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(Received for publication, May 4, 1995; and in revised form, August 7, 1995) From the
A computer program for predicting DNA bending from nucleotide
sequence was used to identify circular structures in retroviral and
cellular genomes. An 830-base pair circular structure was located in a
control region near the center of the genome of the human
immunodeficiency virus type I (HIV-I). This unusual structure displayed
relatively smooth planar bending throughout its length. The structure
is conserved in diverse isolates of HIV-I, HIV-II, and simian
immunodeficiency viruses, which implies that it is under selective
constraints. A search of all sequences in the GenBank data base was
carried out in order to identify similar circular structures in
cellular DNA. The results revealed that the structures are associated
with a wide range of sequences that undergo recombination, including
most known examples of DNA inversion and subtelomeric translocation
systems. Circular structures were also associated with replication and
transposition systems where DNA looping has been implicated in the
generation of large protein-DNA complexes. Experimental evidence for
the structures was provided by studies which demonstrated that two
sequences detected as circular by computer preferentially formed
covalently closed circles during ligation reactions in vitro when compared to nonbent fragments, bent fragments with
noncircular shapes, and total genomic DNA. In addition, a single T
Sequence-directed bending of DNA is one factor that causes local
variations in the structure of genomes (reviewed by Crothers et
al.(1990), Trifonov(1991), Hagerman(1992), and Harrington(1992)).
The major determinant of bent DNA arises from oligo(A) tracts, which
deflect the axis of the helix from a straight line. In bent sites, the
tracts are spaced near the helical repeat of DNA, which causes their
deflections to sum producing curvature of the DNA axis. Studies of bent
DNA have most often focused on simple curved segments that are less
than 100 bp ( Small planar DNA circles produced by ligation of synthetic curved
oligonucleotide precursors have served as model systems for the study
of one complex higher order structure in DNA (Ulanovsky et
al., 1986; Koo et al., 1990; Crothers et al.,
1992). To our knowledge, the only comparable structures that have been
found in natural DNA are in kinetoplast minicircles of the trypanosome Crithidia fasciculata. These sequences have been shown to
exist as circular structures by electron microscopy (Griffith et
al., 1986). In this report, computer programs for calculating
bending from nucleotide sequence were used to identify circular
structures of various sizes in cellular and retroviral genomes.
Ligation studies were then carried out in order to provide experimental
evidence for the structures. The results show that large circular
structures are associated with a wide range of DNA recombination
systems and we suggest that this unusual structure plays a general role
in DNA looping.
Figure 1:
Predicted DNA bending along the HIV-1
genome. The map at the bottom of panelB shows the
positions of the long open reading frames (Gag, Pol, Env) and the
regions within Pol: protease (P), reverse transcriptase (Rt), and integrase (I). The expanded region at the
bottom of the map (bp 4000-5500) gives the positions of AP-1
(
ENDS ratios were computed at the indicated window widths for
sequences from release 73 of the GenBank data base (Benson et
al., 1994). Details of the strategies used for analysis of both
the data base and random sequences have been described previously
(VanWye et al., 1991) and are outlined under
``Results.'' The subdivisions of the GenBank library that
were used in the studies shown in Fig. 3were phage, bacterial,
invertebrates, plants, organelles, nonmammalian vertebrates, rodents,
primates, other mammals, and viruses. Sequences from the structural
RNA, synthetic, and unannotated categories were omitted. ENDS ratio
analyses were performed on all sequences with lengths greater than 1000
bp. A total of 10,823 sequences (37% of the library) were analyzed.
Upon request, the sequence locus names, nucleotide positions, and
lengths of the highest 100 ENDS ratio peaks in each subdivision of the
library will be provided. The accession numbers of the retroviral
sequences used in Fig. 2are given in Table I of Bronson and
Anderson (1994). The accession numbers of sequences A-W from Fig. 4and the base pair positions of ENDS ratio peaks in the
sequences are: A, X62121 (6650); B, J01347(1540); C, M18274(3870); D,
M32345 (1160 and 2720); E, J01801 (520); F, M11774 (291); G,
M11195(1500); H, M65025 (710); I, J00478 (850); J, Z11876 (530); K,
M33720 (2620); L, K00638(1100); M, M36467 (580); N, M57546 (3890); O,
M30176(1450); P, M63034(1374); Q, V00686(1570); R, X14385 (5230); S,
X04658(4020); T, X02451(2070); U, J01426(1850); V, K01979 (550); W,
M18394 (110). Variation is expressed as the standard deviation
(± S.D.) or the standard error (± S.E.) of the mean.
Figure 3:
Characteristics of high ENDS ratio peaks
in GenBank sequences. ENDS ratios of the 10,823 sequences in the
GenBank data base that were greater than 1000 bp in length were
computed at window widths from 20 to 1100 bp as in Fig. 1B. The mean (± S.D.) ENDS ratio values of
all sequences in the data base at each window step (
Figure 2:
Predicted DNA bending in HIV-1, HIV-2, and
SIV. A generalized map of the proviral genome showing positions of Gag,
Pol, Env, P, RT, I, ltr (long terminal repeats) is given at the top of
the figure. The indicated number(N) of full-length sequences
representing the seven viral groups were analyzed for bending as in Fig. 1B. The mean ENDS ratio peak values for each group
are represented. The types and magnitudes of the peaks are represented
by distinguishing symbols at the indicated positions. The values of the
ENDS ratio peaks are as follows: 120-bp window: 1.14-1.18
(
Figure 4:
Two-dimensional projections of the helical
axis of high ENDS ratio peaks. Each peak sequence (A-W)
is represented by two helix projections that are rotated 90 degrees.
All sequences were detected as high ENDS ratio peaks (>15) in the
search from Fig. 3except the C-shaped structures in E and F and the structures in G and U,
which are longer than 1100 bp. Toppanels, sequences
from inversion and looping systems: A, min system
from plasmid 15 B of E. coli (Sandmeier et al.,
1991); B, FLP system for inversion of the 2-µm plasmid of Saccharomyces cerevisiae (Cox, 1989); C, FLP system
for inversion of a plasmid from Zygosaccharomyces bailii (Utatsu et al., 1987); D, pilin inversion system
from M. bovis (Fulks et al., 1990); E, hin system for flagellar variation in S. typhimurium (Szekely and Simon, 1983); F, fim system for
fimbrial variation in E. coli (Abraham et al., 1985); G, genes A and B coding for the phage Mu transposase and
accessory gene (Harshey et al., 1985); H, plasmid R6K
replication-looping system (Kelly and Bastia, 1991); I, the
C
Gel-purified EcoRI fragments were dephosphorylated with calf alkaline
phosphatase and then end-labeled with
[ The circular structure that
was detected by computer in the phage
Figure 11:
Analysis of ligation products of long DNA
molecules. EcoRI digests of plasmids containing the 1192-bp
Figure 5:
Characteristics of the DNA molecules. The
preparation of the DNA is described in ``Experimental
Procedures.'' The intron D segment in pJA46-13 carries a
point mutation that converts a T
Figure 8:
Ligation analysis of C. elegans intron segment (pJA46-2) in the presence of C. elegans genomic DNA. A, EcoRI-digested C. elegans genomic DNA (1 mg/ml) and
Figure 10:
Predicted structures along the phage
lambda genome. ENDS ratios computed at window widths from 20 to 1000 bp
along the entire genome are plotted in C as a function of
sequence position in kilobase pairs. The positions of the genes
encoding head proteins, tail proteins and the major transition in
genome base composition (&cjs0435;) are indicated. Two-dimensional
projections of the helix axis of the first 22 kbp are shown in B. The break in the projection at bp 7963 lies at the
head-tail junction. The position of the circular structure (C)
at bp 870-1750 is indicated. Projections of this 880-bp circular
structure are shown in A.
Computer-generated helical
projections of the HIV-1 proviral DNA are shown in Fig. 1A. The standard program that produced the
projections is based on a model of intrinsic curvature, which assumes
that DNA bending arises solely from AA/TT stacks. Deflections from
linearity are quantified by the ENDS ratio, which is defined as
ℓ/d, where ℓ is the contour length of a DNA segment
and d is the shortest distance between its ends (Eckdahl and
Anderson, 1987). In panelB, ENDS ratios were
computed at window widths of 20-200 bp (top), 20-400 bp
(middle), and 20-1000 bp (bottom) so that the corresponding
short, intermediate, and long bent segments could be characterized in a
single analysis. At the shorter window widths, the major site of
bending occurred in the first 100 bp of reverse transcriptase. Helical
projections of this segment are shown in Fig. 1C. The
structure is characterized by an ENDS ratio value of 1.30 at a window
width of 120 bp. This value is greater than 4 standard deviations above
the mean of random and GenBank data base sequences (Fitzgerald et
al., 1994). The site consists of two 40-bp regions of bending
centered at nucleotides 2689 and 2757. The bending elements in the two
regions are out of phase with each other by about 5 bp, which produces
an S-shaped molecule. This structure is similar to the S-shaped
configuration that is characteristic of nucleosome positioning DNA
(Fitzgerald et al., 1994). Minor DNase I-hypersensitive sites,
indicative of phased nucleosomes, have been mapped immediately adjacent
to this structure on the 3` side (Verdin, 1991). At the intermediate
window widths, regions of bending are noted at the gag-pol overlap, and at the 3` ends of pol and env.
These deflections, as well as minor deflections at the 5` ends of gag and env, can readily be seen by the helical
projections shown in panel A. A single unusually high ENDS ratio peak
with a value of 73 is seen at the 830-bp window. The computer-generated
structure corresponds to a circle of 830 bp (Fig. 1, A and D). The circular structure encompasses most of the
integrase gene at the end of pol, and its central position at
nucleotide 4800 is 22 bp away from the internal polypurine tract. The
circular structure was also seen when the sequence was analyzed by a
computer program that incorporates wedge angles for each of the 16
dinucleotides (Fig. 1E). In order to assess the
conservation of the bent structures in retroviruses, the 60 full-length
retroviral sequences from release 73 of the GenBank data base were
examined as in Fig. 1. In this collection, each of the known
retroviral lineages is represented by multiple sequences (Bronson and
Anderson, 1994). Fig. 2A shows the positions and types
of bending elements along the genomes of the primate lentiviruses
HIV-1, HIV-2, and SIV. The ENDS ratio peaks that characterize each
bending element are more than 2 S.D. above the mean of randomized
sequences (Fig. 2, see legend). Bending occurs most often at the
beginnings and ends of the major open reading frames in essentially all
of the sequences and this preference is especially prominent at the
intermediate window widths. Likewise, the bent sites in the genomes of
other retroviral lineages not shown in the figure tended to occur at
the beginning and/or at the ends of the major open reading frames,
although the positions of the sites varied in the different viral
groups (data not shown). The most striking result in Fig. 2is
the conservation of the circular structure in the integrase gene. The
structure was seen in all isolates of HIV-1, HIV-2, and SIV, although
the magnitudes of the ENDS ratio peaks in some SIV isolates were not as
high as those seen in the human immunodeficiency viruses (Fig. 2A). Helical projections of the integrase
sequence from a virus representing each group are presented in Fig. 2(B-D), which shows that each structure
displays relatively smooth planar bending throughout its length. Near
planar bending was also noted in the homologous regions of integrase in
some nonprimate lentiviruses including the prototype virus visna (data
not shown). No comparable structures were observed in the 17 sequences
from oncoviruses or spumaviruses, which are the other two subfamilies
of retroviruses. The circular structures in the primate lentiviruses
had an average ENDS ratio of 50 with circumferences that ranged from
780 to 1060 bp. The center of the structure in every genome was within
60 bp of the internal polypurine tract. The conservation of the
predicted structure was of interest because there is as much as 40%
divergence in the nucleotide sequence of the circular regions shown in Fig. 2(B-D; see legend), and the divergence is
even greater among the integrase sequences from the viruses depicted in Fig. 2A. This is significant because insertions or
deletions of as few as a single bp in the central 200-bp regions, or a
small number of random substitutions (less than 5%), can abolish the
computer-generated circular projections of the sequences shown in Fig. 2(B-D) (data not shown). In addition, the
structures are heterogeneous in size and shape as seen in Fig. 2(B-D). Consequently, the exchange of
homologous segments of the integrase gene from HIV-1, HIV-2, and SIV
failed to generate circular structures of the types shown in Fig. 1and Fig. 2(data not shown). These results imply
that selective pressures have maintained the integrity of the circular
structure despite extensive sequence divergence.
Three groups of sequences are overrepresented
in Fig. 3when compared to their frequencies in the GenBank data
base. First, 20 of the 135 sequences are found in the integrase region
of primate immunodeficiency viruses and the highest data base peak
(ENDS ratio = 392) was from this region of HIV-1 (MN isolate).
Second, 11 of the peak sequences were found in untranslated DNA from C. elegans and these sequences were particularly prevalent
among the shorter peak sites uncovered by the search. This
overrepresentation is consistent with the observation that the genome
of this nematode is highly bent (VanWye et al., 1991). Third,
a significant fraction (17/50) of the remaining peak sequences longer
than 800 bp were associated with a class of molecules which includes
known mobile elements and related sequences. Helical projections of
sequences that constitute this group are shown in Fig. 4. Alterations in gene expression caused by inversion of DNA segments
has been extensively studied in bacteria and in yeast (reviewed by Cox
(1988), Glasgow et al.(1988), Stark et al. (1992),
and Van de Putte and Goosen(1992)). In these systems, a recombinase
promotes strand cleavage and rejoining within inverted repeat sequences
that flank the invertible element. Efficient inversion in bacteria also
requires a recombinational enhancer. The enhancer and the gene for the
recombinase are typically located within, or adjacent to, the
invertible element. Fig. 4(A-D) shows helical
projections of four invertible systems that were detected as ENDS ratio
peaks in the data base search. In each system, the circular structure
terminates within 200 bp of at least one inverted repeat sequence and
the structures in the two bacterial sequences terminate within 200 bp
of a recombinational enhancer. The 2.1-kbp invertible DNA segment that
controls pilin phase variation in Moraxella bovis is
particularly striking since it consists of two circular structures
containing the two pilin genes (Q and I), which are separated by a
straight stretch of DNA (Fig. 4D). In order to
determine if DNA circularity is a characteristic of invertible systems,
we examined the inversion regions responsible for the well
characterized phase variation of flagellin genes in Salmonella
typhimurium and fimbrial protein genes in E. coli.The
invertible DNA segments in both systems appear as C-shaped structures
as shown in Fig. 4(E and F). The cin invertase gene in phage P1 and P7 and the pin invertase
gene from E. coli also appeared as C-shaped structures when
viewed by the computer program for bending (data not shown). DNA
looping is thought to facilitate the assembly of complex nucleoprotein
structures during synapse formation in inversion systems (reviewed by
Schleif(1988), Wang and Giaever(1988), Echols(1990), and
Matthews(1992)). An analogous looping mechanism has been implicated in
the transposition of the phage Mu transposon (Surette et al.,
1989; Heichman and Johnson, 1990; Mizuuchi, 1992; Surette and Chaconas,
1992). As shown in Fig. 4G, the A gene for the Mu
transposase and the B accessory transposition gene are contained within
a circular structure, which is adjacent to the transpositional
enhancer. Initiation of replication in plasmid R6K utilizes a DNA
looping mechanism, which is mediated by the P1 initiator protein
(Mukherjee et al., 1988a, 1988b). The protein binds to
multiple enhancer sites in the Recombination events near
the ends of linear chromosomes are responsible for antigen variation in
the African trypanosome (Van der Ploeg et al., 1992; Pays et al., 1994), in the spirochete Borrelia hermsii (Barbour, 1993), and in the African swine fever virus (Almendral et al., 1990; de la Vega et al., 1990;
González et al., 1990). These organisms
presumably evade the immune system of their mammalian hosts by
periodically switching the expression of members of multigene families
that code for surface antigens. In the trypanosome and spirochete, a
transcriptionally silent copy of a surface protein gene is typically
duplicated and transferred unidirectionally from a distal site in the
genome to a transcriptionally active telomeric expression locus.
Similarly, duplication and translocation of sequences located near the
left end of the 170-kbp swine viral genome is thought to have played a
role in generating the antigen variation seen among different isolates
of the virus (Gonzalez et al., 1990). As shown in Fig. 4(J and K), circular structures were
noted in the expression locus several kilobase pairs upstream from the
surface antigen genes in the spirochete and the trypanosome. Comparable
O-shaped structures (Fig. 4L), as well as C-shaped
structures (data not shown), were also noted within 1 kbp upstream of
the antigen genes in both systems. Both locations have been implicated
in recombinational events (Pays et al., 1989, 1994; Barbour et al., 1991; Barbour, 1993). Multiple unusual structures also
characterized the left end of the swine virus genome. O-shaped (Fig. 4, M and N) and C-shaped (data not
shown) structures were seen in this region at locations corresponding
to sequences that are the most variable among viral isolates (see Fig. 7in De La Vega et al.(1990)).
Figure 7:
Cyclization kinetics of wild type and
mutant intron segments. Leftpanel, labeled wild-type
(
Type II introns
encoding reverse transcriptase (RT introns) are common in the
organelles of lower eukaryotes. These sequences are thought to be
mobile elements, although the mechanism of their transposition is
unclear (Lambowitz and Belfort, 1993). The data base search in Fig. 3detected four RT introns from organelles, which are shown
in Fig. 4(O-R). The RT sequence from the
carnation etched virus also displayed a circular structure as seen in Fig. 4S. This virus is thought to be more closely
related to transposons than to plant viruses (Doolittle et
al., 1989). Helical projections of the 34 RT introns and related
sequences analyzed by Mohr et al.(1993) were assessed in order
to determine whether DNA circularity is a conserved feature of this
group. The results revealed that about 80% of the sequences displayed
either O-shaped or C-shaped structures (data not shown). Organelles
from plants and lower eukaryotes also contain mobile elements in the
form of small plasmids whose origins are poorly understood (Ray et
al., 1987; Kempken et al., 1992). The data base search
identified four of these sequences with circular structures in the
organelle subdivision of GenBank (Fig. 4, T-W). It is unlikely that the association of circular structures with
mobile elements and related systems is coincidental since the known
sequences that make up this group are expected to represent only a
small fraction of the total data base entries. The preferential
detection of these elements in the computer search can be clearly seen
by considering the identity of the sequences with the highest ENDS
ratio peaks from Fig. 3and Fig. 4. Nineteen of the
circular structures shown in Fig. 4have ENDS ratio values that
are greater than 40. This number is significant since there were only
48 sequences with ENDS ratio peaks greater than 40 in the entire
GenBank data base and 8 of these were found in the integrase region of
the primate immunodeficiency viruses (Fig. 3). The specificity
of the computer search for a discrete subset of the data base can be
further illustrated by a consideration of all peak sequences that were
detected among the 3643 sequence entries that constitute the viral,
organelle, and bacterial subdivisions of GenBank. There were 11 viral
sequences and 5 sequences from organelles with ENDS ratio peaks >40.
All of these structures are shown in Fig. 2(toppanel) and Fig. 4(O, P, T, V, and W). Five of the 11 peak structures
from the bacterial data base with ENDS ratio peaks >40 are also
shown in Fig. 4. Of the remaining six structures from bacteria
that are not shown in the figure, two were associated with ori sequences, one with a toxin gene, and three with genes that
encoded pilin-like proteins. (
A
comparison of the cyclization of the DNA sequences shown in Fig. 5should provide a stringent test for the predictive power
of the computer program used in this study. In Fig. 6, DNA
fragments were end-labeled and then incubated with T4 DNA ligase at 4
°C for the indicated times in the presence of an excess of
nonradioactive digested plasmid DNA. Following incubation, samples were
electrophoresed on agarose gels in the presence of ethidium bromide in
order to resolve circular and linear species (Shore et al.,
1981). Inspection of the resulting autoradiograms revealed that the
control and satellite DNA molecules preferentially formed linear
multimers during the ligation, and nearly all of the radioactivity was
sensitive to ExoIII digestion upon termination of the experiment. In
contrast, both the wild-type and mutant circular structures from intron
D preferentially formed closed monomer circles that were resistant to
ExoIII digestion. In addition, the rate of cyclization of the wild-type
sequence was 3.2 ± 0.4-fold faster than that of the mutant
sequence, as revealed by four replicate gel experiments of the type
shown in Fig. 6, and 2.7-fold faster, as seen from the ExoIII
experiments shown in Fig. 7.
Figure 6:
Analysis of the ligation products. The
panels show autoradiograms of the ligation products of the restriction
fragments shown in Fig. 5. Ligation conditions are described
under ``Experimental Procedures.'' Samples were removed at
the indicated times after addition of ligase and electrophoresed on a
2% agarose gel. The samples at time 0 were removed prior to the
addition of the ligase, and those designated as E were treated
with exonuclease III following 15 min of ligation. LT, linear
trimer; LD, linear dimer; DC, dimer circle; LM, linear monomer; MC, monomer
circle.
Additional evidence for the
preferential cyclization of the intron segment was obtained by carrying
out ligations in the presence of EcoRI-digested C. elegans genomic DNA (Fig. 8). In panelA, the The
torsional alignment of the two ends of a DNA molecule can influence the
rate of cyclization (Shore and Baldwin, 1983a, 1983b). However, it is
unlikely that this effect is responsible for marked differences in
cyclization efficiencies reported in Fig. 6Fig. 7Fig. 8. For example, the wild-type and
mutant intron segments displayed clear differences in cyclization rates
as shown in Fig. 6and Fig. 7, but both sequences have
the same number of nucleotides and essentially the same number of
helical turns as calculated from dinucleotide twist values (18.87 versus 18.85 turns). In addition, the failure of the control
and satellite fragments to readily cyclized in the studies shown in Fig. 6was expected since noncircular molecules in this size
range resist cyclization, especially under the conditions of high DNA
concentrations which favor bimolecular associations (Shore and Baldwin,
1981). To further rule out this possibility, ligation reactions were
carried out in the presence of varying concentrations of ethidium
bromide in order to alter the twist of control and circular molecules
(Shore and Baldwin, 1983b). As shown in Fig. 9A,
ethidium bromide had no noticeable effect on the preferential
cyclization of the intron sequence when high DNA concentrations were
used in the reaction mixtures. Likewise, cyclization of the intron
sequence but not the control exon fragment was seen at all levels of
the drug when the DNA concentrations were reduced by about 350-fold (Fig. 9B). We also note that ethidium bromide did not
reduce the R
Figure 9:
Effects of ethidium bromide and
temperature on ligation reaction products. Labeled exon (pJA71-2) and
intron (pJA46-2) segments were ligated in the presence of
ethidium bromide (A and B) or at various temperatures (C) for 15 min. A, the labeled DNAs (1 µg/ml)
were ligated in the presence of nonradioactive EcoRI-cut
recombinant plasmids (350 µg/ml). The concentrations of ethidium
were: 0, 0, 10, 20, 40, and 200 ng/ml. Ligase (5 units/ml) was added to
samples in lanes 2-6. B, the procedure
described in A was used, except the nonradioactive DNA was
omitted from the ligation mixture. C, samples were ligated in
the presence of nonradioactive plasmids but in the absence of ethidium
at 5, 10, 20, 25, and 37 °C for lanes 1-6,
respectively. Higher concentrations of ligase were used (50 units/ml)
to ensure complete ligation at all temperatures. Positions of the
linear and circular monomers (LM, CM) are
indicated.
The studies in Fig. 10and Fig. 11focused on a large circular structure from phage Non-bent molecules that are
shorter than the DNA persistence length of about 150-200 bp
resist cyclization in a ligation reaction (Shore et al.,
1981). Consequently, the rate of cyclization of small DNA molecules
with intrinsic curvature can be several orders of magnitude greater
than the cyclization rate of a nonbent molecule of the same length (Koo et al., 1990; Crothers et al., 1992). The effects of
sequence-dependent curvature on the cyclization of larger molecules
should be more difficult to demonstrate since nonbent large fragments
will readily cyclize during a ligation reaction (Shore et al.,
1981). In order to investigate the cyclization of the predicted
circular structure from The standard computer program used in this work for
predicting DNA structure from nucleotide sequence assumes that AA/TT
steps are the major determinants of bending as has been proposed from
numerous experimental findings (Crothers et al., 1990;
Diekmann et al., 1992; Hagerman, 1992; Haran et al.,
1994; Wang et al., 1994; Sprous et al., 1995). A
precondition for predicting structure by this program is that bending
possibly caused by non AA/TT stacks should not significantly alter the
interpretations of the analysis. This is apparently the case since the
circular structure in the HIV-1 integrase gene (Fig. 1E), as well as other selected circular
structures (data not shown), were also observed when the sequences were
analyzed by a program that incorporated predicted wedge values for all
dinucleotides. In addition, we have recently shown that programs which
assume that AA/TT is the only source of bending were better predictors
of electrophoretic data than a program based on all wedge values (Wang et al., 1994). Recent electrophoretic studies by Haran et
al.(1994) have also stressed the relative unimportance of non
AA/TT stacks in bending and critical discussions of non-A tract bending
models for curvature are provided by Haran et al.(1994), Wang et al.(1994) and Sprous et al., 1995). The standard
program has previously been shown to reproduce the shapes of short DNA
molecules seen in electron micrographs and to accurately predict the
planarity of curvature as determined from electrophoretic studies
(Eckdahl and Anderson, 1987; Fitzgerald et al., 1994; Wang et al., 1994) and from ligation analysis of small synthetic
circular DNAs (data not shown). The results of this study provide
additional support for the predictive power of the program and
consequently for the model of A tract bending since the circular intron
segment from C. elegans preferentially cyclized in ligation
reactions when compared to nonbent fragments, bent fragments with
noncircular shapes, and total genomic DNA (Fig. 5Fig. 6Fig. 7Fig. 8). In addition,
the wild-type intron sequence cyclized significantly faster than a
mutant sequence with a single base substitution and the difference was
reflected in differences in electrophoretic anomaly, ENDS ratio values,
and helical projections ( Fig. 5and Fig. 6). Ring closure
probabilities ( DNA inversion systems in prokaryotes have served as models for the
study of site-specific recombination (Glasgow et al., 1988;
Heichman and Johnson, 1990; Stark et al., 1992; Van de Putte
and Goosen, 1992; Merker et al., 1993). Typically, a
recombinase binds as a dimer to each of two inverted repeat sequences
and dimers of Fis interact with two sites within a transpositional
enhancer located nearby. Protein-induced DNA bending and
Fis-recombinase interactions then facilitate the looping of intervening
DNA to form a large multi-protein complex called the invertasome.
Accessory DNA-bending proteins such as the histone-like proteins HU and
IHF also aid in particle formation by facilitating the folding of the
DNA (Surette et al., 1989; Harrington, 1992). Thus, the
ability of DNA to loop in the invertasome should not only depend on the
position and orientation of specific protein binding sites but also on
the physical properties of the intervening DNA including its
flexibility and intrinsic curvature. Support for this idea has been
derived from the observation that intrinsically bent DNA can substitute
for protein-induced bending in many systems including inversion
reactions (Bracco et al., 1989; Goodman and Nash, 1989).
Consequently, the short segments of bent DNA found in recombinational
enhancers have been viewed as factors that facilitate protein-induced
bending and thus the formation of the invertasome (Johnson et
al., 1987; Glasgow et al., 1988;
Hübner et al., 1989). The preferential
association of high ENDS ratio peaks with a variety of inversion and
long-range looping systems (Fig. 4, A-H) makes it
likely that the large circular structures play an analogous but perhaps
more global role in particle formation. This model could also be
applied to the sequences in Fig. 4(I-N), which
are thought to display regional recombinational specificity. In these
systems, the large circular structures may direct recombinational
complexes to the regions where strand cleavage and rejoining take
place. The circular structures described in this report may play
some role in recombination in addition to a general packaging function.
A consideration of such a role is particularly relevant since the
requirement for a highly ordered DNA topology in the invertasome has
led to the view that the DNA molecules are active participants in
recombination rather than merely passive substrates for protein action.
An important characteristic of inversion systems is the requirement of
a negatively supercoiled DNA substrate that facilitates protein-protein
interaction and the formation of the invertasome. Restriction nuclease
studies and electron microscopy have revealed that bent DNA is
positioned within end loops of supercoiled molecules (Silver et
al., 1986; Laundon and Griffith, 1988). Thus, bent DNA can order
the global structure of superhelical DNA (Silver et al., 1986;
Laundon and Griffith, 1988; Kremer et al., 1993). It seems
likely that this organizing effect would depend on the configuration of
the bent segment since O- and C-shaped structures should preferentially
reside at the ends of interwound molecules while bent structures such
as S-shaped regions should be preferentially excluded from these sites.
These considerations imply that the circular DNA shapes have the
potential to align DNA sites both within and adjacent to protein
complexes on domains of supercoiled DNA and this could be important in
controlling DNA topology during recombination and other long range
looping events. The circular structures in the primate
immunodeficiency viruses were detected as the highest ENDS ratio
sequence set in the entire GenBank data base (Fig. 1Fig. 2Fig. 3). This structure is not found
in all retroviruses, but is apparently more conserved than sequence
(see Fig. 1and Fig. 2and ``Results''), which
implies that selective pressures have maintained the circular form. To
our knowledge, this region is not a hot spot for recombination,
although a role for the structure in this process cannot be excluded.
Perhaps more likely, the structure functions in a DNA looping mechanism
which serves to bring together the protein binding sites located in the
two distinct subdomains of the intragenic transcriptional enhancer (see Fig. 1). According to this view, the structure may have a
packaging function analogous to the circular elements in Fig. 4(A, D, E, G, and H). The presence of multiple functional AP-1 recognition
sequences in the upstream enhancer subdomain of HIV is of interest
since these sequences are frequently components of complex regulatory
elements, which contain binding sites for multiple transcription
factors (Sonnenberg et al., 1989; Kerppola and Curran, 1991).
Several potential binding sites for transcription factors other than
AP-1 are indeed found in this region of the HIV genome (Van Lint et
al., 1991). Thus, the DNase I-hypersensitive site that resides at
this location in HIV-1 chromatin (Verdin, 1991) may be a reflection of
a complex regulatory particle assembled onto the circular DNA segment.
The DNA structure is expected to be regulated since unintegrated linear
viral DNA has a single-stranded gap at this site, which would prevent
circle formation prior to integration. In addition, the enhancer and
the nuclease-hypersensitive site are regulated by cellular factors
following the integration of the HIV genome into host cell chromatin
(Verdin et al., 1990; VanLint et al., 1991; Verdin,
1991). Although the function of this highly unusual conformation in
regulating the HIV life cycle is not yet known, the structure may
provide a model system for the study of a regulatable looping mechanism
that is dependent on the intrinsic circularity of the DNA.
Volume 270,
Number 40,
Issue of October 06, pp. 23570-23581, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
C substitution in one of these sequences rendered it less planar
as seen by computer analysis and significantly reduced its rate of
ligase-catalyzed cyclization. These results permit us to speculate that
intrinsically circular structures facilitate DNA looping during
formation of the large protein-DNA complexes that are involved in site-
and region-specific recombination and in other genomic processes.
)in length. However, A tract bending has the
potential to give rise to complex higher order structures, which may be
important in regulating genome packaging and function. For example, the
spatial organization of bending elements and straight DNA segments in
replication origins and promoters is thought to be an important feature
in the initiation of replication (Eckdahl and Anderson, 1987; 1990) and
transcription (McAllister and Achberger, 1989;
Pérez-Martin, 1994). Similarly, a multi-domain
structure of nucleosome positioning DNA has been proposed to be a
determinant for the deposition of nucleosomes at specific sites in
chromatin DNA (Drew and Travers, 1985; Fitzgerald et al.,
1994). It is generally assumed that intrinsic curvature facilitates the
wrapping of DNA around regulatory and structural protein complexes, and
it seems likely that the intrinsic shape of the curved DNA plays some
role in the assembly mechanism. Thus, a description of higher order DNA
structures beyond the level of simple curved loci may be needed for a
complete understanding of the biological relevance of bent DNA.
Computer Analysis
The standard program for
calculating DNA bending from nucleotide sequence has been previously
described (Eckdahl and Anderson, 1987). The program incorporates the
dinucleotide twist angles reported by Kabsch et al.(1982) and
uses the parameters of the wedge model for bent DNA (Ulanovsky and
Trifonov, 1987). The program assumes that bending occurs only at AA/TT
and uses wedge values estimated by Ulanovsky and Trifonov,(1987). A
derivative program, which incorporates wedge values for all 16
dinucleotides (Bolshoy et al., 1991), has also been described
previously (Wang et al., 1994) and was used for the analysis
in Fig. 1E. The programs calculate three-dimensional
coordinates of the helical axis along a sequence and then compute the
ENDS ratio from these coordinates. The ENDS ratio is used to quantify
predicted bending and is defined as the ratio of the contour length of
a DNA segment to the shortest distance between the ends of the segment.
) and NF-
B binding sites (
), which are located within
the two subdomains of the intragenic transcriptional enhancer (Verdin et al., 1990; Van Lint et al., 1991). Also shown are
the positions for the internal DNase I-hypersensitive site ( 
) and the central polypurine tract ( 
). The position of the circular structure
(see below) is indicated by the heavyline above I. A, two-dimensional projections of the helical axis
of the HIV-1 proviral DNA. Each successive projection is rotated by 60
degrees. The positions of the 5` end of the proviral DNA, the start (S) and end (E) of Gag (G), Pol (P), and Env (E), and the circular structure at the
end of Pol (C) are indicated along the projections. B, ENDS ratios were computed at window widths of 20-200
bp (top), 20-400 bp (middle), and 20-1000
bp (bottom) at a window step of 10 bp along the sequence. C, two-dimensional projections of the S-shaped bend at the
beginning of RT (nucleotides 2639-2807). D and E, two-dimensional projections of the circular structure in I. Each projection in C-E is rotated by 36
degrees. The projections and ENDS ratios in A-D were
calculated by the standard program, while the projections in E were calculated by a program that incorporates all 16 dinucleotide
wedge angles.
) and the
mean of the highest ENDS ratio peaks per data base sequence (+)
are shown for the indicated window widths. At a window width of 1500
bp, the corresponding ENDS ratios were 1.7 and 2.9. Each of the 135
sequences that displayed an ENDS ratio peak >15 is indicated by a
symbol in the top portion of the graph, and the window width at which
the peak maximum was observed is given on the x axis. Symbols
represent peak centers found in untranslated DNA (), protein
coding DNA (), coding DNA within 200 bp of the end of a gene
(
), and the integrase regions of HIV-1, HIV-2 or SIV (*). The
two structures indicated by the arrows are described under
``Results.''
), 1.19-1.25 (
), and >1.25 (
); 400-bp
window: 1.35-1.60 (
), >1.60 (); windows from 400 to
1100 bp: 3-20 (openbox) and >20 (bold
open box). The mean ± S.D. ENDS ratio peaks for randomized
sequences at window widths of 120 and 400 bp were 1.05 ± 0.03
and 1.14 ± 0.10, respectively. B-D,
two-dimensional projections of the helical axes of the integrase
regions of HIV-1 (RF isolate), HIV-2 (ISY isolate), and SIV (sooty
mangebey). The length, central positions, and ENDS ratios of the
depicted sequences are, respectively: (B) 820, 4320, 86; (C) 1000, 4620, 100; (D) 1060, 5070, 36. There is
60-65% nucleotide sequence identity between B and C and between B and D.
switch region (Sµ) from mouse (Sakano et
al., 1980). The maps adjacent to each set of projections show the
positions of the circular elements (-), recombinase cleavage
sites ( ), enhancers (), and genes
(). Heavilylinedboxes indicate
genes for site-specific recombinases. The recombinational enhancers
bind either Fis (A, D, and E) or Mu
transposase A (G), and the replication enhancer in H binds the P1 initiator protein. Note that a circular structure
terminates within 200 bp of each enhancer. Multiple recombination sites
in I are located in the region between the twoarrows (Matsuoka et al., 1990). Scale bars are
given in the rightlowercorner of each
panel. Middlepanels, sites within telomeric
recombination systems located: J, in a VMP expression site of Borrelia hermsii (Barbour et al., 1991); K and L, in a VSG expression site of Trypanosoma brucei (Pays et al., 1990; Boothroyd and Cross, 1982); M and N, in the left-terminally located variable region of
the swine fever virus (Almendral et al., 1990;
González et al., 1990). Bottompanels, mitochondrial plasmids and other reverse
transcriptase coding entities from: O-R, Type II (RT)
introns (Wahleithner et al., 1990; Wissinger et al.,
1991; Lazowska et al., 1980; Siemeister et al.,
1990); S, ORF5 of the carnation etched virus (Hull et
al., 1986); T and U, S-1 and S-2 plasmids from
CMS-S Zea mays mitochondria (Paillard et al., 1985;
Levings and Sederoff, 1983); V and W, kinetoplast
plasmid minicircles of Leishmania tarentolae and Crithidia
fasciculata (Kidane et al., 1984; Ray et al.,
1986). The coordinates of the ENDS ratio peaks in sequences A-W are given in ``Experimental
Procedures.''
General DNA Techniques and
Electrophoresis
Standard molecular biological techniques were
used for DNA manipulations (Sambrook et al., 1989).
Double-stranded DNA sequencing was performed by the dideoxy chain
termination method (Sanger et al., 1977) using a Sequenase kit
from U. S. Biochemical Corp. Oligonucleotides were synthesized by the
Purdue University Oligonucleotide Synthesis Facility. Unless otherwise
indicated, DNA fragments were analyzed on 6% native polyacrylamide gels
(29:1 acrylamide/bisacrylamide) run at 4 °C in TAE (0.04 M Tris acetate (pH 8.3), 3 mM EDTA) or on agarose gels run
at room temperature in TAE in the presence of 1 µg/ml ethidium
bromide. The apparent size of fragments on polyacrylamide was estimated
by comparing mobilities of fragments to a 123-bp ladder (Life
Technologies, Inc.) and to HaeIII digests of pUC18.Preparation of DNA Molecules and Ligation
Reactions
An EcoRI fragment corresponding to the
circular structure detected by computer in intron D of the RNA
polymerase II gene from Caenorhabditis elegans (accession no.
M29235) was prepared by PCR. Plasmid DR36 (Bird and Riddle, 1989)
containing this segment was kindly provided by Dr. David Bird
(University of California, Riverside). An XhoI/BamHI
fragment from the plasmid was used as a template to amplify the 192-bp
segment (bp 3809-4000), and the primers were designed such that
the PCR product could be inserted into the EcoRI site of
pUC18. EcoRI-digested recombinant plasmids isolated from
transformed Escherichia coli (DH5-
) were analyzed by
electrophoresis on polyacrylamide gels and then by DNA sequencing in
order to identify wild-type and mutant sequences. Of the 32
characterized plasmids, one point mutation was recovered; we presume
that it was introduced during PCR. The mutation differs from the
wild-type sequence by a single T C substitution at position
3856. Ligation products of wild-type and mutated fragments were
compared to the ligation products of EcoRI fragments from
plasmid pSV2 cat (Gorman, 1985) (bp 4752 to 5006) and PJA71-2. PJA71-2
is a 213-bp fragment from an exon of the RNA polymerase II gene (bp
6332 to 6530). Ligations were also carried out with bent satellite
monomers from Tenebrio molitor (Plohl et al., 1990), Panagrellus redivivus (de Chastonay et al., 1990),
and Columbia riscoria (Fitzgerald et al., 1994).
Satellite monomers were isolated from EcoRI-digested genomic
DNA and cloned in pUC18. Genomic DNA was isolated as described
previously (Van Wye et al., 1991).
-
P]ATP using T4 polynucleotide kinase as
described by Drak and Crothers(1991). Unless indicated otherwise,
labeled DNAs (1.0 µg/ml) mixed with nonradioactive EcoRI-digested recombinant plasmids (350 µg/ml) were
equilibrated at 5 °C in 30 mM Tris (pH 7.8), 10
mM MgCl
, 10 mM dithiothreitol, and 1
mM ATP. These high DNA concentrations favor multimerization of
noncircular fragments (Zahn and Blattner, 1987). Unless otherwise
indicated, ligation was initiated by the addition of T4 DNA ligase
(Promega) to a final concentration of 5 units/ml. Samples were removed
at the indicated times and added to stop buffer (66 mM EDTA,
5% glycerol, 0.05% bromphenol blue) in order to quench the reaction.
One ligated sample set was treated with exonuclease III (ExoIII; 0.5
units/µl) for 1 h at 37 °C prior to its addition to stop
buffer. Ligated products were resolved on 2% agarose gels in TAE
containing ethidium bromide (Shore et al., 1981). Gels were
subsequently dried and autoradiographed. genome (bp 870-1750) was
amplified and cloned as described above using BamHI-digested
DNA as the template in the PCR. The ligation products of this
880-bp segment were compared to the ligation products of control
(noncircular) EcoRI fragments from the troponin I (Koppe et al., 1989) and
-prothymosin (Schmidt and Werner, 1991)
genes, which are 700 and 1192 bp in length, respectively. EcoRI-digested recombinant plasmids (20 µg/ml) were
equilibrated at 0-1 °C in 30 mM Tris (pH 7.8), 10
mM MgCl, 10 mM dithiothreitol, 1
mM ATP, and 1.8 M sucrose. Ligation was initiated by
the addition of T4 DNA ligase. The final concentration of ligase was 10
units/ml unless otherwise indicated. In some experiments, 10-µl
samples of the ligation reaction were removed at 3-120 min after
the addition of ligase, mixed with stop buffer, and electrophoresed on
1.3% agarose gels containing ethidium bromide. DNA in the gels was
transferred to nitrocellulose, and the blots were probed with
nick-translated insert DNAs. In other experiments including the one
shown in Fig. 11, ligation reactions contained all three
digested plasmids (20 µg/ml each) and 0.5-ml samples were removed
at the indicated times and added to 1 ml of ethanol. Following
precipitation, DNA was dissolved in electrophoresis sample buffer and
resolved on 1.3% agarose gels containing ethidium bromide. Photographic
negatives of the gels and blots were scanned by computer, and DNA forms
were quantified using the NIH image 1.52 program. Open circular, closed
circular, and linear forms were identified by comparing the gel
mobilities of DNA samples treated and not treated with ExoIII, by the
restriction nuclease test described in Fig. 2of Shore et
al.(1981), and by polyacrylamide gel electrophoresis (Ulanovsky et al., 1986) (data not shown). Photographic negatives of
ethidium bromide-stained gels are presented in Fig. 5, Fig. 8, and Fig. 11.
-prothymosin gene (A), the 880-bp segment from phage
shown at the top of Fig. 10(B), and the 700-bp
troponin I gene (C) were incubated with T
DNA
ligase for the indicated times. Samples were electrophoresed on a 1.3%
agarose gel in the presence of ethidium bromide, and a photographic
negative of the gel is shown on the left. An enlargement of the bottom
portion of the negative is shown in the center of the figure. L, linear monomer; OC, open circular monomer; CC, closed circular monomer. The rightpanel shows the ratio of circular (OC + CC) to
linear monomers as a function of ligation time for A (), B (), and C ().
tract to TCT.
Relative length (R
) is defined as the ratio of the
apparent length to the actual length. Apparent lengths were determined
by electrophoresis on 6% polyacrylamide gels. ENDS ratios were
calculated at a window width that corresponded to the actual length of
the fragment. Each successive projection of the helix is rotated by 36
degrees. The 5% gels at the bottom of the figure were run at the
indicated temperatures. Lanes A-E are, respectively:
pSV2cat, pJA670, pJATM5, pJA46-2, and pJA46-13. LaneM shows markers.
P-labeled intron segment (1
µg/ml) were incubated together in the absence (lanes1 and 2) or in the presence (lanes3 and 4) of ligase (30 units/ml) for 30 min at 4 °C and then 30
min at 22 °C. Samples in lanes2 and 4 were treated with Exo prior to electrophoresis on a 2% agarose
gel. A negative of the ethidium-stained gel and its autoradiogram are
shown (B). The
P-labeled intron segment (1
µg/ml) was incubated alone (lanes1 and 2) or with size fractionated C. elegans genomic DNA
(100 µg/ml, lanes 3-8) in the absence (odd-numbered lanes) or in the presence (even-numbered
lanes) of ligase as in A. The sizes of the genomic DNA
fragments were 150-200 bp (lanes3 and 4), 200-300 bp (lanes5 and 6), and 300-500 bp (lanes7 and 8). C, procedures are the same as in A,
except
P-labeled genomic DNA (1 µg/ml) was mixed with
an EcoRI-digested plasmid containing the intron (500
µg/ml) prior to ligation. Arrows indicate the positions of
the 198-bp nonligated intron segment.
Computer Analysis of Retroviruses
A map of the
human immunodeficiency virus type I (HIV-1, strain HXB2) is shown at
the bottom of Fig. 1B. Given are the positions of the
three long open reading frames (gag, pol, and env) and the locations of regulatory elements in the vicinity
of the integrase gene at the end of pol. These elements
include a transcriptional enhancer with two subdomains containing AP-1
and NF-B binding sites (Verdin et al., 1990; Van Lint et al., 1991) and the central polypurine tract which serves as
an initiation site for synthesis of the plus strand HIV DNA (Hungnes et al., 1991; Charneau et al., 1992; Hungnes et
al., 1993). A single-stranded gap that flanks this tract is seen
in unintegrated copies of the linear double-stranded viral DNA
(Charneau and Clavel, 1991). A major DNase I-hypersensitive site is
found between the enhancer subdomains when the provirus is packaged
into cellular chromatin (Verdin, 1991).Computer Analysis of Cellular Sequences
A search
of all sequences in the GenBank data base containing >1000
nucleotides was carried out as in Fig. 1B in order to
estimate the frequency of high ENDS ratio peaks in cellular DNA. The
results in Fig. 3show ENDS ratio values of the highest peaks
plotted against the window widths that gave the maximal peak values.
Data base sequences were scanned at window widths from 20 to 1100 bp,
and an ENDS ratio value >15 was used as the criterion for a peak.
This value is greater than 20 standard deviations above the mean of the
data base sequences and greater than 10 standard deviations above the
mean of the highest ENDS ratio peak in each data base sequence (Fig. 3). No ENDS ratio peaks >10 were seen when 1500 kbp of
randomized sequences were subjected to the analysis, which also
illustrates the unusual nature of the peak structures in natural DNA. A
total of 135 peaks were observed in the 10,823 data base entries, but
this frequency is an overestimate of circular structures of the type
shown in Fig. 2since visual inspection of the helical
projections revealed that only about half of the peak sequences
displayed planar bending throughout their lengths (data not shown). All
18 peak sequences with lengths between 160 and 580 bp were found in
untranslated regions of the genome. We presume these elements are
excluded from coding DNA because the closely spaced oligo(A/T) tracts
needed to generate such peaks would impose severe coding constraints.
Peak sequences with lengths from 620 to 1100 bp also displayed a
preference for untranslated DNA, although significant fractions resided
in sites that overlap coding and noncoding regions or in more central
areas of coding DNA.-ori, and the resulting
DNA-protein complex then loops to and activates the
-ori located 1.2 kbp downstream. A circular structure is seen at
the site of loop formation in the intervening DNA between the
-
and
-oris and its 5` end terminates in the -enhancer
element (Fig. 4H). Class switching of immunoglobulin
heavy chain constant regions (C
) occurs through a
recombinational event in switch regions located 5` to each C (reviewed by Coffman(1993)). The most common form of switching
involves the looping out and excision of chromosomal DNA between the
most 5` switch region (Sµ) and one of the switch regions located
further downstream along the chromosome. Switch recombinational sites
are clustered within a region at the 5` edge of the Sµ segment and
this region is immediately downstream of a circular structure in the
mouse genome (Fig. 4I).
) and mutant () intron segments from Fig. 5were
incubated with 5 units/ml ligase for the indicated times. Exo-resistant
radioactivity was then measured following acid precipitation. Results
are expressed as the means ± S.E. of the percentages of the
valves for DNA not treated with Exo. Rightpanel,
data replotted as in Crothers et al.(1992) where (D)t is the concentration of Exo-sensitive
radioactivity (unreacted monomer) at time t. Exo-sensitive
radioactivity is equated with unreacted monomer because autoradiograms
of these samples following electrophoresis revealed >98% of the
radioactivity was either unreacted or circular monomer (not shown).
Cyclization rate constants k were 5.9
10 and 2.2
10 s
for the wild-type and mutant sequences,
respectively.
)Three of the four genes were
from pathogens, as were most of the bacterial recombinational systems
shown in Fig. 4. To our knowledge, these genes and ori sequences have not been tested for recombinational or looping
activities. Many of the bacterial sequences with ENDS ratio peaks
between 15 and 40 were also from pathogens, and the circular structures
were most often found in or near genes that encode toxins, membrane
proteins, or proteins that form filamentous appendages. Pathogenic
bacteria frequently display phenotypic variations of their surface
components and toxins and genetic recombination is a prevalent
mechanism that mediates these phenotypic changes (reviewed by Robertson
and Meyer(1992)). Thus, it is conceivable that these systems also
exhibit phenotypic variation mediated by recombination and the circular
DNA structures could play a role in the process.Experimental Analysis of Selected Structures
Two
high ENDS ratio peaks with lengths of less than 200 bp were identified
in the search of the GenBank data base (see Fig. 3). One
sequence was from kinetoplast minicircles of C. fasciculata,
and this K-DNA has been visualized as a circular structure in electron
micrographs (Griffith et al., 1986). This sequence appeared as
a nearly perfect circle when analyzed by the computer program (Fig. 4W). The other sequence was found within intron D
of the RNA polymerase II gene of C. elegans. Experiments were
performed using this intron segment in order to provide additional
evidence for the existence of small circles in natural DNA. Fig. 5gives some of the characteristics of the EcoRI
fragments used in the work. Two of the fragments (pJA71-2 and pSV2CAT)
have electrophoretic properties and ENDS ratio values that are within
the ranges of those seen for the bulk of natural DNA (Anderson 1986;
Eckdahl and Anderson, 1987; VanWye et al., 1991; Fitzgerald et al., 1994). The three satellite sequences display
significant bending, and the sequence from dove has nearly as many
phased oligo(A) tracts as are found in K-DNA and in the C. elegans intron segment (Fitzgerald et al., 1994). However, the
dove satellite monomer is composed of two bent loci, which are out of
phase with each other by approximately one-half helical turn. This
gives rise to the computer-generated S-shaped structure shown in the
figure (Fitzgerald et al., 1994). The 198-bp intron segment
has an R value (apparent chain length/real chain
length) which is similar to that of the K-DNA sequence (Kitchin et
al., 1986). The magnitude of the electrophoretic anomaly displayed
by these segments is greater than those reported for all other natural
sequences of this length. The temperature dependence of the gel anomaly
was also similar to K-DNA. The R values for both
sequences declined by about 50% upon increasing the temperature from 5
°C to 35 °C (Diekmann and Wang, 1985; Fig. 5). Mutations
in the intron sequence were recovered following PCR amplification and
cloning. The computer-generated structure of one sequence carrying a
point mutation that converts a T tract to TCT is shown in the figure. The helical projections of this sequence
are less planar then those displayed by the wild-type sequence, and
this difference is reflected in a reduction in the ENDS ratio and a
small but reproducible reduction in electrophoretic anomaly.
P-labeled intron segment was mixed with a 1000-fold mass
excess of C. elegans DNA prior to ligation. Gel analysis of
the ligation products revealed cyclization of the intron segment
persisted even in the presence of excess genomic DNA. A similar
analysis in panel B showed that the cyclization of the intron segment
was not noticeably effected by 100-fold excess levels of genomic DNA
fragments that ranged in size from 150 to 500 bp. PanelC shows the reverse approach where trace amounts of
P-labeled genomic DNA were ligated in the presence of an
excess of an EcoRI-digested plasmid containing the intron
sequence. The preferential cyclization of the intron sequence when
compared to the genomic DNA is also apparent from this analysis.
valve of the intron segment when the
fragment was electrophoresed in the presence of 0.3 and 0.7 µg of
this drug/ml (data not shown). Cons and Fox(1990) have similarly noted
that ethidium bromide failed to alter the anomalously slow gel mobility
of K-DNA. Preferential cyclization of the intron sequence was also
observed at ligation temperatures ranging from 5 °C to 37 °C in
the absence of ethidium bromide (Fig. 9C). Temperature
variations within this range, like ethidium, alter the helical twist of
DNA molecules of this size (Shore and Baldwin, 1983b). Taken together,
these results and the observations mentioned above strongly suggest
that differences in the orientation of the ends of DNA molecules in Fig. 5are not responsible for the preferential ligation of the
intron sequence.
.
The 880-bp circular structure is found in the A gene which codes for
the major subunit of the
DNA terminase. The enzyme cleaves the
terminal cos site and also plays an active role in the
packaging of DNA into proheads (Cue and Feiss, 1993). The
sequence was characterized by an ENDS ratio peak of 18 which was the
highest value observed for sequences in the phage subdivision of
GenBank. Non-overlapping fragments from this region displayed a
pronounced electrophoretic anomaly which was attributed to phased
oligo(A) tracts that were found throughout this segment of the genome
(Anderson, 1986). The sequence appears to belong to a set of relatively
high ENDS ratio peaks which lie within the G+C-rich left arm of
the genome. As noted in Fig. 10, major structural variations are
seen near the beginning and end of the left arm which contains the head
and tail structural genes, while minor variations are observed in the
vicinity of the head-tail junction.
shown in Fig. 10, the rate of
cyclization of an EcoRI fragment that corresponds to the
structure was compared to the rates of cyclization of two noncircular
molecules of similar lengths. In Fig. 11, plasmids containing
each of the sequences were digested with EcoRI and mixed
together prior to ligation. The reaction was carried out using low DNA
concentrations in order to favor cyclization of all DNA molecules. In
an attempt to minimize random motions of the molecules, ligations were
carried out at 0-1 °C in the presence of 1.8 M sucrose. The results revealed that the cyclization of the
segment (designated B) occurred faster than the cyclization of the
control molecules A and C. This can be seen by the preferential loss of
linear monomer in B and the selective increase in the corresponding
circular monomer during the course of the reaction. Results similar to
those shown in the figure were seen in all five experiments where this
procedure was used, and in all seven experiments where the three
plasmids were ligated in separate reactions and the products detected
either by ethidium bromide staining or by blot hybridization analysis
(data not shown). The preferential cyclization of the
sequence
also occurred using varying amounts of ligase (1-20 units/ml) but
was not observed in the absence of sucrose in the ligation mixture
(data not shown).
)could not be accurately determined for any
of the molecules shown in Fig. 5because the intron segments
displayed very low rates of bimolecular association, which could not be
quantified reliably, while the noncircular fragments did not cyclize or
cyclized very slowly under all reaction conditions tested (Fig. 6, Fig. 7, and Fig. 9; data not shown).
However, relative cyclization efficiencies could be estimated by making
assumptions that have made by others who have encountered similar
problems with short synthetic DNA molecules (Crothers et al.,
1992). We estimate that the relative cyclization efficiency of the
wild-type intron segment was about 3-fold greater than the mutant
intron segment and more than 100-fold greater than all of the
noncircular molecules shown in Fig. 5. These estimates were not
affected by variation in DNA concentration, ligase concentration,
ethidium bromide, or temperature (Fig. 6Fig. 7Fig. 8Fig. 9; data not shown).
The sequence encoding the phage terminase also cyclized
significantly faster than noncircular molecules of similar lengths and
this sequence displays predicted planar bending throughout its 880-bp
length ( Fig. 10and Fig. 11). Additional studies along
these lines are needed in order to further characterize the shapes and
stabilities of such large structures in solution. The identification of
an ensemble of sequences with circular helical projections as in Fig. 1Fig. 2Fig. 3Fig. 4provides a rich
source of natural molecules that could be used for such an analysis.
)))
We thank Dr. David Bird, University of California,
Riverside, California, for plasmid DR36, Dr. B. J. Taparowsky for PSV2
cat, and Sonia Nasr for the plasmid containing the satellite monomer of T. molitor. We also thank Drs. D. Asai, B. Wanner, I. Tessman,
S. Nasr and P. T. Gilham for reviewing the manuscript, Joy Sheridan for
assistance with the manuscript, and Dr. P. T. Gilham for the idea of
using sucrose in ligation reactions shown in Fig. 11.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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