Vol.

172,

October

No. 30,

2, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages 402-408

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.

402

Vol.

172.

No.

2,

BIOCHEMICAL

1990

DNA bending per se been demonstrated

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

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

Vol. 172, No. 2, 1990

BIOCHEMICALAND BIOPHYSICALRESEARCHCOMMUNICATIONS

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

BIOCHEMICALAND BIOPHYSICALRESEARCHCOMMUNICATIONS

Vol. 172, No. 2, 1990

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

Vol.

172, No. 2, 1990

BIOCHEMICAL

BSSHII WTINV

AVLI WT

BStNI

INV

WT

AND BIOPHYSICAL

SfaNI INV

WT

SphI

ECORI IN”

WT

IN”

RESEARCH COMMUNICATIONS

PstI

WTIN”

WT

IN”

-396

-344

-298

-220

-154

12

3

4567

8

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

Vol.

172, No. 2, 1990

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

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

Vol.

172, No. 2, 1990

DNA fragments

containing

BIOCHEMICAL

AND BIOPHYSICAL

these sequences

RESEARCH COMMUNICATIONS

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.

408

Induction of an altered DNA conformation by an inversion rearrangement in the 5'-flanking DNA of a U1 RNA gene.

The anomalous electrophoretic behavior of a 686 base pair restriction fragment containing an in vitro-generated inversion mutation within the enhancer...
2MB Sizes 0 Downloads 0 Views