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1990
INDUCTION OF AN ALTERED DNA CONFORMATION BY AN INVERSION REARRANGEMENT IN THE S-FLANKING DNA OF A Ul RNA GENE Kenneth Department San Diego Received
A. Roebuck*
and William
of Chemistry and Molecular State University, San Diego,
September
6,
E. Stumph Biology Institute California 92182
1990
The anomalous electrophoretic behavior of a 686 base pair restriction fragment containing an in vitro-generated inversion mutation within the enhancer region of a chicken Ul RNA gene was investigated. This DNA fragment migrated with an abnormally slow mobility in polyactylamide gels but migrated normally in agarose gels relative to the wild type fragment of identical size and base composition. In polyacrylamide gels, the degree of retardation was enhanced at low temperature, a phenomenon associated with bent DNA. A putative site of bending was localized at or near one end of the inverted region. These data suggest that the altered DNA conformation results from the juxtaposition of two normally remote DNA sequences. 0 1990Academic press, 1°C. The local structure of the DNA double helix and its path through space is modulated
by the nucleotide
sequence
of the molecule. Duplex DNA containing
repeated stretches of dA.dT at ten to eleven base pair (bp) intervals exhibit an anomalous migration in polyacrylamide gels (1). This unusual property was first observed for kinetoplast DNA and was suggested to result from curvature of the DNA helix (2). This intrinsic bending of the DNA helix is presumed to hinder the reptation of the molecule through the pores of polyacrylamide fragments
matrix (3). In contrast, most bent
migrate through the more porous matrix of agarose gels at rates close to
that expected for their size (2, 4, 5). Several elegant studies have convincingly demonstrated that adenine tracts, typically 4-6 nucleotides in length and phased along the same side of the DNA helix, can result in intrinsic DNA bending (5-8). A variety of physicochemical techniques including electric dicroism (9), hydroxyl radical cleavage (lo), circularization (1 l), and electron microscopy (12) have since proven the existence of bent DNA. Moreover, bends in DNA are associated with a number of biological functions chromatin
including site-directed
organization
recombination
(13), DNA replication
(15), and gene regulation (16). Only recently, however, has
* To whom correspondence should be addressed. Present address: Department of Medicine M-023-D, University of California, San Diego, La Jolla CA 92093. pbbreviations: bp, base pair(s); CAT, chloramphenicol acetyltransferase; WT, wild type; INV, inversion. 0006-291X/90 Copyright All rights
(14),
$1.50
0 1990 by Acudemic Press, Inc. of reproductiorl in my fbrm reserved.
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DNA bending per se been demonstrated
AND
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to be directly involved in DNA functions (17,
18). In this report, we have investigated 686 bp restriction fragment enhancer
containing
the anomalous
electrophoretic
an inversion rearrangement
behavior of a within the
region of a chicken Ul RNA gene. Based upon its unusual gel migration
properties, we conclude that the mutant fragment has an altered DNA conformation consistent with DNA bending. Because the altered structure mapped to a region near one end of the inverted segment, our results suggest that the altered DNA conformation arose from the juxtaposition of two normally remote DNA sequences. Moreover, the sequences
at or near the junction indicate that a mechanism
(or in addition to) phased adenine tracts must be responsible apparent bend.
MATERIALS
AND
other than
for the formation
of the
METHODS
Gel electroohoresis Polyacrylamide gels were run under native conditions and had an acrylamide concentration of either 4% or 6% and an acrylamide:#, AI’methylenebisacrylamide ratio of 19:l or 29:1, respectively. They were run in 40 mM Tris-acetate (pH 8.3) 20 mM Na-acetate, 2 mM EDTA at either 6V or 2V per cm. Agarose gels were run in the same buffer at 5V per cm. DNA fraoments The two 686 bp Pvull fragments were derived from two plasmids previously described (19). Briefly, pSP64 vectors contained Ul promoter sequences (positions -388 to +3 relative to the transcription initiation site of the Ul-52a RNA gene [20]) attached to the 5’ end of CAT (chloramphenicol acetyltransferase) coding sequences. In both chimeric constructions Ul sequences from -369 to -314 had been deleted. In the plasmid containing the inversion mutation, the segment from -309 to -204 (bounded by natural BssHII sites) had been deleted and then recloned in the opposite orientation. The structures of the two Pvull fragments, which are identical outside of the inversion, are shown in Fig. 1.
RESULTS A DNA fraament electroohoretic
containina
an internal inversion exhibits an anomalous
mobilitv. During the course of characterizing
the promoter region of a
chicken Ul RNA gene (20, 21), we noticed that a Pvull restriction fragment a short inversion
rearrangement
containing
within the Ul enhancer exhibited an anomalous
electrophoretic
mobility. This mutant fragment
polyacrylamide
gels than the corresponding
wild type and mutant Pwll fragments,
migrated more slowly in
wild type fragment.
designated
The structures
WT and INV respectively,
of the
are
shown in Figure 1. It should be noted that these two 686 bp fragments are identical in size and base composition, but the mutant fragment contains an inversion of the 105 bp segment flanked by the two BssHlI restriction sites. When plasmids containing
the
mutant and wild type configurations were digested with Pvull and electrophoresed together in an agarose gel under standard conditions, the Pvull fragments exhibited an indistinguishable
electrophoretic
mobility (Fig. 1, compare lanes 1 and 2). 403
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WT pspsvector __* f Fvun
enhancer I Ul
promter
CAT 685 bP
[ Pvull Bsthd
Awl
Sfml
INJ$!Y.cE psptjvector b
ui pmmter
CAT -
bP
Pull 1
2
3
4
EstM
S&l
Aval
Fiaure 1. Structures and gel mobility properties of the wild type and mutant Pvull fragments. Plasmids containing the wild type configuration (WT) or the inversion mutation (INV) were digested with Pvull and electrophoresed in a 1.5% agarose gel (lanes 1 and 2) or in a 4% polyacrylamide gel (lanes 3 and 4) under standard conditions (i.e., room temperature; 5-6V/cm). The DNA bands were visualized by ethidium bromide staining. (The 686 bp Pvull fragments migrate near the bottom of the gel.) At the right, the structures of the WT (top) and INV (bottom) 686 bp fragments are diagrammed along with a restriction enzyme map. Solid boxes represent chicken Ul RNA gene sequences; open boxes represent non-U1 DNA sequences (i.e., vector and CAT DNA); and hatched boxes represent Ul DNA sequences that have been inverted in the INV fragment by in vitro mutagenesis. However, in polyactylamide different electrophoretic
gels the two smaller Pvull fragments
pattern. Under these conditions,
displayed
the fragment
a quite
containing the
inverted segment (INV) exhibited a markedly slower electrophoretic mobility relative to the wild type (WT) fragment (Fig. 1, compare lanes 3 and 4). This differential mobility suggests that the DNA fragment altered DNA conformation, The anomalous
presumably
containing the inversion mutation has an a bend.
mioration of the mutant fraament is temperature
been shown that DNA bending is enhanced temperatures
tend to reduce the abnormal
To study the nature of the phenomenon the mutant fragment,
at lower temperatures,
dependent.
whereas higher
migration in polyacrylamide
responsible
It has
for the anomalous
gels (5, 8, 22). migration of
we digested the mutant and wild type fragments with Psfl and
Sphl, which both cleave 3’ of the inverted segment. We then compared of the resulting subfragments
in 6% polyacrylamide
the migration
gels run at 5OC and at 37OC, to
assess whether the abnormally slow migration varies with temperature. The double digestion produced three matching pairs of subfragments (designated fl , f2 and f3 in Figure 2), but only the one subfragment containing the inverted BssHII segment (fl) exhibited a retarded mobility relative to the wild type. The other two subfragments (f2 and f3) each co-migrated
normally under these conditions.
In addition, the migration
of the subfragment containing the inverted segment was noticeably slower (relative to wild type) at 5OC than at 37OC, indicating that the anomaly is temperature dependent, as would be expected for bent DNA. The locus responsible for the anomalous
migration maps near the upstream end of
the inversion rearranoement. For fragments containing a locus of DNA bending, it has been shown that their relative mobility in polyacrylamide gels is dependent upon the 404
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50 W-f
37O
INV
WT
INV
396 344
-
298
fl
-
f2
-
396
220 -220
13 -
123
4
123
A Fiaure 2. Electrophoretic high and low temperature.
4
B mobilities
of the
WT and
INV fragments
at
The WT (lanes 2) and INV (lanes 3) Pvull fragments were co-digested with Sphl and Psfl and electrophoresed in a 6% polyacrylamide gel run at 2V/cm for 120h at 5OC (panel A) or at 37OC (panel B). Markers (pBR322 digested with Hinfl) were included in lanes labeled 1 and 4. Note that three subfragments (fi, f2, and f3) were produced by the double digestion, but only the 11 subfragment contained the inverted region.
location of the bend within the DNA molecule (4, 5). Fragments near the center tend to migrate slower in gels than fragments
containing
in which the bend is
positioned close to one of the ends. To map the DNA sequences anomalous
a bend
associated with the
migration, we digested the mutant and wild type Pvull fragments
with
various restriction enzymes to generate a series of subfragments that contained the inverted DNA segment at different internal positions. The resulting subfragments were then analyzed pair-wise in a 6% polyacrylamide gel run at low temperature. From the autoradiogram shown in Figure 3, it is apparent that for each pair of restriction digestions an anomalous mobility was observed only in the case of the specific subfragments containing the inverted BssHII segment. All other subfragments migrated normally in these gel conditions. Moreover, the most retarded bands were those in which the upstream boundary of the inverted segment was positioned at or near the center of the fragment. Fragments generated by digestions with Pstl (lanes 13 and 14), SfaNl (lanes 7 and 8), and Sphl (lanes 11 and 12), each of which contain the upstream end of the inversion near the center, migrated substantially slower than the corresponding wild type fragments of identical length. In contrast, nearly normal electrophoretic
mobility was exhibited by fragments
generated
with BsrNl (lanes 5
and 6), EcoRl (lanes 9 and lo), and Aval (lanes 3 and 4), each of which contain the 405
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AVLI WT
BStNI
INV
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PstI
WTIN”
WT
IN”
-396
-344
-298
-220
-154
12
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4567
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9
10
11
12
13
14
Fiaure 9. Mapping the location of the DNA sequences associated with the anomalously slow migration. The WT (lanes 1, 3, 5, 7, 9, 11, and 13) and INV (lanes 2, 4, 6, 8, 10, 12, and 14) Pvull fragments were end-labeled with [32P-r) ATP and T4 polynucleotide kinase, gel purified, and digested with various restriction enzymes as indicated above each lane. (The conditions of the BssHll digestion were chosen to yield incomplete digestion products). The resulting subfragments were resolved by electrophoresis in a 6% polyacrylamide gel run at 5OC for 120h at 2V/cm. Positions of size markers (MM-digested pBR322) are indicated to the right. The digestions in each case produced two labeled bands except for the partial BssHll digestion which produced four labeled bands in addition to the uncut Pvull fragment. The small labeled subfragments generated by the EMNI and SfaNl digestions ran off the gel.
upstream end of the inversion site near the end of the restriction fragment. Interestingly, in the BssHll partial digestion (lanes 1 and 2) both types of subfragments were produced, i.e. a subfragment containing the upstream end of the inversion near the middle and one with it near the end, but again only the one 406
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containing this site near the center of the fragment exhibited an altered electrophoretic
mobility (second fragment from bottom in lane 2). Taken together,
these results localize the sequences associated with the anomalous migration to a region at or near the upstream end of the inversion mutation. This implies that the juxtaposition
of specific DNA sequences
the formation
at the upstream
BssHll site has resulted in
of a bend in the DNA.
DISCUSSION We have investigated
the anomalous
migration of a 686 bp restriction fragment
that contains an inversion rearrangement
generated
in vitro within the promoter
region of a chicken Ul RNA gene. Because this mutant fragment abnormally
slow mobility in polyacrylamide
and because the degree of retardation that the fragment Furthermore,
presented sequences
was enhanced
at low temperature,
we believe
with the inversion mutation contains a site of DNA bending.
the locus of DNA bending maps to the upstream
inversion rearrangement. natural sequence
migrated with an
gels relative to its wild type counterpart
boundary of the
In several cases, it has been shown that mutations in a
can reduce or abolish DNA bending (16). However, in the work
here, a novel site of DNA bending was apparently within a DNA fragment
created by rearranging
that does not exhibit anomalous
mobility in its wild
type configuration. How might this bend structure form? In several well studied examples,
DNA
bending results from the periodic phasing of multiple adenine tracts (5-8). It is conceivable
that an inversion of DNA sequences
could bring together
such phased
adenine tracts. However, Figure 4 shows that such a pattern is not obvious in the mutant fragment.
In fact, the region encompassing
the upstream inversion junction is
very GC rich and contains only a few sporadic adenine/thymine tracts none of which is longer than three residues. Nevertheless, from the results presented in Figs. 1-3,
Fiaure 4. DNA Sequence of the region containing the inversion rearrangement in the INV fragment. The sequence of the INV fragment from the EcoRl restriction site to the Sphl site is shown in approximate relation to the turn of the DNA helix (i. e., 10 bp per turn). The two BssHll sites, which form the boundaries of the inverted region, are highlighted by diagonal boxes. Adenine and thymine tracts three residues or greater in length are in bold print. The positions of EcoRI, Aval, and Sphl restriction sites are pointed out by the diagonal underlines. Note that the sequence is very GC rich and contains no phased adenine or thymine tracts. 407
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DNA fragments
containing
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behave anomalously
gels. Thus, in the example reported here, a novel mechanism addition to) adenine tracts appears to be responsible
in polyacrylamide
other than (or in
for the induction of the bend.
ACKNOWLEDGMENTS We thank John C. Weng for preparing the graphics used in Figs. 1 and 4. This work was supported
by a grant (GM3351 2) from the National Institutes of Health and in part
by the California Metabolic Research Foundation. predoctoral
KAR at the time of this work was a
student receiving support from the SDSU Department
of Biology.
REFERENCES 1. Diekmann, S. (1987) in: Eckstein, F., and Lilly, D. (Eds.) Nucleic Acids and Molecular Biology, vol 1, Springer, Berlin, pp 138-l 56. 2. Marini, J. C., Levene, S. D., Crothers, D. M., and Englund, P. T. (1982) Proc. Natl. Acad. Sci. USA 79, 7664-7668. 3. Levene, S. D., and Zimm, B. H. (1989) Science 245, 396-399. 4. Wu, H.-M., and Crothers, D. M. (1984) Nature 308, 509-513. 5. Diekmann, S., and Wang, J. C. (1985) J. Mol. Biol. 186, l-1 1. 6. Koo, H. S., Wu, H.-M., and Crothers, D. M. (1986) Nature 320, 501-506. 7. Hagerman, P. J. (1985) Biochemistry 24, 7033-7037. 8. Diekmann, S. (1986) FEBS Letters 195, 53-56. 9. Levene, S. D., Wu, H.-M., and Crothers, D. M. (1986) Biochemistry 25, 39883995. 10. Burkoff, A. M., and Tullius, T. D. (1987) Cell 48, 935-943. 11. Kotlarz, D., Fritsch, A., and But, H. (1986) EMBO J. 5, 799-803. 12. Griffith, J., Bleyman, M., Rauch, C. A., Kitchin, P. A., and Englund, P. T. (1986) Cell 46, 717-724. 13. Hagerman, P. J. (1986) Nature 321, 449-450. 14. Zahn, K., and Blattner, F. R. (1985) EMBO J. 4, 3605-3616. 15. Ebralidse, K. K., Grachev, S. A., and Mirzabekov, A. D. (1988) Nature 331, 365367. 16. Bossi, L., and Smith, D. M. (1984) Cell 39, 643-652. 17. Synder, U. K., Thompson, J. F., and Landy, A. (1989) Nature 341, 255-257. 18. Goodman, S. D., and Nash, H. A. (1989) Nature 341, 251-254. 19. Roebuck, K. A. (1989) Ph.D. Thesis, University of California, San Diego and San Diego State University. 20. Roebuck, K. A., Walker, R. J., and Stumph, W. E. (1987) Mol. Cell. Biol. 7, 418541 93. 21. Roebuck, K. A., Szeto, D. P., Green, K. P., Fan, Q. N., and Stumph, W. E. (1990) Mol. Cell. Biol. 10, 341-352. 22. Diekmann, S. (1987) Nucleic Acids Res. 15, 247-265.
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