Investigation of the Influence of Cytosine Methylation on DNA Flexibility
1995; Elsevier BV; Volume: 270; Issue: 1 Linguagem: Inglês
10.1074/jbc.270.1.197
ISSN1083-351X
AutoresYvonne Hodges-Garcia, Paul J. Hagerman,
Tópico(s)RNA modifications and cancer
ResumoTo test the influence of pyrimidine methyl groups on DNA flexibility and helix repeat, two sets of 14 mixed sequence DNA molecules, spanning a range of lengths from 158 to 180 base pairs, were cyclized with T4 DNA ligase. The two sets differed only in that the Cyt-5 positions of all cytosines (80-90 cytosine residues per molecule) were fully methylated in the members of one set. Determination of the molar cyclization factors, persistence lengths, helix repeats, and torsional elastic constants revealed no significant differences between the two sets. These results imply that, at least for mixed sequence DNA, the biological consequences of cytosine methylation are likely to derive from either local structural distortions in the helix, which do not propagate as altered twist, or from direct protein-methyl cytosine interactions. To test the influence of pyrimidine methyl groups on DNA flexibility and helix repeat, two sets of 14 mixed sequence DNA molecules, spanning a range of lengths from 158 to 180 base pairs, were cyclized with T4 DNA ligase. The two sets differed only in that the Cyt-5 positions of all cytosines (80-90 cytosine residues per molecule) were fully methylated in the members of one set. Determination of the molar cyclization factors, persistence lengths, helix repeats, and torsional elastic constants revealed no significant differences between the two sets. These results imply that, at least for mixed sequence DNA, the biological consequences of cytosine methylation are likely to derive from either local structural distortions in the helix, which do not propagate as altered twist, or from direct protein-methyl cytosine interactions. INTRODUCTIONThe Cyt-5 methylation of cytosine bases in DNA is known to play an important role in gene expression, in part through its modulation of transcriptional activation(1.Cedar H. Cell. 1988; 53: 3-4Abstract Full Text PDF PubMed Scopus (727) Google Scholar, 2.Cedar H. Razin A. Biochim. Biophys. Acta. 1990; 1049: 1-8Crossref PubMed Scopus (150) Google Scholar). Moreover, cytosine methylation is believed to contribute to both inactivation of the X chromosome (3.Migeon B. Am. J. Med. Genet. 1993; 47: 685-686Crossref Scopus (3) Google Scholar) and genomic imprinting(4.Li E. Beard C. Janenisch R. Nature. 1993; 366: 362-365Crossref PubMed Scopus (1743) Google Scholar), wherein the phenotype of a particular mutant allele depends on the parent of origin of the allele. As an example of the latter, the fragile X syndrome(5.Hagerman R.J. Silverman A.G. Fragile X Syndrome: Diagnosis, Treatment, and Research. Johns Hopkins University Press, Baltimore1991Google Scholar), the leading stably heritable form of mental retardation, is believed to express its most severe phenotype only when an abnormally expanded triplet-repeat sequence (CGG)n is fully methylated (6.McConkie-Rosell A. Lachiewicz A.M. Spiridigliozzi G.A. Tarleton J. Schoenwald S. Phelan M.C. Goonewardena P. Ding X. Brown W.T. Am. J. Hum. Genet. 1993; 53: 800-809PubMed Google Scholar, 7.Hagerman R.J. Hull C.E. Safanda J.F. Carpenter I. Staley L.W. O'Connor R. Seydel C. Mazzocco M.M. Snow K. Thibodeau S. Kuhl D. Nelson D.L. Caskey C.T. Taylor A. Am. J. Med. Genet. 1994; 51: 298-308Crossref PubMed Scopus (181) Google Scholar, 8.Vincent A. Heitz D. Petit C. Kretz C. Oberle I. Mandel J. Nature. 1991; 349: 624-626Crossref PubMed Scopus (173) Google Scholar) on an active X chromosome. In addition to the functional consequences of cytosine methylation, it is known that methylation can influence the formation of DNA cruciforms (9.Murchie A.I. Lilley D.M.J. J. Mol. Biol. 1993; 205: 593-602Crossref Scopus (33) Google Scholar) and triple helices(10.Xodo L.E. Manzini G. Quadrifoglio F. van der Marel G.A. van Boom J.H. Nucleic Acids Res. 1991; 19: 5625-5631Crossref PubMed Scopus (203) Google Scholar, 11.Strobel S.A. Dervan P.B. Nature. 1993; 350: 172-174Crossref Scopus (129) Google Scholar). Methylation can also modulate the B to Z transition in DNA (12.Zacharias W. Jost J.P. Saulz H.P. DNA Methylation: Molecular Biology and Biological Significance. Birkhauser Verlag, Basel, Switzerland1993: 27-38Crossref Scopus (29) Google Scholar) as well as the apparent curvature of DNA containing A-tracts(13.Hagerman P. Biochemistry. 1990; 29: 1980-1983Crossref PubMed Scopus (38) Google Scholar, 14.Hodges-Garcia Y. Hagerman P. Biochemistry. 1992; 31: 7595-7599Crossref PubMed Scopus (56) Google Scholar).The physical mechanisms by which cytosine methylation affects gene expression (or even DNA structure) are unknown. It has been demonstrated that methylation stabilizes Z-DNA and changes the helix twist of poly(dG•dC) copolymers from 10.5 to 10.7 base pairs (bp) 1The abbreviations used are: bpbase pair(s)PCRpolymerase chain reaction.per turn(12.Zacharias W. Jost J.P. Saulz H.P. DNA Methylation: Molecular Biology and Biological Significance. Birkhauser Verlag, Basel, Switzerland1993: 27-38Crossref Scopus (29) Google Scholar). Moreover, an x-ray crystallographic analysis of synthetic oligonucleotides containing single cytosine (Cyt-5) methyl groups suggested that the methyl moiety induces a small, local structural perturbation of the involved base pair that includes a slight displacement of the base pair toward the minor groove(15.Heinemann U. Hahn M. J. Biol. Chem. 1992; 267: 7332-7341Abstract Full Text PDF PubMed Google Scholar). It has also been observed, by our lab and by others (13.Hagerman P. Biochemistry. 1990; 29: 1980-1983Crossref PubMed Scopus (38) Google Scholar, 14.Hodges-Garcia Y. Hagerman P. Biochemistry. 1992; 31: 7595-7599Crossref PubMed Scopus (56) Google Scholar, 16.Diekmann S. EMBO J. 1987; 6: 4213-4217Crossref PubMed Scopus (73) Google Scholar), that methylation of the Cyt-5 position in pyrimidines (dC→Me5dC; dU→dT) can either decrease or enhance curvature within oligo(purine)-oligo(pyrimidine) tracts, depending upon the location of the methylated bases. Those studies, which employed electrophoretic mobility as the assay for curvature, were not able to distinguish between changes in axial curvature and alterations in helix twist. Small differences in A-tract mobility could thus be due, in part, to the latter effect. Therefore, a more direct measurement of the influence of cytosine methylation on twist and intrinsic flexibility is warranted and constitutes the rationale for the current investigation.At present, the most sensitive method for measuring both intrinsic flexibility and helix twist is the T4 DNA ligase-catalyzed cyclization assay, originally described by Shore et al.(17.Shore D. Langowski J. Baldwin R. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 4833-4837Crossref PubMed Scopus (408) Google Scholar), and later developed further by our lab (18.Taylor W.H. Hagerman P.J. J. Mol. Biol. 1990; 212: 363-376Crossref PubMed Scopus (190) Google Scholar, 19.Hagerman P.J. Ramadevi V.A. J. Mol. Biol. 1990; 212: 351-362Crossref PubMed Scopus (73) Google Scholar) and by others(20.Shore D. Baldwin R.L. J. Mol. Biol. 1983; 170: 957-981Crossref PubMed Scopus (325) Google Scholar). A particular form of the cyclization assay, developed by Taylor and Hagerman(18.Taylor W.H. Hagerman P.J. J. Mol. Biol. 1990; 212: 363-376Crossref PubMed Scopus (190) Google Scholar), allows one to determine directly, in a single ligation reaction, the molar cyclization factor, JM. In this approach, one exploits the fact that for a given DNA concentration, the ratio of the initial rates of formation of linear dimer and monomer circle species is directly proportional to JM. The "ratio" approach is particularly suited for comparisons involving small changes in JM, as in the current instance.In the current investigation, cyclization assays were performed on two sets of DNA molecules ranging in length from 158 to 180 bp, the sets differing only in the methylation status of the cytosine residues, which constitute approximately 26% of the bases in the molecules (≈52% GC). The DNA concentrations were adjusted so that both bimolecular and cyclization reactions were always observed. The principal conclusion of this study is that, for mixed sequence DNA, cytosine methylation appears to induce almost no change of either helix twist or intrinsic flexibility.MATERIALS AND METHODSSynthesis and Purification of OligonucleotidesOligodeoxyribonucleotides were synthesized using a Biosearch model 8750 automated oligonucleotide synthesizer. All phosphoramidites were purchased from Milligen/Biosearch. The oligonucleotides were cleaved from their supports, deprotected, and purified as described elsewhere(21.Hagerman P.J. Biochemistry. 1985; 24: 7033-7037Crossref PubMed Scopus (220) Google Scholar).EnzymesTaq polymerase and EcoRI and MboI restriction endonucleases were obtained from Promega; calf intestinal alkaline phosphatase was purchased from Boehringer Mannheim; T4 DNA ligase and polynucleotide kinase were purchased from New England Biolabs. Ligase-catalyzed reactions, both for constructs and for cyclization assays, contained 25 mM Hepes, pH 7.5, 50 mM potassium glutamate, 10 mM MgCl2, 1 mM ATP, and 1 mM NaEDTA. The New England Biolabs unit of ligase activity is used herein. 1 unit is defined as the amount of enzyme required to ligate 50% of HindIII DNA fragments of bacterial phage lambda in 30 min at 16°C.Plasmid Construction, Purification, and Sequence VerificationA set of 14 plasmids was produced, with each member of the set containing a distinct EcoRI fragment inserted into the EcoRI site of pGEM-7Zf(+) (Promega). The inserts ranged in size from 185 to 202 bp (Fig. 1). Plasmid-containing DH5αF′ cells were grown on L-broth supplemented with ampicillin at 100 μg/ml. Plasmid purification proceeded as described elsewhere(22.Hagerman P.J. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 4632-4636Crossref PubMed Scopus (190) Google Scholar). The plasmids were sequenced using the chain termination method of Sequenase version 2 from U. S. Biochemical Corp.Polymerase Chain Reaction (PCR) Amplification and Purification of ProductsPCR amplifications were carried out in a Perkin-Elmer DNA Thermal Cycler 480. Reactions (200 μl) employed the following buffer: 50 mM KCl, 10 mM Tris-HCl, pH 9.0, 1.5 mM MgCl2, 0.1% Triton X-100, and 200 μM of each dNTP. 5-methyl-2′deoxycytidine-5′triphosphate (Boehringer Mannheim) was substituted for dCTP when making methylated DNA. The DNA-primer concentration was 5 μM, and the template DNA concentration was approximately 8 nM (1 μg/ml). Primers L and R (see Fig. 1 and Table 1) were used to generate the majority of the PCR DNA fragments. Primers L1 and R1 each contain one additional nucleotide. Primers L1 and R were used to make the 164-bp PCR product, and primers L1 and R1 were used to make the 165-bp PCR product. These primers introduce MboI sites at each end of the PCR DNA products. The reactions were denatured at 94°C for 60 s, annealed at 60°C for 60 s, and elongated at 72°C for 180 s. PCR products were phenol extracted twice, followed by extraction with diethyl ether and ethanol precipitation. The PCR DNA was cut with MboI, followed by preparative electrophoresis on 7% polyacrylamide gels in E buffer (40 mM Tris acetate, pH 7.9, 20 mM sodium acetate, 1 mM EDTA). DNA was eluted from gel slices using a preparative electroelution system designed in this laboratory(22.Hagerman P.J. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 4632-4636Crossref PubMed Scopus (190) Google Scholar). DNA concentrations were determined on a Cary 219 spectrophotometer.Tabled 1View Large Image Figure ViewerDownload Hi-res image Download (PPT) Open table in a new tab Preparation of Radiolabeled DNADNA molecules were treated with calf intestinal alkaline phosphatase (in 50 mM Tris-HCl, pH 8.5, 0.1 mM EDTA) for 30 min at 37°C. The reaction mixture was phenol/ether extracted and ethanol precipitated. The dephosphorylated DNA was 5′-end-labeled with 20 units of T4 polynucleotide kinase and [γ-32P]ATP (3000 Ci/mmol) for 40 min at 37°C. The reaction mixture contained 0.5 μg of DNA and 50 μCi of [γ-32P]ATP in 50 mM Tris-HCl, pH 7.6, 10 mM MgCl2, 5 mM dithiothreitol. The label was then chased with cold ATP (0.1 mM final ATP concentration) and an additional 20 units of T4 polynucleotide kinase to fully phosphorylate the DNA. Labeled linear monomer was added to cold (non-labeled) DNA to the extent of ≤5% total DNA, thus permitting the accurate determination of DNA concentration through the measured A260 values of cold DNA(18.Taylor W.H. Hagerman P.J. J. Mol. Biol. 1990; 212: 363-376Crossref PubMed Scopus (190) Google Scholar).Cyclization ReactionsThe cyclization buffer comprised 25 mM Hepes, pH 7.5, 50 mM potassium glutamate, 10 mM MgCl2, and 1 mM ATP. Cyclization reactions contained either 0.05 or 0.1 μg/ml of a linear DNA fragment having MboI ends and either 200 or 400 units/ml T4 DNA ligase. Reactions were performed at 30°C, and the reaction mixtures were preincubated for 10 min at 30°C before the addition of ligase. Cyclization reactions were terminated at various times with the addition of NaEDTA and sodium dodecyl sulfate to final concentrations of 12.5 mM and 0.5%, respectively. Individual cyclization reactions were always carried out at least three times.Analysis of the Products of Cyclization ReactionsProducts of the timed cyclization reactions were run on 11.5-cm vertical polyacrylamide gels (7% acrylamide; monomer:bisacrylamide ratio, 30.5:1). The gels were run at room temperature in E buffer. Following electrophoresis, the gels were dried under vacuum at 80°C and were used to expose phosphoimaging screens. The image screens were analyzed using a Molecular Dynamics PhosphorImager and ImageQuant 1.31 software. Volume integration was used to determine the intensities of individual linear and circular species. Standard errors were computed using Sigmaplot (Jandel Scientific). The standard error for the determination of DNA concentrations was approximately 18%.RESULTS AND DISCUSSIONThe Use of a PCR-based Approach for Generating Sets of DNA Molecules for Cyclization StudiesOne of the prerequisites for a detailed study of the flexibility and helical properties of DNA via ligase-catalyzed cyclization is a set of substrate molecules that span at least one turn of the helix. These substrate molecules can be obtained through the direct isolation of cloned DNA fragments(18.Taylor W.H. Hagerman P.J. J. Mol. Biol. 1990; 212: 363-376Crossref PubMed Scopus (190) Google Scholar). However, a convenient alternative, once a suitable DNA sequence has been chosen, is to generate the actual substrate molecules through the use of PCR-based methods(23.Saiki R. Scharf S. Faloona F. Mullis K.B. Horn G.T. Erlich H.A. Arnheim N. Science. 1985; 230: 1350-1354Crossref PubMed Scopus (6691) Google Scholar). In particular, PCR primers can be used to generate appropriate restriction sites (24.Arnheim N. Erlich H. Annu. Rev. Biochem. 1992; 61: 131-156Crossref PubMed Scopus (174) Google Scholar) and can also add to the number of DNA lengths available for cyclization through alterations in the number of nucleotides in the initially non-complementary primer sequence(25.Kahn J.D. Crothers D.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6343-6347Crossref PubMed Scopus (158) Google Scholar). 2G. Bellomy and P. J. Hagerman, manuscript in preparation.In the current study, a PCR-based approach has been used to produce the desired (MboI-derived) ends for cyclization (25.Kahn J.D. Crothers D.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6343-6347Crossref PubMed Scopus (158) Google Scholar) and to introduce specific base modifications (Me5C)(26.Colasanti J. Sundaresan V. Nucleic Acids Res. 1991; 19: 391-394Crossref PubMed Scopus (8) Google Scholar).The general outline of the current approach is described in Fig. 1 and Table 1. The plasmids (pGEM) depicted in Fig. 1 contain various members of a set of DNA fragments cloned earlier as EcoRI-HindIII fragments (pESL series) in this laboratory(18.Taylor W.H. Hagerman P.J. J. Mol. Biol. 1990; 212: 363-376Crossref PubMed Scopus (190) Google Scholar). For each fragment, the HindIII-generated end was filled and subsequently converted to an EcoRI end by ligation with a linker containing an EcoRI site. The resultant plasmids, with phasing inserts as indicated in Table 1, were used as templates for PCR with R, R1, L, and L1 primers (Fig. 1, Table 1). The products of the PCR reactions, following cleavage with MboI, comprised sets of 14 DNA molecules ranging in length from 158 to 180 bp, slightly more than two turns of helix. The length spacing within each set was generally 2 base pairs, with additional species generated using the L1 and R1 primers. The two sets used in the current study thus differed only in the state of the cytosine residues (modified or non-modified).Determination of the Influence of Cytosine Methylation on the Intrinsic Flexibility and Helix Repeat of Mixed Sequence DNAIn the current study, the molar cyclization factors (JM) for each DNA molecule were determined from the ratios of the band intensities for cyclized monomer (c) and linear dimer (d) species using the following formula (18.Taylor W.H. Hagerman P.J. J. Mol. Biol. 1990; 212: 363-376Crossref PubMed Scopus (190) Google Scholar):JM=4MoLimt→0 [c/d[c/d]],(Eq. 1) where Mo is the initial DNA concentration (molar monomer fragment) and c and d are the band intensities of the cyclic and linear dimer species, respectively. The phosphorimage of one experiment (174 bp) is displayed in Fig. 2; the corresponding c/d ratios (for both methylated and non-methylated 174-bp species) are plotted as a function of time in Fig. 3.Figure 2Phosphorimage of early time points in a cyclization reaction of the 174-bp methylated DNA fragment. m, monomer fragment; d, linear dimer; c, covalently-closed, circular (monomer) species. Lanes represent time points (minutes). DNA concentration, 0.1 μg/ml; T4 DNA ligase, 400 units/ml; buffer: 25 mM Hepes, pH 7.5, 50 mM potassium glutamate, 10 mM MgCl2, 1 mM ATP. Although approximately 5% of the total number of counts remain in the wells, this material is present in equal amounts in all wells including the preligation control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Plot of the time dependence of the ratio (c/d) of the band intensities from phosphorimages for 174-bp circular monomer (c) and linear dimer (d) species (see "Materials and Methods"). Extrapolation of c/d to zero time gives the quantity, Lim[c/d], used in for the determination of JM. Solidcircles, methylated; opencircles, non-methylated. There is a greater uncertainty in the first two points on the c/d curve due to background. For the solidcircles, an extrapolation excluding the first two points changes the value of log10JM from −9.84 to −9.57.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The results of forty-two separate cyclization experiments for molecules possessing Me5C residues (14 DNA lengths between 158 and 180 bp) are expressed as log10JM values in Fig. 4, with a corresponding set of experiments for the non-methylated species displayed in Fig. 5. The log10JMversus length curves have each been analyzed by subjecting the log10JM data to a global least-squares analysis, utilizing equation 73 of Shimada and Yamakawa(27.Shimada J. Yamakawa H. Macromolecules. 1984; 17: 689-698Crossref Scopus (296) Google Scholar), with the persistence length P, the torsional elastic constant C, and the helix repeat h as independent parameters. The results of both analyses are presented in Table 2. It is clear from these results (Figure 4, Figure 5, Figure 6) that cytosine methylation has no significant effect on either the helix repeat (identical maxima for the log10JM curves) or the intrinsic flexibility (equal average values and peak trough variations for the two curves) of the mixed sequence DNA molecules employed in the current study.Figure 4Plot of experimental values for log10JM for nonmethylated C residues as a function of length. The solidline represents a best-fit log10JM curve computed using the P, C, and h parameters from Table 2 and equation 73 of Shimada and Yamakawa(27.Shimada J. Yamakawa H. Macromolecules. 1984; 17: 689-698Crossref Scopus (296) Google Scholar). The dashedline represents a computed log10JM curve for p = 450 Å.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Plot of experimental values for log10JM for 5 MeC residues as a function of length. The solidline represents a best-fit log10JM curve computed as in Fig. 4.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Tabled 1View Large Image Figure ViewerDownload Hi-res image Download (PPT) Open table in a new tab Figure 6Plot of the difference in the experimental log10JM values for the methylated and non-methylated species. The filledcircles represent the difference, log10JM (methyl) - log10JM (nonmethyl).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Previous investigations (13.Hagerman P. Biochemistry. 1990; 29: 1980-1983Crossref PubMed Scopus (38) Google Scholar, 14.Hodges-Garcia Y. Hagerman P. Biochemistry. 1992; 31: 7595-7599Crossref PubMed Scopus (56) Google Scholar, 16.Diekmann S. EMBO J. 1987; 6: 4213-4217Crossref PubMed Scopus (73) Google Scholar) have demonstrated that methylation can alter the mobilities of DNA molecules on polyacrylamide gels. In particular, for molecules possessing short homopurine-homopyrimidine tracts, both the extent of methylation and the locations of methyl groups within and adjacent to the tracts can influence the relative mobilities of the methylated species relative to their non-methylated counterparts. Although the methylation-dependent modulation of gel mobility was believed to be due to local changes in the direction of the helix axis, consistent with the notion of local structural perturbations suggested by crystallographic work(15.Heinemann U. Hahn M. J. Biol. Chem. 1992; 267: 7332-7341Abstract Full Text PDF PubMed Google Scholar), one could not rule out contributions due to changes in either the helix repeat or the intrinsic flexibility of DNA. The current results have addressed this issue, providing strong support for a model in which the structural alterations introduced by the methyl groups are, in fact, due primarily to local changes in the direction of the helix axis rather than changes in either twist or flexibility.Although the persistence lengths determined in the current work are only slightly smaller (10-15%) than values previously determined by ligase-catalyzed DNA cyclization (450-490 Å)(18.Taylor W.H. Hagerman P.J. J. Mol. Biol. 1990; 212: 363-376Crossref PubMed Scopus (190) Google Scholar, 20.Shore D. Baldwin R.L. J. Mol. Biol. 1983; 170: 957-981Crossref PubMed Scopus (325) Google Scholar, 28.Levene S.D. Crothers D.M. J. Mol. Biol. 1986; 189: 61-72Crossref PubMed Scopus (94) Google Scholar), we regard these differences as significant (Fig. 4). In particular, for molecules in the 158-180-bp range, an 11% increase in the persistence length (e.g. 400 Å→450 Å) would be accompanied by a 3.7-fold reduction in JM (Fig. 4), well outside of the current limits of error. One possible origin of this difference is a partial phasing of regions of curvature within the monomer fragments which, while not reflected in reduced gel mobilities, may increase the propensity for cyclization. In this regard, the current DNA fragments do possess several short A-tracts (Fig. 1). Moreover, we have observed 3Y. Hodges-Garcia and P. J. Hagerman, unpublished observations.that for DNA molecules closely related to those employed in the current study, monomer fragments are electrophoretically normal whereas dimers of those fragments display slight shifts in mobility, depending on the orientation of the monomers within the dimer. We have not investigated this latter issue in detail since it does not affect the central conclusions regarding the methyl effect. However, the current observations underscore the importance of considering "static" components of the persistence length (i.e. those due to stable bends) (30.Zhurkin V.B. Ulyanov N.B. Gorin A.A. Jernigan R.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7046-7050Crossref PubMed Scopus (137) Google Scholar, 31.Trifonov E.N. Tan R.K.-Z. Harvey S.C. Olson W.K. Sarma M.H. Jundaralingam J. DNA Bending and Curvature. Adenine Press, Schenectady, NY1987: 243-253Google Scholar) 4J. A. Schellman and S. C. Harvey, submitted for publication.when discussing the intrinsic, elastic properties of DNA.The current values for the torsional elastic constant, C (Table 2), are well within the range of values for C (2-3.8 × 10−19 erg-cm) obtained in recent work (see (32.Hagerman P.J. Annu. Rev. Biophys. Biophys. Chem. 1988; 17: 265-286Crossref PubMed Scopus (925) Google Scholar) for review, also Refs. 18, 28, 33, and 34). As pointed out by Crothers et al.(35.Crothers D.M. Drak J. Kahn J.D. Levene S.D. Methods Enzymol. 1992; 212: 3-29Crossref PubMed Scopus (203) Google Scholar), differences within the above range may reflect real differences among DNA sequences employed for cyclization experiments, and although those authors "prefer" the value of 3.4 × 10−19 erg-cm, a unique value for C may not exist; rather, it may vary with sequence. In fact, in more recent work, Kahn et al.(33.Kahn J.D. Yun E. Crothers D.M. Nature. 1994; 368: 163-166Crossref PubMed Scopus (173) Google Scholar) have observed a value of 2.1 × 10−19 erg-cm from cyclization measurements, within 5% of the value obtained earlier by Taylor and Hagerman(18.Taylor W.H. Hagerman P.J. J. Mol. Biol. 1990; 212: 363-376Crossref PubMed Scopus (190) Google Scholar). In this regard, the small, albeit reproducible deviations of the 159 and 169-bp fragments from the log10JM curves may again reflect the partial phasing of regions of curvature, leading to a more complex pattern for the length dependence of the log10JM curves. Preliminary Monte Carlo simulations 5Y. Hodges-Garcia and P. J. Hagerman, unpublished results.are consistent with this latter proposal.CONCLUSIONIn the current work, the DNA cyclization assay was used to demonstrate that cytosine methylation has little, if any, influence on either the intrinsic flexibility or the helix repeat of mixed sequence DNA. The values obtained for the torsional elastic constant and the helix repeat are consistent with published results, while the current values for the persistence length are slightly reduced, possibly due to contributions from regions of curvature within the molecules used for these studies. This latter issue warrants further investigation.The observation (29.Brukner I. Dlakic M. Savic A. Susic S. Pongora S. Suck D. Nucleic Acids Res. 1993; 21: 1025-1029Crossref PubMed Scopus (62) Google Scholar) that GGGCCC elements give rise to curvature in the absence of A-tracts, coupled with earlier observations of the influence of methyl groups in specific GC elements(13.Hagerman P. Biochemistry. 1990; 29: 1980-1983Crossref PubMed Scopus (38) Google Scholar, 14.Hodges-Garcia Y. Hagerman P. Biochemistry. 1992; 31: 7595-7599Crossref PubMed Scopus (56) Google Scholar), raises the possibility that the effects of methylation on flexibility and helix repeat may be different in such elements. This last issue also warrants further study. INTRODUCTIONThe Cyt-5 methylation of cytosine bases in DNA is known to play an important role in gene expression, in part through its modulation of transcriptional activation(1.Cedar H. Cell. 1988; 53: 3-4Abstract Full Text PDF PubMed Scopus (727) Google Scholar, 2.Cedar H. Razin A. Biochim. Biophys. Acta. 1990; 1049: 1-8Crossref PubMed Scopus (150) Google Scholar). Moreover, cytosine methylation is believed to contribute to both inactivation of the X chromosome (3.Migeon B. Am. J. Med. Genet. 1993; 47: 685-686Crossref Scopus (3) Google Scholar) and genomic imprinting(4.Li E. Beard C. Janenisch R. Nature. 1993; 366: 362-365Crossref PubMed Scopus (1743) Google Scholar), wherein the phenotype of a particular mutant allele depends on the parent of origin of the allele. As an example of the latter, the fragile X syndrome(5.Hagerman R.J. Silverman A.G. Fragile X Syndrome: Diagnosis, Treatment, and Research. Johns Hopkins University Press, Baltimore1991Google Scholar), the leading stably heritable form of mental retardation, is believed to express its most severe phenotype only when an abnormally expanded triplet-repeat sequence (CGG)n is fully methylated (6.McConkie-Rosell A. Lachiewicz A.M. Spiridigliozzi G.A. Tarleton J. Schoenwald S. Phelan M.C. Goonewardena P. Ding X. Brown W.T. Am. J. Hum. Genet. 1993; 53: 800-809PubMed Google Scholar, 7.Hagerman R.J. Hull C.E. Safanda J.F. Carpenter I. Staley L.W. O'Connor R. Seydel C. Mazzocco M.M. Snow K. Thibodeau S. Kuhl D. Nelson D.L. Caskey C.T. Taylor A. Am. J. Med. Genet. 1994; 51: 298-308Crossref PubMed Scopus (181) Google Scholar, 8.Vincent A. Heitz D. Petit C. Kretz C. Oberle I. Mandel J. Nature. 1991; 349: 624-626Crossref PubMed Scopus (173) Google Scholar) on an active X chromosome. In addition to the functional consequences of cytosine methylation, it is known that methylation can influence the formation of DNA cruciforms (9.Murchie A.I. Lilley D.M.J. J. Mol. Biol. 1993; 205: 593-602Crossref Scopus (33) Google Scholar) and triple helices(10.Xodo L.E. Manzini G. Quadrifoglio F. van der Marel G.A. van Boom J.H. Nucleic Acids Res. 1991; 19: 5625-5631Crossref PubMed Scopus (203) Google Scholar, 11.Strobel S.A. Dervan P.B. Nature. 1993; 350: 172-174Crossref Scopus (129) Google Scholar). Methylation can also modulate the B to Z transition in DNA (12.Zacharias W. Jost J.P. Saulz H.P. DNA Methylation: Molecular Biology and Biological Significance. Birkhauser Verlag, Basel, Switzerland1993: 27-38Crossref Scopus (29) Google Scholar) as well as the apparent curvature of DNA containing A-tracts(13.Hagerman P. Biochemistry. 1990; 29: 1980-1983Crossref PubMed Scopus (38) Google Scholar, 14.Hodges-Garcia Y. Hagerman P. Biochemistry. 1992; 31: 7595-7599Crossref PubMed Scopus (56) Google Scholar).The physical mechanisms by which cytosine methylation affects gene expression (or even DNA structure) are unknown. It has been demonstrated that methylation stabilizes Z-DNA and changes the helix twist of poly(dG•dC) copolymers from 10.5 to 10.7 base pairs (bp) 1The abbreviations used are: bpbase pair(s)PCRpolymerase chain reaction.per turn(12.Zacharias W. Jost J.P. Saulz H.P. DNA Methylation: Molecular Biology and Biological Significance. Birkhauser Verlag, Basel, Switzerland1993: 27-38Crossref Scopus (29) Google Scholar). Moreover, an x-ray crystallographic analysis of synthetic oligonucleotides containing single cytosine (Cyt-5) methyl groups suggested that the methyl moiety induces a small, local structural perturbation of the involved base pair that includes a slight displacement of the base pair toward the minor groove(15.Heinemann U. Hahn M. J. Biol. Chem. 1992; 267: 7332-7341Abstract Full Text PDF PubMed Google Scholar). It has also been observed, by our lab and by others (13.Hagerman P. Biochemistry. 1990; 29: 1980-1983Crossref PubMed Scopus (38) Google Scholar, 14.Hodges-Garcia Y. Hagerman P. Biochemistry. 1992; 31: 7595-7599Crossref PubMed Scopus (56) Google Scholar, 16.Diekmann S. EMBO J. 1987; 6: 4213-4217Crossref PubMed Scopus (73) Google Scholar), that methylation of the Cyt-5 position in pyrimidines (dC→Me5dC; dU→dT) can either decrease or enhance curvature within oligo(purine)-oligo(pyrimidine) tracts, depending upon the location of the methylated bases. Those studies, which employed electrophoretic mobility as the assay for curvature, were not able to distinguish between changes in axial curvature and alterations in helix twist. Small differences in A-tract mobility could thus be due, in part, to the latter effect. Therefore, a more direct measurement of the influence of cytosine methylation on twist and intrinsic flexibility is warranted and constitutes the rationale for the current investigation.At present, the most sensitive method for measuring both intrinsic flexibility and helix twist is the T4 DNA ligase-catalyzed cyclization assay, originally described by Shore et al.(17.Shore D. Langowski J. Baldwin R. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 4833-4837Crossref PubMed Scopus (408) Google Scholar), and later developed further by our lab (18.Taylor W.H. Hagerman P.J. J. Mol. Biol. 1990; 212: 363-376Crossref PubMed Scopus (190) Google Scholar, 19.Hagerman P.J. Ramadevi V.A. J. Mol. Biol. 1990; 212: 351-362Crossref PubMed Scopus (73) Google Scholar) and by others(20.Shore D. Baldwin R.L. J. Mol. Biol. 1983; 170: 957-981Crossref PubMed Scopus (325) Google Scholar). A particular form of the cyclization assay, developed by Taylor and Hagerman(18.Taylor W.H. Hagerman P.J. J. Mol. Biol. 1990; 212: 363-376Crossref PubMed Scopus (190) Google Scholar), allows one to determine directly, in a single ligation reaction, the molar cyclization factor, JM. In this approach, one exploits the fact that for a given DNA concentration, the ratio of the initial rates of formation of linear dimer and monomer circle species is directly proportional to JM. The "ratio" approach is particularly suited for comparisons involving small changes in JM, as in the current instance.In the current investigation, cyclization assays were performed on two sets of DNA molecules ranging in length from 158 to 180 bp, the sets differing only in the methylation status of the cytosine residues, which constitute approximately 26% of the bases in the molecules (≈52% GC). The DNA concentrations were adjusted so that both bimolecular and cyclization reactions were always observed. The principal conclusion of this study is that, for mixed sequence DNA, cytosine methylation appears to induce almost no change of either helix twist or intrinsic flexibility.
Referência(s)