(Received for publication, August 12, 1994)
From the
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.
The 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, 2) . Moreover, cytosine
methylation is believed to contribute to both inactivation of the X
chromosome (3) and genomic imprinting(4) , 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) , 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) is fully methylated (6, 7, 8) 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) and triple
helices(10, 11) . Methylation can also modulate the B
to Z transition in DNA (12) as well as the apparent curvature
of DNA containing A-tracts(13, 14) .
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(dGdC) copolymers from 10.5 to 10.7 base pairs (bp) (
)per turn(12) . 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) . It has also been observed, by our lab and by
others (13, 14, 16) , that methylation of the
Cyt-5 position in pyrimidines (dC
Me
dC; 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) , and
later developed further by our lab (18, 19) and by
others(20) . A particular form of the cyclization assay,
developed by Taylor and Hagerman(18) , allows one to determine
directly, in a single ligation reaction, the molar cyclization factor, J. 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 J
. The ``ratio''
approach is particularly suited for comparisons involving small changes
in J
, 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.
Figure 1:
a, outline of the PCR-based
method for producing the DNA fragments used in the current study. The
methylation status of the PCR products is determined by the use of
either dCTP or d(Me)CTP in the reaction mix. The insert
sequence is as listed in Table 1. b, sequence of the
parent DNA sequence used for the cyclization reactions (the boldunderlinedsequence is the 14-bp insert phasing
sequence in Table 1).
The general outline of the current approach is described in Fig. 1and Table 1. The plasmids (pGEM) depicted in Fig. 1contain various members of a set of DNA fragments cloned earlier as EcoRI-HindIII fragments (pESL series) in this laboratory(18) . 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).
where M 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 2:
Phosphorimage 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 MgCl, 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.
Figure 3:
Plot 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 J. 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 log
J
from -9.84 to
-9.57.
The results of forty-two separate cyclization experiments for
molecules possessing MeC residues (14 DNA lengths between
158 and 180 bp) are expressed as log
J
values
in Fig. 4, with a corresponding set of experiments for the
non-methylated species displayed in Fig. 5. The
log
J
versus length curves have each
been analyzed by subjecting the log
J
data to a
global least-squares analysis, utilizing equation 73 of Shimada and
Yamakawa(27) , 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 (Fig. 4Fig. 5Fig. 6) that cytosine methylation has
no significant effect on either the helix repeat (identical maxima for
the log
J
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 4:
Plot of experimental values for
logJ
for nonmethylated C residues as a
function of length. The solidline represents a
best-fit log
J
curve computed using the P, C, and h parameters from Table 2and equation 73 of Shimada and Yamakawa(27) . The dashedline represents a computed
log
J
curve for p = 450
Å.
Figure 5:
Plot of experimental values for
logJ
for 5 MeC residues as a function of
length. The solidline represents a best-fit
log
J
curve computed as in Fig. 4.
Figure 6:
Plot of the difference in the experimental
logJ
values for the methylated and
non-methylated species. The filledcircles represent
the difference, log
J
(methyl) -
log
J
(nonmethyl).
Previous investigations (13, 14, 16) 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) , 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, 20, 28) , 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 Å450 Å) would
be accompanied by a 3.7-fold reduction in J
(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 (
)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, 31) (
)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
erg-cm) obtained in recent work (see (32) for review,
also Refs. 18, 28, 33, and 34). As pointed out by Crothers et
al.(35) , 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
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) have observed a value of
2.1
10
erg-cm from cyclization
measurements, within 5% of the value obtained earlier by Taylor and
Hagerman(18) . In this regard, the small, albeit reproducible
deviations of the 159 and 169-bp fragments from the
log
J
curves may again reflect the partial
phasing of regions of curvature, leading to a more complex pattern for
the length dependence of the log
J
curves.
Preliminary Monte Carlo simulations (
)are consistent with
this latter proposal.
In 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) 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, 14) , 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.