentration -6 gM) were dissolvedin 0.01 Msodiumphosphate buffer containing 1.0 M NaCl and 0.05 mM NazEDTA at pH 7.0. Melting data were collected in triplicate with a temperature rise of 1 “C/min. The temperature range was 10-80 O C . For NMR spectroscopy, the modified duplex was prepared at a concentration of 2 mM. For observation of nonexchangeable protons, the sample was dissolved in 0.5 mL of 0.01 M sodium phosphate buffer containing 0.1 M NaCl and 0.05 mM NazEDTA at pH 7.0. The sample was exchanged 3 times with 99.9% DzO and dissolved in 99.96 % DzO. For observation of exchangeable protons, the samplewas dialyzed against 50mL of 0.001 Msodium phosphate buffer containing 0.5 M NaC1,0.05 mM NaZEDTA, pH -8.4, and 9:l HzO/DzO containing Chelex (Bio-Rad Laboratories, Richmond, CA) for 3 exchanges. 1H NMR spectra were recorded at 500.139 MHz. The temperature was controlled to h0.5 “C. One-dimensional (1D) spectra in HzO were obtained using either the 1-1 or the 1-3-3-1 (33,34)binomial pulse sequences. Two-dimensional(2D)spectra were obtained by replacing the final 90° pulse of the NOESY experiment by a 1-1 binomial pulse (35,36). Convolution difference was used during processing to minimize the residual signal arising from water (37). NOESY experimentsutilized the TPPI phase sequence. TOCSY experiments used the standard pulse sequence, with a 120-ms MLEV-17 spin lock (38). Data
Chem. Res. Toxicol., Vol. 7, No. 3, 1994 321
PdG Opposite a Two-Base Deletion Table 1. Optical Melting Temperatures ("C;Strand Concentration = -6 pM) for Oligodeoxynucleotides Derived from the hisD3062 Gene of S. typhimurium abbreviation sequence T, unmodified duplex modified duplex
PdG-11-9 PdG-13-11 PdG-2BD
5'-CGCGCGGCATG-3' 3'-GCGCGCCGTAC-5' 5'-CGCXCGGCATG-3' 3'-GCGCGCCGTAC-5' 5'-CGCXCGGCATG-3' 3'-GCG-CCGTAC-5' 5'-CGCGCXCGGCATG-3' 3'-GCGCG- CCGTAC-5' 5'-ATCGCXCGGCATG-3' 3'-TAGCG---CCGTAC-S'
65
41 40
50 45
processing was carried out using FELIX 2.10 (Biosym Technologies, Inc., San Diego, CA), running on Iris workstations (Silicon Graphics, Inc., Mountain View, CA). Chemical shifts were referenced internally to DSS. Oligodeoxynucleotide Ligation and Gel Electrophoresis. Five A2m units of the oligodeoxynucleotideswere phosphorylated on the 5'-ends with T4 polynucleotide kinase (PNK) (BRL, Bethesda, MD) for 1.5 h at 35 "C. Two units of enzyme were added at time 0, and 2 units after 45 min under standard PNK buffer conditions [0.06M Tris-HC1 (pH 7.8),10 mM MgC12, 15 mM 2-mercaptoethanol,and 0.33 gM ATP in a 25-gL volume]. An aliquot containing -0.4 Am unit of the phosphorylated oligodeoxynucleotideswas then ligated with 4 units of T4 DNA ligase (BRL) at 20 O C for 2 h [0.05 M Tris-HC1(pH 7.6),50 mM MgC12, 5 mM ATP, 5 mM dithiothreitol, and 25% w/v poly(ethyleneglycol)80001. A 15%acrylamide gel [19:1acrylamide/ bis(acry1amide)lwas run with TBE running buffer (0.089 M Tris base, 0.089 M boric acid, and 0.002 M NazEDTA, pH 8) with 0.2 A2m unit of sample per well at room temperature at 10 W/cm for 12 h. The gels were stained in running buffer with 1 gg/mL ethidium bromide for 20 min followed by destaining in TBE buffer for 1 h. A pBR322 Hue111 digest was run as a standard and was used to calculate the apparent lengths of the oligodeoxynucleotides of interest.
Results Thermodynamic Stability of PdG Opposite a TwoBase Deletion. Table 1 compiles results of thermal denaturation assays for PdG-2BD and a series of related oligodeoxynucleotides. Introduction of PdG into the hisD3052 oligodeoxynucleotide resulted in a 24 "C drop in T,, from 65 to 41 "C (30). We observed that deletion of CG from the complementary strand opposite PdG did not substantially alter T , as compared to that of the fully complementary modified duplex (compare "modified duplex" with PdG-11-9). In an effort to increase the T , of the oligodeoxynucleotide containing the CG deletion, we added two C-G base pairs at the 5'4erminal; this resulted in a 10 "C increase in T,. However, the resulting oligodeoxynucleotide PdG-13-11 did not provide satisfactory lH NMR spectra, so the sequence was again modified by replacing the two terminal C-G base pairs with A.T base pairs. The resulting oligodeoxynucleotidewas named PdG-2BD (Chart 1). Figure 1s in the supplementary material shows representative melting curves for PdG2BD. The optical melting temperature (T,) of PdG-2BD was 45 f 1 "C. The temperature dependence of the base imino protons of PdG-2BD is shown in Figure 1. At 0 "C, four resonances from thymine imino protons were observed between 13.2 and 14 ppm, which indicated formation of a stable duplex on both sides of the unpaired bases. Upon raising the temperature, exchange broadening of the thymine imino protons was observed; the thymine imino protons that resonate at highest and lowest field, assigned as the
I
14.0
I
13.0
PPm Figure 1. Expanded plots of one-dimensionallH NMR spectra for PdG-2BD in HzO buffer at 5 "C (A), 15 "C (B), 25 "C (C), and 35 "C (D). terminal thymines T-l and T22,respectively, were the first to disappear from the spectrum. lH NMR Spectral Assignments for PdG-2BD. (A) NonexchangeableDNA Protons. The sequential NOE connectivities between aromatic and anomeric DNA protons at 25 "C are shown in Figure 2, panels A and B, for the modified strand and the complementary strand, respectively. The sequential NOES (39, 40) for the modified strand (Figure 2A) were uninterrupted except at the X4 C5 step, where the cross-peak between X4H1' and C5 H6 was missing. The cross-peak between C5 H1' (5.77 ppm) and G6 H8 (7.82 ppm) was weak, indicating a greater-than-normal distance between these two protons. For the complementary strand (Figure 2B) the sequential NOESwere uninterrupted. The deoxyribose spin systems were assigned using standard methods, via a combination of NOESY and TOCSY data (see supplementary material, Figure 2s). This confirmed the assignments from the NOESY experiments and enabled the complete assignment of the H2', H2" protons, 22 H3' protons (C' and C17 H3' were not assigned), and 14 H4' protons. The H5',5" protons were assigned for only the 5'-terminal nucleotides A-2 and C12. The assignments are tabulated in Table 2. (B) Exchangeable DNA Protons. To minimize buffer-catalyzed proton exchange, the sample used for assignment of the exchangeable protons was prepared in sodium phosphate buffer diluted 10-fold as compared to that for the samples in D2O-containing buffer. In addition, the NaCl concentration was increased from 0.1 to 0.5 M to minimize any contribution to exchange caused by strand dissociation (41). Figure 3 shows the assignments for the non-hydrogen-bonded (subscript a) and hydrogen-bonded (subscript b) cytosine amino protons along the D1 axis of the spectrum. The assignments were made on the basis of NOE connectivity to the respective cytosine H5 protons, which occur from 5 to 6.4 ppm along the D2 axis of the spectrum. All of the cytosine amino protons were ob-
-
322 Chem. Res. Toxicol.,
Moe et al.
VoZ. 7,No. 3,1994
Table 2. Chemical Shifts (ppm) of the Nonexchangeable Protons from PIG-2BD
A. Modified Strand
b
0
e
i"
Q
I
I
7.5
8.0 D1 (PPm
B. ComplementaryStrand b
0
H8a H6 A-2 8.28 T-' 7.45 C' 7.51 G2 7.93 c3 7.35 X4 7.66 c5 7.38 G6 7.82 GI 7.77 C8 7.39 A9 8.34 TO ' 7.14 G1' 7.89 C2 ' 7.72 A13 8.42 T14 7.16 G15 7.86 C ' 6 7.38 C7 ' 7.53 G18 8.01 C ' 9 7.38 G20 7.89 A2' 8.23 T22 7.24 a
H5 H2 H1' H2' 8.04 6.27 2.74 6.07 2.23 5.70 5.74 2.03 5.84 2.62 5.52 5.88 1.90 5.75 2.39 5.47 5.77 1.92 5.56 2.63 5.93 2.59 5.37 5.69 2.11 7.73 6.28 2.70 5.83 1.89 6.15 2.36 5.97 5.71 2.04 7.86 6.32 2.78 5.78 2.05 5.90 2.63 5.30 5.98 2.04 5.65 6.01 1.92 5.99 2.64 5.51 5.59 1.99 5.63 2.70 8.02 6.32 2.69 6.12 2.16 ~-
H2" 2.90 2.53 2.39 2.62 2.21 2.39 2.29 2.69 2.72 2.46 2.93 2.33 2.59 2.46 2.98 2.45 2.70 2.48 2.01 2.70 2.34 2.77 2.89 2.16
H3' H4' H5,5" CHa 4.89 4.31 3.84 4.92 4.29 1.41 4.98 4.72 4.85 4.88 4.90 4.99 4.89 5.04 4.86 4.68 4.73 5.04 4.71 4.99 4.82
4.13 4.16 4.13 4.21 4.16 4.19 4.08 3.75
1.52
4.21
1.45
4.21
4.96 4.84 4.12 5.01 5.01 4.46 4.53 4.04
1.50
The imidazole proton for the PdG base is designated H2.
Figure2. Expanded plot of a phase-sensitive NOESY spectrum in D2O buffer with a mixing time of 250 ms for PdG-2BD showing the sequential NOEs from the aromatic to H1' protons. (A) Connectivities are traced for the modified strand. (B) Connectivities are traced for the unmodified strand. The base positions are indicated at the intranucleotide aromatic to sugar H1' NOE cross-peak.
servable except for C5NH2. The amino protons for this position may be broadened due to exchange with solvent H20 (see Discussion). The amino proton resonances for C3 were closely superimposed with those arising from C19, but could be partially resolved at lower temperatures, as shown in Figure 3s in the supplementary material. In Figure 4, the guanine imino protons were assigned from their strong NOE connectivities to the hydrogen-bonded cytosine amino protons, observed at the bottom of the figure, and weaker NOEs to the non-hydrogen-bonded amino protons, observed at the top of the figure. The thymine imino protons were assigned on the basis of their NOEsto adenine H2 protons. At 20 "C,the imino protons for T-l and T22were broadened, and cross-peakswere only observed for the internal TIOand T14imino protons. The assignment of the guanine and thymine imino protons was confirmed by observation of internucleotide NOEs between adjacent guanine and thymine imino protons (data not shown). The assignments of the terminal imino protons (T22N3H and G1 N1H) and the imino protons adjacent to the bulge (G18 N1H) were facilitated by examiningthe exchange cross-peaksin a NOESY spectrum run in H20 buffer (the exchange cross-peaks can be seen in Figure 6 at 6 4.81 ppm). The comparison of the exchange peaks and a 1D spectrum at the same temperature are illustrated in Figure 4s of the supplementary material with
8.0
7.0
6.0
D1 ( P P 4
Figure 3. Expanded plot of a NOESY spectrum in H2O buffer for PdG-2BD showing the assignments for the exchangeable cytosine amino proton resonances, on the basis of their connectivities to the cytosine H5protons. The H5 and N4H, (nonbonded cytosine amino proton) and N4Hb (hydrogen-bonded cytosine amino proton) are indicated. the assignments of the base imino protons indicated. The assignments of the exchangeable proton resonances are tabulated in Table 3. (C)PdG Protons. Table 4 lists the assignments of protons unique to PdG. The assignment of the PdG methylene resonances was facilitated by successful observation of the exchangeable proton N5H on the PdG ring, which was located at 6.4 ppm, as shown in Figure The N5H proton showed a strong NOE to H6a and H6b, which were overlapped at 2.66 ppm. H7,b, located at 1.28 and 1.36 ppm, were assigned on the basis of strong NOEs to both H6a,b and H8a,b. It was not possible to individually assign H7a and H7b, since no NOE was observed to either of these protons. H8a and H8b overlapped at 3.28 ppm 3 The definitions of the diastereotopic protons at C6, C7, and C8 are based upon the Cahn,Ingold, and Prelog nomenclature. H6a is the pro-S proton at C6, and H6b is the pro-R proton at C6. H7a is the pro-R proton at C7, and H7b is the pro-S proton at C7. H8a is the pro-R proton a t C8,and H8b is the p r o 4 proton at C8. Protons H6a, H7a, and H8a face in the 5'-direction, whereas protons H6b, H7b, and H8b face in the 3'direction.
PdG Opposite a Two-Base Deletion
Chem. Res. Toxicol., Vol. 7, No. 3, 1994
I
PdG HBa.6
'dG N5H
14.0
I
I
I
I
13.5
13.0
12.5
6.0
Figure 4. Expanded plot of a NOESY spectrum in H20 buffer for PdG-2BD, showing the connectivities between aminoprotons, adenine H2 protons, and base imino protons.
Table 3. Chemical Shifts (ppm) of the Exchangeable Protons from PdG-2BD N1H A-2 T-1
C' G2
N3H
N4H,
N4Hb
4.0
8.53
c3 x 4
6.73
b
CS G6 G7 C8
b
b
in H2O buffer, showing the assignment of the PdG methylene protons. The exchangeable N5H proton from the propano ring is observed in a NOESY spectrum run in H20, which enables conclusive assignments for the three sets of PdG methylene protons.
A.
PdG H8 a,b
6.43
8.28
6.96
8.03
6.45 6.99
8.22 8.36
6.71
8.49
+&
12.99
G 8
I
I I I I I I
12.86 12.95
I
I
13.67 12.700
T14
I I C8
13.51 12.69
C" G18 C'9
13.03*
G20
12.93
13.0
12.0
rigure 6. Expanded plot of a NOESY spectrum of PdG-2BD
13.W
Assignment based on the exchange peak with H2O (Figure 3s). Resonance not observed due to exchange with H20. a
Table 4. Chemical Shifts (ppm) of the Exchangeable Proton N5H and the Methylene Protons from PdG in the Modified Nucleotide X4 6.4
14.0
D1 (PPm
A21
T=
x4
C17NHb tpdGH8a'b
6
A13
*
2.0
Figure 5. Expanded plot of a NOESY spectrum for PdG-2BD
A9
GlS C'6
3.0
13.76" 6.97
TO ' GI' C2 '
5.0
D l (PPm)
D1 ( P P 4
2.66
1.28,1.36
3.28
and were assigned by observation of strong NOEs to H7a,b and a weaker NOE to H6a,b. The assignments of the methylene protons were confirmed from TOCSY data (not shown). Significant increases in shielding were observed for each of the PdG methylene protons, as compared to their chemical shifts in the PdG monomer (22). H6,,b shifted upfield by -0.9 ppm, H7,b shifted upfield by -0.75 ppm, and H8,,b shifted upfield by 0.7 ppm. Several NOEs were observed between the PdG methylene protons and DNA protons. Two cross-strand NOES were observedfrom H64b to H2' (1.82 ppm) and H2" (2.01 ppm) of CY7, as indicated in Figure 5. Weak NOES were observed between H%b (3.28 ppm) and two imino protons. Figure 6A shows an NOE observed to G18 N1H (13.03 ppm) (which is on the 5'-side of the lesion) and a weaker NOE observed to G6N1H (12.86 ppm) (which is on the 3'-side of the lesion).
in H20 buffer. (A) Internucleotide NOEs between iminoprotons G18 NlH, G6 NlH, and X4 H8,b. Higher order NOEs from cytosine H5 protons to G N1H protons via the cytosine amino protons are observable at long mixing times (bottom of panel). Note the absence of an NOE transferred through the aminogroup of Cs. (B) NOE from X4H8,b to the hydrogen-bonded amino proton of Cl7 (NHb).
Chart 2. Base Sequences of the Oligodeoxynucleotides Used in the DNA Bending Assay 21mer (Control DNA)
5'-AT G T A C G C G G C A T G A C G C G G C-3' 3'-GC C G T A C A T G C G C C G T A C T G C-5' PdG 23.21mer (Bulge DNA) 5'-A T G T A C G C X C G G C A T G A C G C G G C-3' 3'-G C C G T A C A T G C G - - --C C G T A C T G C-5'
In addition, a weak NOE was seen from PdG H84b to C17 NHb (8.36 ppm), the hydrogen-bonded amino proton, shown in Figure 6B. Electrophoretic Mobility of Oligodeoxynucleotide Multimers. Chart 2 shows the sequences of two oligodeoxynucleotide multimers, derived from PdG-2BD. The
324 Chem. Res. Toxicol., Vol. 7,No. 3, 1994
Moe et al.
1 2 3 4
-
DNA duplex. The largest fragment migrated with an 2). apparent size almost twice the actual size (12
Discussion
dimer
+
monomer-
0
0
0 0
*
11
0
I
I
I
I
2
4
6
8
10
Multiples of Monomer
Figure 7. Nondenaturing polyacrylamide gel electrophoresisof the ligation ladders for the control DNA and for the PdG-2321-mer (bulge DNA). (Top) A 15%acrylamide gel was run with TBE running buffer at room temperature. Lanes 1 and 4, molecular weight standards (pBR322 Hue111 digest); land 2, ligation ladder from the control DNA; lane 3, ligation ladder of the PdG bulge DNA. (Bottom) Plot of the apparent size us the actual size for each of the oligodeoxynucleotides.
control ligation utilized a fully complementary 21-mer sequence, as shown in the top panel. The PdG-23-21-mer (bottom panel) contained two unpaired bases at the lesion site,spaced at 21-base-pairintervals (2 helicalrepeat units). Figure 7A shows the results of nondenaturing PAGE of the respective ligation ladders. The electrophoretic mobility of the bulge multimers (lane 3) was strongly retarded as compared to the control multimers (lane 2) which contained no bulge. The apparent length of the fragments was calculated relative to the migration of the molecular markers (pBR322 HaeIII digest, lanes 1and 4). The plot of the apparent length/actual length (k) us multimeric number is illustrated in Figure 7B. For the control multimers, k remained at a value of 1-1.2, whereas for the PdG-2BD multimers, the value of k increased from 1to -1.8 as a function of multimeric unit. No plateau was observed for the longer fragments (up to n = 7), indicating that the bulges were in phase with the helical repeat of the
The alternating (CG)4 repeat sequence from the hisD3052 gene represents a hotspot for adduct-induced frameshift mutations, commonly observed to be deletions of CG (I0, I I,29). These mutations could occur via adductinduced strand slippage at the primer template complex. We previously reported structural studies on PdG inserted into this sequence, in which the modified guanine was located opposite cytosine in the complementary strand (30). At pH 5.8, PdG was oriented such that the propano moiety faced into the major groove. PdG was in the syn conformation at the glycosyl bond and formed a Hoogsteen base pair with N3-protonated cytosine. lH NMR revealed the orientation of PdG to be dependent upon pH. This extended work by Pate1 and co-workers, who first demonstrated rotation of PdG from the anti conformation to the syn conformation about the glycosyltorsion angle (2325). One intriguing finding in our previous study was the observation of a second pH-dependent conformational equilibrium, in which the 3’-neighbor base pair equilibrated between Hoogsteen and Watson-Crick base-pairing. The exocyclic lesion thus perturbed two base pairs, which represented a single repeat unit in the iterated (CpG)3 sequence (30). The PdG-2BD oligonucleotide was designed from the same sequence of the hisD3052gene as used in the previous study, but it places the lesion opposite a two-base deletion (Chart 1),the putative frameshift mutagenesis intermediate (29,30). PdG-2BD differsfrom the actual replication complex, which does not contain a fully complementary duplex in the 3’-direction from the putative deletion site. Two A.T base pairs were added to the 5’-terminus of the oligodeoxynucleotide. Initial NMR studies on the corresponding oligonucleotides lacking the A*T base pairs revealed broadened resonances, indicative of equilibria involving multiple conformational states. While these equilibria were not characterized, they were believed to result from slippage of the iterated repeat sequence, resulting in formation of a 5’ overhanging end, possibly stabilizedvia formation of a protonated PdG(syn)C+(anti) Hoogsteen base pair at the lesion site. Inclusion of the terminal A=Tbase pairs eliminated this problem. Conformation of the Unpaired Nucleotides. The observation of four thymine imino protons demonstrated that PdG-2BD formed a stable duplex with two unpaired bases (Figure 1).An intrahelical orientation for PdG was established from NOE data. An NOE was observed from the methylenic protons H8a,b of X4to the imino proton of G18,which is located in the 5’-direction. Additionalweaker NOEs were observed to the imino proton of G6 (Figure 6A) and to C17 NH2 (Figure 6B), both located in the 3’direction. Cross-strand NOEs were also observable from X4 H6a,b to C17 H2’ and H2” (Figure 5). The NOEs were consistent with the glycosyl torsion angle of PdG being in the anti conformation. A NOESY experiment with a mixing time of 50 ms was run to determine whether any of the glycosyl torsion angles were in the syn conformation; no evidence for this was observed. Chemical shift data also supported an intrahelical location for PdG. The methylene protons were shifted upfield relative to the PdG monomer; these upfield shifts were as large as 0.9 ppm for H6a,b, as shown in Figure 8. This large upfield shift was attributed to ring current
Chem. Res. Toxicol., Vol. 7, No. 3, 1994 325
PdG Opposite a Two-Base Deletion 0.7
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El PdG monomer - PdG-2BD @ 75 O C
Figure 8. Chemical shift changes for the propano methylene protons relative to their chemical shifts in the monomer. A6 values represent differences in chemical shift as indicated in the legendbelow the figure. Solutionconditions: monomer dissolved in CDaOD;for the modified singlestrand 0.01 M phosphate buffer, 0.1 M NaC1, and 0.05 mM NaZEDTA, pH 7.0 at 55 O C ; for the modified oligodeoxynucleotide,0.01 M phosphate buffer, 0.1 M NaC1, and 0.05 mM NaZEDTA, pH 5.8 at 10 O C ; and for PdG2BD, 0.1 M NaCl and 0.05 mM NazEDTA, pH 7.0 at 25 O C , and 75 O C . Scheme 1. Possible Base-Pairing Orientation for PdG-2BD C3X4 bulge X4C5 bulge 2 3 4 5 6 1
...G C X
C G G...
19 18
11 16
...C G
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effects from the neighboring base pair; similar effects were observed for methylene protons from the PdG(anti)-A(anti) base pair at high pH (24). In contrast, the PdGtsyn). A(anti) (at low pH) (24) and PdG(syn)-Wanti) (30) adducts in which PdG was in the syn conformation about the glycosyl bond placed the methylene protons in the major groove,where they were not observed to shift upfield. The chemical shifts of the methylene protons in the modified single strand [5'-d(CGCXCGGCATG)-3'] and in PdG-SBD, under denaturing conditions (spectra run at high temperature), were similar to those of the monomer PdG (22, 29). For an iterative repeat sequence in which the complementary strand contains a deletion of one dinucleotide repeat unit, two possible base-pairing orientations are possible, which result in either a C3X4 or an X4C6bulge (Scheme 1). Exchange between a C3X4and an X4C5bulge represents the shortest possible limit of "bulge migration", proposed to stabilize strand slippage in iterated repeats (42). Only one set of resonances was observed in the lH NMR spectra at 25 OC. The line widths of the C3 and C6 H5 and H6 resonances were comparable to other cytosines in the oligodeoxynucleotide. This was consistent with a single conformation for the unpaired bases, or a fast average between two structures. If rapid bulge migration occurred between C3X4and X4C5,one might predict the observation of transferred NOEs, with intensity weighted by the position of the equilibrium and the inverse sixth power of the respective internuclear distances for each of the potential conformations. Instead, the NOES were con-
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Figure 9. Chemical shift changes (A6ppm) of selected protons from PdG-2BDrelativeto the unmodified duplexwhere positions 1-20 are compared (see Chart 1). (A) Chemical shift differences for the aromatic H8 (open bars), H6 (solid bars), and H5 (crosshatched bars) proton resonances. (B)Chemical shift differences for the sugar H1' (open bars), H2' (solid bars), and H2" (crosshatched bars) proton resonances. sistent with X4C5being unpaired, or alternatively, rapid equilibrium between unpaired C3X4 and X4C6 favoring the latter, as shown in Scheme 1. The NOEs from X4H8,b to two imino protons, G18N1H and G6 NlH, and to the hydrogen-bonded amino proton of C17 (Figure 6) were diagnostic of an unpaired conformation for X4C5. The observation of these NOEs argues that G18 was base paired with C3 and not Cs; in the case of C5-Gl8base-pairing, an NOE between X4 H8,b and G6 N1H would not be predicted. This conclusion was corroborated by the failure to observe the NH2 protons of C5. These were presumed to be broadened by chemical exchange with H20, consistent with an extrahelical location for this nucleotide. In contrast, the C3 NH2 protons were observable, although they were partially superimposed with those of C19 in the spectra shown in Figures 3 and 6. In Figure 6A, a second-order NOE was observed between G18 N1H and C3 H5, consistent with C3-G18base-pairing. This pattern of NOESwas consistent with the observation that the sequential NOESY connectivities were interrupted between X4 and C5 (Figure 2A). There was an NOE cross-peak from C5 H1' to G6 H8, but it was quite weak, indicating a greater-than-normalseparation between these two protons. The chemical shift perturbations observed for PdG2BD at 25 "C are shown in Figure 9.4 For the aromatic protons, the chemical shifts showed differences at both C3 and C6 on the modified strand, and C17 and C19 on the unmodified strand (Figure 9A). The magnitude of these chemical shift changes, however, was of the order of those seen at positions C1 and G20 (A6 I 0.2 ppm) as a result of the differences in flanking sequences for the two oligodeoxynucleotides (the unmodified oligodeoxynucleotide contains a C.G base pair adjacent to G20, whereas PdG4 Base positions 1-20 are compared. Chemical shift differences at C1 and Gm are due to the presence of the flankng sequences A-2T-1 and in the PdG-2BD oligodeoxynucleotide.
A21T22
326 Chem. Res. Toxicol., Vol. 7, No. 3, 1994
Moe et al.
2BD contains flanking A-T base pairs). In a C3X4bulge, C19 would be adjacent to the bulged bases, C17 would be two base pairs removed, and the deoxyribose of G18would be located across from the bulge. In the X4C5bulge, the C17 base would be adjacent to the bulge and the deoxyribose would be located opposite the bulge. For the anomeric protons, pronounced chemical shift perturbations were observed at CI7,on the complementary strand. C17H1’ was shifted downfield by almost 0.5 ppm, and C17 H2’ and H2” were shifted upfield by 0.1 and 0.3 ppm, respectively (Figure 9B). In contrast, no changes greater than 0.1 ppm were observed for the corresponding protons of C19or Gl8. The large chemical shift perturbations seen for C17 Hl’, H2’, and H2” were supportive of an X4C5 bulge. Chemical shift data also showed small downfield changes for C5 H5 and H6. Observation of an X4C5 bulge and not a C3X4 bulge (Scheme 1) is noteworthy. In our previous NMR study which located PdG in the fully complementary hisD3052 oligodeoxynucleotide opposite cytosine, it was also base pairs X4C5 and not C3X4which were perturbed (30). This observation suggests the possibility that this pattern of structural perturbation may be characteristic of PdG adducts embedded within this iterated repeat sequence. Perhaps it reflects more favorable base-staqking interactions in the case of the X4C5 bulge as compared to the C3X4 bulge. Alteration of Electrophoretic Mobility. Insertion of one or more unpaired bases into the B-form helix would be predicted to induce a deformation of the double helix (43-46). To probe for such a structural perturbation, the electrophoretic mobilities of a series of multimeric oligonucleotides derived from the PdG-2BD sequence were investigated. The PdG-23-21-mer contains the lesion opposite a two-base deletion such that when ligated into multimers, the unpaired bases are a t 21-base-pair intervals (two helical repeat units). The corresponding control oligonucleotide (21-mer control), contains the same sequence without any unpaired bases. The electrophoretic mobility was retarded in the multimeric oligonucleotides which incorporated the two-base-pair deletion opposite PdG. This was consistent with the notion that the PdG bulge induced a perturbation in the DNA helix (k 2). Previous reports have indicated unpaired bases induce DNA bending (43-45). Previous Studies of Bulged Nucleotides. The PdG2BD oligonucleotiderepresents the fint case in which PdG forms part of a bulge, although the Pate1group previously examined PdG opposite an apurinic site (27). Oligodeoxynucleotides containing unpaired bases or bulges of various sequence context and length have been examined using both NMR spectroscopy (28, 46-52) and X-ray crystallography (53). Thermodynamic measurements examined A-T-rich DNA containing unpaired bases (54, 55). DNA successfully accommodates unpaired bases in both intrahelical and extrahelical environments (511,the conformation being dependent both upon the identity of the unpaired base and upon the specific DNA sequence flanking the site (50). Unpaired purines generally adopt an intrahelical conformation in solution as evaluated by NMR spectroscopy (56, 5 3 , although in one instance crystallographic studies reported an extrahelical orientation for an unpaired adenine (53). For unpaired pyrimidines both intra- (58,591 and extrahelical conformations (48-50,60) have been observed. The recent observation that three additional bases can simultaneously adopt an
-
S-CGCXCGGCATG-3 CGCCGTAC-5’
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Figure 10. Possible mechanisms for two-base deletions involving adduct-induced strand slippage of a DNA template strand.
intrahelical conformation illustrates the conformational flexibility of B-form DNA (46, 51). Models for Strand-Slippage Deletions. Adductinduced frameshifts that arise in the (CG)4repeat sequence of hisD3052 are consistent with mechanisms whereby the modified guanine and adjacent cytosine undergo transient strand slippage. Figure 10 illustrates two mechanisms whereby this could occur. In the Streisinger model (I), pausing of the replication apparatus at the lesion site enables slippage,resultingin the formation of two unpaired nucleotides,from which DNA polymerization can continue. More recently, Kunkel and co-workers demonstrated that insertion of a nucleotide opposite the lesion could also occur prior to strand slippage;in that instance, the slippage occurs as a result of failure to extend the primer a t the site of the modified nucleotide (3). Thus, response to PdG in the template could affect the kinetics of either nucleotide insertion or strand extension, or both. Structural studies cannot distinguish between these two mechanisms, but do provide a basis for understanding how transient strand slippage can be accommodated by the double helix. The relative frequency of adduct-induced deletions at particular sites in DNA as well as the precise mechanism by which they occur is modulated by multiple factors, including adduct structure (5,61-63),DNA sequence (64, 65),and the identity of the particular polymerase or repair enzyme (5,661. The N-acetyl-2-aminofluorene(AAF) and deacetylated 2-aminofluorene (AF) adducts at C8 of deoxyguanosine are perhaps the most studied DNA adducts in the context of two-base deletions. The acetylated adduct produces frameshifts, while the deacetylated product produces point mutations ( 5 , 6 1 4 3 ) . The AAF adduct only caused deletions in vitro in the presence of the Klenow fragment of E . coli DNA polymerase I lacking 3f+5’-exonuclease activity, suggesting that the proofreading ability of the enzyme acted to suppress frameshift mutations. DNA sequence modulates the induction of two-base deletions by AAF.5 Independent investigations established that the frequency of two-base deletions correlated with the identity of the nucleotide inserted at the adduct site and the identity of the 5’-neighbor nucleotides. Two-base deletions were promoted by the sequence [5’-d(GCGC)-3’], when AAF was located at the second guanine in the dinucleotide repeat, where the 5‘5 DNA sequence can also influence base-pair substitutions. Adducts of styrene oxide at adenine N6 in the codon 61 rm protooncogenesequence (70) showed dramatic differences in base-pair substitution mutations dependingupon position (71). The (S)-styreneoxide diastereomerinduced A G transitions at the f i t adenine in the codon 61 sequence, but when moved one nucleotide downstream, the same adduct was found to be nonmutagenic. Similarly, the (R)-styrene oxide adduct at adenine N6 waa found to create a lethal block to replication a t the fmt adenine in codon 61,but when moved one nucleotide downstream, waa also found to be nonmutagenic.
-
PdG Opposite a Two-Base Deletion flanking sequence was GC (5). Likewise, deletions in the NurI restriction sequence [5’-d(GGCGCC)-3’1 only occurred when AAF was located at the third guanine (64, 65). Both observations are consistent with the transient slippage model formulated by Kunkel and co-workers (3, 67),which postulates enhanced slippage due to difficulty in extending polymerization from the adducted nucleotide in the template strand. For the AF adduct opposite adenine in the complementary strand, NMR structural studies revealed that rotation of the glycosyl bond of the adducted guanosine from anti tosyn occurred, thus placing AF into the minor groove (68). A different study of an AAF adduct opposite cytosine caused similar rotation of the modified deoxyguanosine from anti to syn, with the predominant conformation having the AAF moiety stacked into the helix and the guanosine base displaced into the major groove (69). Those studies established that AF and AAF adducts can induce different conformational perturbations into DNA. Whether those conformational differences were caused by the differences in the structure of the two adducts, or by different DNA sequence contexts, or both factors, is not established. Recent studies on sitespecificallymodified oligodeoxynucleotidescontaining the dG-(+)- and dG-(-)-anti diastereomers of benzo[alpyrene at the exocyclic amino group of guanine have also revealed one-, two-, and three-base deletions which may be explained by transient strand slippage (66). Our NMR studies reveal the formation of a stable structure in which PdG is inserted opposite a two-base deletion in the complementary strand. We find that this dinucleotide bulge containing PdG results in a localized structural perturbation, involving the modified base pair and its 3’-neighbor base pair. Structural distortions involve both the modified and complementary strand. The data indicate that PdG adopts an anti conformation with respect to the glycosidic bond and that it is intrahelical. In contrast, the unpaired 3’-neighbor cytosine adopts a poorly stacked or extrahelical conformation. The bulged bases induce an apparent bend or kink in the DNA as evidenced by alteration of electrophoretic mobility in nondenaturing gels. The refinement of the solution structure of this modified oligodeoxynucleotide is currently in progress and should provide a more quantitative determination of the stacking patterns and overall structural perturbations involved in the X4C5 bulge.
Acknowledgment. This work was supported by NIH Research Grants CA-55678 (M.P.S.) and CA-47479 (L.J.M.) and NIH Instrumentation Grant RR-05805 (NMR spectrometer). Additional support for laboratory core facilities, including NMR spectroscopy, was provided by the Vanderbilt Center Grant in Molecular Toxicology, ES-00267. Supplementary Material Available: UV melting profiles of PdG-2BD (Figure Is), an expandedplot of aTOCSY spectrum of PdG-2BD showing sugar spin systems (Figure 2s), an expanded plot of a NOESY spectrum in H20buffer of PdG-2BD showing t h e partial resolution of the C3NH,-C3H5 and C19NHa-C19H5 cross-peaks (Figure 3s), a n d a comparison of the exchange crosspeaks from a NOESY spectrum in H2O buffer with a 1D spectrum also in H2O buffer for PdG-2BD (Figure 4s) (4pages). Ordering information is given o n a n y current masthead page.
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