J. Am. Chem. Sot 1992,114,6562-6563
6562
I.
Quantitative evaluation of the N, effect requires introducing position-dependent a values into models of the helix-coil equiA rough estimate can be obtained by assuming that libri~m.~.'~ the Nap interaction affects a,the helix nucleation constant, rather than s, the helix propagation constant: and fitting a new a value to the CD data.14 Substitutionsin the middle of the sequence are taken to reflect changes in s values, rather than a.14 Table I shows that Ala stabilizes a helix more than Ser by 0.55 kcal/mol in the middle, as in other models,2b*c while Ser is 0.74 kcal/mol more stable than Ala at the N,, position. The availability of a and s values specific for the N, C, and middle positions of a-helices should lead to improved predictions of helix structure.'
,
5 ' -d (AATTCGCGCGC)-3 ' Und.c"
assembly
5'-d( 3'-d(
insertion/ ligation
...AATTCGCGCGC
...TTAA
AATTCGCGCGC AATTCGCGCGC AATT. . . ) CGCGCGCTTAA CGCGCGCTTAA CGCGCGCTT A A . . . ) M-88wfnbbd undrcrmr 1. m'dGTP. !3equenase 2. T4 DNA ligase.ATP
...A A T T C G CUG C G C m T T C G C0G C G C m T T C G CU G C G C m T T ...)
3'-d(
...TTmCGCGCGCTTqCGCGCGCTTA4ECGCGCGCGCTT A A . . . polydado" Fnu D II endonuclease
)
i 0
B
5 ' -d (CGCEAATTCGCG) 3'-d ( G C G C T T A W G C )
Acknowledgment. This research was supported by grants from the NIH (GM 40746) and the NSF (DMB 88-15998). P.C.L. is an NRSA postdoctoral fellow of the NIH (GM 14803). N.J. was supported by a MOST summer research fellowship.
DNA
ligase (14) Gans, P. J.; Lyu, P. C.; Manning, M. C.; Woody, R. W.; N. R. Biopolymers 3991, 31, 2283.
-
5'-d(
Kallenbach,
-
H
O.Jo'
0
O."p ,O-y-O-y-OH
9
0- 0-
);. m'dGTP
'"c6 A
II
Aberrantly Methylated DNA Site-Specific Introduction of N7-Methyl-2'-deoxyguanosine into the Dickerson/Drew Dodecamer Khosro Ezaz-Nikpay and Gregory L. Verdine* Department of Chemistry Harvard University Cambridge, Massachusetts 02138 Received March 25, 1992
Whereas the products of enzymatic DNA methylation are essential for proper genetic function, most products of nonenzymatic DNA methylation are genotoxic.' Adducts arising from aberrant methylation are believed to present a persistent challenge, since most organisms maintain high concentrations of a methylating agent2*3-S-adenosyl-~-methionine-in the solution containing their DNA? The predominant adduct, N7-methyl-2'deoxyguanosine (m7dG),5v6remains poorly understood at the
RpO
-
m7dG: R,, R, = H or DNA m7dGTP: R, = HP,OS, R2 = H
molecular level, in part because it has heretofore not been incorporated into DNA site-specifically. M7dG is not only encountered frequently as a biological lesion but is also widely used as a probe for specific protein-DNA interaction^;^.^ this has in-
* To whom correspondence should be addressed.
(1) Topal, M. D. Carcinogenesis 1988, 9, 691-696. (2) Rydberg, B.; Lindahl, T. EMBU J. 1982, I , 21 1-216. (3) Barrows, L. R.; Magee, P. N. Carcinogenesis 1982, 3, 349-351. (4) See also: Vaughan, P.; Sedgwick, B.; Hall, J.; Gannon, J.; Lindahl, T. Carcinogenesis 1991, 12, 263-268. (5) Lawley, P. D.; Brooks, P. Biochem. J. 1963,89,127-138. (6) Singer, B.; Grunberger, D. Molecular Biology of Mutagens and Carcinogens; Pleunum Press: New York, 1983. (7) Gilbert, W.; Siebenlist, U. Proc. Natl. Acad. Sci. U.S.A. 1980, 77, 122-1 26.
T Figure 1. Synthesis of a Dickerson/Drew dodecamer specifically mod-
ified with m7dG. (a) Reaction scheme. Arrows denote sites at which blunt-ended cleavage yields the desired product. Boxes directly under sequence denote overlapping FnuDII sites: unmodified site (unshaded); m7dG-containing sites (shaded). All oligonucleotides shown in this scheme and elsewhere in the text possess a 5'-phosphoryl substituent; 3'-phosphoryl substituents, if any, are shown explicitly. (b) Structure of the single-nucleotide gaps into which an m7dG nucleotide unit (bold structure) is inserted enzymatically. The orientation of the strands is reversed from that shown in part a. Arrows denote atoms that are joined by the enzymes indicated. Table I. Thermal Denaturation of Duplex Oligonucleotidesa 2 ssDNA s dsDNA
T,,,(OC) AGO AHO T U o sequence 5'-d(CGCGAATTCGCG) 63.0 -14.7 -66.8 -52.1 58.5 -14.9 -74.5 -59.6 5'-d(CGCCAATTCGCG) a AGO, AHo, and T U o are in units of kcal&l-'. T = 298 K.
tensified the need for its chemical and structural characterization in DNA. Here we report the site-specific insertion of m7dGinto a prototypical duplex oligodeoxynucleotide, the Dickerson/Drew d~decamer.~ An undecamer, 5'-d(AATTCGCGCGC), was designed to self-assemble (by base-pairing) into a multimeric array with single-nucleotidegaps (Figure la). The gaps possess all of the functionality required for enzymatic insertion of a single m7dG residue: 3'-hydroxyl and 5'-phosphate groups, to participate in phosphodiester linkages with an m7dG nucleotide unit (in bold); and a cytosine (on the opposite strand) to Watson-Crick pair with m7dG(Figure 1b). Incubation of 5'-d(AATTCGCGCGC) with m7dGTPI0and modified T7 polymerase' ("Sequenase") afforded the dodecamer 5'd(AATTCGCGCGCQ (c = m7dG). This was ligated to yield a polydodecamer (Figure la) containing repeating units of the desired product; all that remained was to cleave the (8) Hayashibara, K. C.; Verdine, G. L. J. Am. Chem. Soc. 1991, 113, 5104-5 106. (9) Drew, H.R.; Wing, R. M.; Takano, T.; Broka, C.; Tanaka, S.;Itakura, K.;Dickerson, R. E. Proc. Natl. Acad. Sci. U.S.A. 1981, 78,2179-2183. (IO) Hendler, S.; Fiirer, E.; Srinivasan, P. R. Biochemistry 1970, 9, 414 1-4 153. (1 1) Tabor, S.;Richardson, C. C. J. Biol. Chem. 1989,264,64476458.
0002-7863/92/ 15 14-6562%03.00/0 0 1992 American Chemical Society
J . Am. Chem. SOC.1992, 114, 6563-6564
6563
+
+
polydodecamer at a specific site in each repeating unit (Figure A [4 31 Transition State for a [4 21 Cycloaddition. la, open arrows). The restriction endonuclease FnuDII catalyzes A New Secondary Orbital Interaction in Diels-Alder blunt-ended cleavage at 5’-d(CGCG) sites, three of which overlap Reactions in each repeating unit (Figure l a ) of the polydodecamer: two Daniel A. Singleton possess m7dG residues (shaded), whereas one is native (unshaded). FnuDII cleavage took place only at the unmodified site, thereby Department of Chemistry, Texas A&M University generating t h e Dickerson/Drew dodecamer, 5’-dCollege Station, Texas 77843 (CGC&L4TTCGCG).12-’4 The presence and amount of m7dG Received April 17, I992 in the dodecamer were confirmed by nucleoside composition Recent reports from this laboratory have detailed the excepanalysis, and its location within the sequence was confirmed by tional and unusual characteristics of Diels-Alder reactions of specific cleavage at the m7dG site with piperidine.I4 vinylboranes.’ We describe here some surprising results of a b In thermal denaturation experiments, the duplex half-melting initio calculations of transition structures for these reactions which transition (T,)of the Dickerson/Drew dodecamer was lowered shed light on the unusual reactivity and selectivity of vinylboranes slightly by the presence of m7dG (Table I). However, analysis and suggest the importance of a new secondary orbital interaction in terms of AGO, A I - Z O , and AS’ revealed that, at room temperfor Diels-Alder reactions in general. ature, duplex stability in 5’-d(CGCGAATTCGCG) is indistinFully optimized endo and exo transition structures for the guishable from that in 5’-d(CGCGAATTCGCG). Moreover, the reactions of butadiene with vinylborane and vinyldimethylborane enthalpy (AH”)of duplex formation was made significantly more were located in ab initio R H F and MC-SCF2 calculations as shown favorable by the presence of m7dG, suggesting that the m7dGdC in Table I. The most striking result of these calculations is the base-pair is more stable than dGdC.IS The less favorable entropy [4 atom 3 atom] character of the endo transition structures change (ASo)for denaturation of 5’-d(CGCCAATTCGCG) is (1-3). In each case, C I is closer to the boron atom (B) than to more difficult to interpret, but may arise from effects on solvation c6. The bonding of C1 and B in 1-3 is apparent from positive and stacking interactions.I6 Whatever their origins, the small Mulliken overlap populations and pyramidalization of C1 toward magnitude of these thermodynamic effects strongly suggests that B instead of C6.3 In sharp contrast, the exo transition structures m7dG does not significantly perturb duplex DNA structure. By (4 and 5) are [4 + 21 in character: c, is closer to c6 than to B, way of comparison, N6-methylation of adenines in DNA reduced and the c&-cS-c6 dihedral angles are only -5.9’ and -4O, T,,, by 6-18 OC,I7 yet was shown by X-ray crystallography to have compared to 19.8’, 18.0°, and 16.7’ for 1-3, respectively. a negligible effect on duplex structure.I8 Although the activation energies at the 6-31G*//3-21G level We have found that m7dG is stable for days in duplex D N A are high by -25 kcal, the predicted endo selectivity and relative at 25 “ C (half-life > 1000 h; K.E.-N., unpublished), which reactivity of vinyldimethylborane correlate well with experimental contrasts with the reported instability of the free n u c l e ~ s i d e . ~ - ~ ~ observations. Calculations correctly predict that butadiene should This observation leads us to conclude that the probe moiety in react faster with vinyldimethylborane than with acrolein (AAGtmld dimethyl sulfate7 and template-directed8 interference footprinting = 2.9 kcal: kre1298= 405), but slower than with maleic anhydride is m7dG and not a product of its decomposition. Furthermore, (AAG*cald = -1.6 kcal,6 k1,1298= 0.067) or Lewis acid-acrolein these findings are consistent with the observed persistence of m7dG complexes,s despite no allowance for solvent and entropy effects residues in genomic DNA.I9 Repair proteins specific for m7dG or zero-point energies. The calculations also predict well the endo are although poorly understood mechanistically. selectivity of vinyldimethylborane (AAGtaId = 2.3 kcal, endo:exo The modified dodecamer reported here now provides a homoge= 9 5 5 with piperylene). These results suggest that the structural neous substrate for studies of recognition and repair by these factors responsible for the relative reactivity and endo selectivity proteins. of vinyldimethylborane are reflected in the calculations (Chart The methodology reported herein should be useful for gener11.9 ating specifically modified DNA molecules that could not be The preference for endo [4 31 and exo [4 21 transition accessed by prior methods.22 structures seems fairly rigid. When the c&-cs-c6 dihedral angle of the ex0 vinylborane transition structure was fixed at 15O, Supplementary Material Available: Detailed experimental the resulting [4 31-like structure (after reoptimization) was 3.3 procedures, polyacrylamide gel electrophoretic analysis of reaction kcal higher in energy at the RHF-3-21G level. An attempt to products, and an HPLC trace showing the results of nucleoside obtain a [4 + 21-like endo transition structure by fixing the composition analysis on 5’-d(CGCGAATTCGCG) (6 pages). c3
