the segment inversion site of herpes simplex virus type 1 adopts

THE .JOURNAL O F BIOLOGICAL CHEMISTRY I C 1987 hy The American Society of Biological Chemists, Inc.

Vol. 262, No. 13, Issue of May 5. pp. 640744115. 1987 Printed in U. S A

The Segment Inversion Site of Herpes Simplex Virus Type 1 Adopts a Novel DNA Structure*

(Received for publication, December 15, 1986)

Franz Wohlrab, Michael J. McLean, and Robert D. Wells From the Department of Biochemistry, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, Alabama 35294

The 12-base pair (bp) tandem direct repeat sequences (DR2) at the joint region (a sequence) of herpes simplex virus type 1 (strain F) adopt a new type of DNA conformation under the influence of negative supercoiling. The novel conformation is dependent on the number of the DR2 repeats; the 19 mer (228 bp total) and the 14 mer (168 bp) readily form the alternate structure whereas pentamer, trimer, and dimer repeats show somewhat different properties. S1 and P1 nuclease studies reveal that the new conformation has a major structural aberration at its center and conformational periodicities which are not identical on the complementary strands. Also, the effect of salt and pH, the location of reaction with bromo- and chloroacetaldehyde, the type of sequence (direct repeat) involved, and the nature and extent of supercoil-induced relaxations dem- onstrate that this structure differs from previously recognized conformations including left-handed 2 helices, cruciforms, bent DNA, and slipped structures. We propose the existence of a novel conformation, anisomorphic DNA, with different structures on the complementary strands which elicit a structural aberration at the physical center of the tandem sequences. Since the oligopurine. oligopyrimidine sequence may be inher- ently inflexible, this supercoil-induced structural change and the physical stress on these inserts in recombinant plasmids tend to deform (crack) the DR2 sequences at their centers. Possible roles for anisomorphic DNA in the functions of this segment of intense biological activity are proposed.

Herpes simplex virus type 1 (HSV-1)’ is a large enveloped DNA virus which replicates in the nucleus of the infected cell (1). Its genome (Fig. 1) consists of approximately 155 kilo- bases of double-stranded DNA and can be divided into two segments, S and L. Both of these segments are flanked by inverted repeats (b and c, respectively), and the ends of the genome carry a set of tandem repeats (designated a) of vari- able length, which is also present at the L-S junction in inverted orientation (2,3). Upon passage of the virus through cells, high frequency inversions of the two segments relative to each other occur, and the progeny virus population thus consists of four isomeric forms of linear virion DNA which differ in the relative orientation of the unique sequence com-

* This work was supported by Grants GM 30822 and P30 CA 13148 from the National Institutes of Health and by Grant PCM 800 2622 from the National Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

’ The abbreviations used are: HSV-1, herpes simplex virus type 1; pur .pyr, oligopurine. oligopyrimidine sequences; bp, base pair(s).

ponents. It has recently been demonstrated that both the signal for packaging and cleavage of the concatemeric repli- cative form of the viral DNA as well as the signal for segment inversions reside within the a sequence (4-6). More precise analysis of this region located the signal for segment inversion in or near a set of directly repeated sequence components within the a region. This set of tandem repeats, designated DR2, is a highly conserved region which, depending on the strain examined, consists of 12 or 11 nucleotides which are repeated 8 to 23 times (7-9). This repeat is very rich in G+C (92%) and shows a high bias for purines on one of the strands (92%); a closely related and neighboring repeat (DR4) shows similar properties.

It is now well established that DNA is a highly polymorphic molecule in which different secondary structures can coexist side by side (for reviews see Refs. 10 and 11). This structural flexibility suggests that DNA conformation carries biological information. It is likely that a number of protein recognition sites on the DNA are defined by DNA structure rather than its primary sequence which can vary from an ideal consensus to more or less closely related sequences with altered abilities to adopt certain secondary structures. The types of non-B DNA structures that have been observed under physiological conditions include: left-handed 2-DNA, which is found prin- cipally at certain alternating purine-pyrimidine sequences (10-14); cruciforms which occur at inverted repeats (15, 16); slipped structures which were postulated to occur at direct repeats (17, 18); and bent DNA (19) detected in fragments from kinetoplast DNAs (20, 21) and certain genetic control regions (22-24); and a new structure found at some oligopurine .ohgopyrimidine sequences (pur. pyr) (25-28). Negative supercoiling at physiological densities stabilizes all of these unusual structures except bent DNA (15, 17, 27, 29, 30).

These types of sequences were identified as non-B structures on the basis of their high sensitivity to attack by certain nucleases ( i e . S1 nuclease), which were originally isolated on the basis of their ability to degrade single-stranded nucleic acids, as well as other determinations. Although the a sequence of HSV-1 does not strictly fit into any of the above mentioned categories, it might be considered as a special case of a polypurine.polypyrimidine sequence.

In the course of an evaluation of the capacity of segments of the HSV-1 genome to adopt unorthodox conformations, we found that the DR2 segment of the joint region, a site of intense biological activity, adopts a novel DNA structure induced by supercoiling with properties unlike those observed previously for left-handed Z-DNA, cruciforms, bent DNA, or pur. pyr sequences.

MATERIALS AND METHODS

Plosmids”pRW790 (13) is a 1990-bp derivative of pBR322 carrying the pUC12 multiple cloning site as an EcoRI-Hind111 fragment

6407

6408 HSV-1 DR2 Sequences: Anisomorphic DNA A L S a b b' a' c1 c 0

I pRW 1201

!-- pRW 1202

p R W 1203

B pRW12Ol

pRW1202

pRW1203

pRWl214

pRW1212

pRW1250

... CGTTTT (CGCTCCTCCCCC),,CGGTCC.

..CGTTTT (CGGTCCTCCCCC),,CGCTCC ...

.CGTTTT (CGCTCCTCCCCC)5 CGCTCC.

... GATCCC (CGCTCCTCCCCC), CGCTCCTCCCCC CGGATC ...

... GATCCC CGCTCCTCCCCC CGCTCCTCCCTC CGATC.. .

... CATCCC CGCTCCTCCCCC CGCC CGCTCCTCCCCC CGATC.

FIG. 1. Structures of the plasmids used in this study. Panel A, structure of the L-S junction region of HSV-1. The top line represents the physical map of HSV-1 (1). L, S, long and short segment, respectively; TRL, TRs, IRL, IRs, terminal and inverted repeats; b, b', c, c', inverted repeats flanking the unique sequences UL and US; a, a' , terminal direct repeats. The second line is a schematic representation of the structure of the L-S junction. The 19 DR2

present in pRW1201 and the deletion mutants pRW1202 and repeats and the 3 DR4 repeats are shown as open boxes. The sequences

pRW1203 are represented at the bottom of the figure as straight lines. The 12-bp sequence of the DR2 repeat is CGCTCCTCCCCC, and the 37-hp sequence of the DR4 repeat is CGCTCCTC4GCTC3GCGGC4- GC,AACGCC. Panel B, DNA sequences of the plasmid inserts used in this work. The insert in pRW1202 is in the opposite orientation compared to the other plasmids.

and has a large deletion encompassing the tetracycline resistance gene. The deletion extends from pBR322 coordinate 45 to 2434.'

pRB601 was a gift of Dr. B. Roizman (University of Chicago) and consists of the HSV-1 (strain F) L-S junction sequence as a HaeII fragment in a pACYC177 vector (7). Subcloning of the 440-bp SrnaI fragment containing the DR2 repeats into the unique SmaI site of pRW790 gave pRW1201 (Fig. 1).

We also characterized two deletion mutants of pRW12Ol which arose spontaneously by passage of the plasmids in Escherichia coli. In both cases, deletions occurred within the DR2 and DR4 regions yielding SmaI junction fragments with lengths of 265 (pRW1202) and 165 bp (pRW1203), respectively (Fig. 1).

For the construction of plasmids pRW1212, pRW1214, and pRW1250, the following strategy was employed. Two dodecameric oligodeoxynucleotides, d(CCCGCTCCTCCC) and (dCGGGGGGAG- GAG), containing the DR2 nucleotide sequence, were synthesized on an Applied Biosystems 380A oligonucleotide synthesizer using the phosphoramidite method. After purification of the single strands on denaturing polyacrylamide gels, they were annealed to each other under conditions described elsewhere (13). The resulting double strands possessed 4-bp overhangs which allowed formation of head- to-tail oligomers. The fragments were then phosphorylated using T4 polynucleotide kinase (Pharmacia P-L Biochemicals). Ligation was performed in a total volume of 20 pl containing 2 pg of 12-mer duplex, 5 mM MgCl,, 10 mM spermidine, 10 mM Tris.HC1 (pH 7.6), 1 mM

~~~ - - F. Wohlrab. unpublished data.

ATP, and 2 units of T4 DNA ligase (Boehringer Mannheim). Reac- tion times were 30 min and 24 h at 15 "C, respectively. After electrophoresis on polyacrylamide gels, the synthetic DR2 oligomers were eluted and purified. The ends were filled in using dCTP and dGTP in the presence of the Klenow fragment of E. coli DNA polymerase (Boehringer Mannheim) and resulting fragments were inserted into the filled-in BarnHI site of pRW790. All recombinant plasmid inserts were characterized by DNA sequencing using the chemical degradation method (31). The sequences of pRW1212, pRW1214, and pRW1250 are shown in Fig. 1B.

Nuclease Digestions-S1 nuclease from Aspergillus oryzae and P1 nuclease from Penicillium citrinum were obtained from Bethesda Research Laboratories or Boehringer Mannheim. Digestions were carried out in a total volume of 50 p1 at a DNA concentration of 30 pg/ml. Reaction conditions for S1 nuclease were essentially identical to those described by Singleton et al. (29), namely 30 min at 37 "C in 40 mM sodium acetate (pH 4.6), 50 mM NaC1, 1 mM ZnS04, and 1.5 units of enzyme/pg of DNA except where noted otherwise. Reactions were stopped by placing the mixtures on ice and adding EDTA to a final concentration of 30 mM. The samples were then microdialyzed against restriction buffer (29), and the position of the SI nuclease cut(s) was mapped by restriction endonuclease digestion. P1 nuclease digestions were performed in 50 mM Tris . HCI (pH 7.5) (or in 50 mM Tris acetate at pHs 6.0, 7.0, 7.5, and 8.0), 50 mM NaCI, 10 mM MgCl,, and 0.1 unit of enzyme/pg of DNA for 5 min a t 37 "C. Reactions were stopped by addition of EDTA to a final concentration of 30 mM, followed by microdialysis into restriction buffer as described above. For mapping purposes, the DNA was then digested with an appropriate restriction endonuclease and analyzed on agarose or polyacrylamide gels alongside appropriate size markers.

Fine Mapping of SI Nuclease-induced Nicks-Supercoiled DNA was digested with S1 nuclease as described above except that 10 units of enzyme/pg of DNA were used, and reaction times were between 0.5 and 2 min. After dialysis, the DNA was cut with an appropriate restriction endonuclease and 5'-end laheled using T4 polynucleotide kinase. Following a second digestion with another restriction enzyme, the labeled fragments were separated on polyacrylamide gels. The location of the S1 nuclease-specific nick was then determined by electrophoresis on denaturing polyacrylamide gels in parallel with Maxam-Gilbert sequencing ladders. Autoradiograms of the gels were scanned with a Joyce-Loebl densitometer.

Generation of Topoisorners-Topoisomers were generated following the procedure of Singleton and Wells (32). Briefly, supercoiled plasmids (40 pg/ml) were treated with wheat germ topoisomerase (gen- erously provided by Dr. R. R. Burgess, University of Wisconsin) or calf thymus topoisomerase (gift of J. E. Larson, this laboratory) in a buffer containing 50 mM Tris. HCI (pH 7.61, 10 mM MgCI,, 0.1 mM dithiothreitol, 2 mM spermidine, and ethidium bromide in concentrations from 0 to 20 pg/ml for 3 h in the dark at room temperature. Bound ethidium bromide was determined by measuring the specific fluorescence enhancement of the samples in a Perkin-Elmer spectro- fluorimeter. After removal of the intercalator by phenol/chloroform extraction, the DNA was ethanol precipitated and dissolved in 10 mM Tris.HCI (pH 7.5), 0.1 mM EDTA to give a final concentration of 250 pg/ml. The calculated superhelical density was confirmed by analysis of the samples on 2% agarose gels containing varying concentrations of chloroquine phosphate (Sigma) (33).

Brornoacetaldehyde Reactions-Bromoacetaldehyde was prepared by a modification of the method described by Kohwi-Shigematsu et al. (34). Briefly, hromoacetaldehyde diethylacetal was added to an equal volume of 0.1 M H2S04 and gently refluxed for 1 h. After cooling, the reaction mixture was distilled and the fractions with boilingpoints between 75 and 80 "C were collected and pooled. NaOH was added to give a final pH of 7.0, and the product was extracted three times with t-butyl-methyl ether. The organic phase was dried with MgS04, filtered, and evaporated under a gentle stream of air to give a final ratio of t-butyl-methyl ether:bromoacetaldehyde of approximately 2:l. This mixture was stored in aliquots at -70 "C. For reactions, the t-butyl-methyl ether was removed under a gentle stream of air, and the residue was determined to be 2 M hromoacetaldehyde. Reaction of bromoacetaldehyde with plasmids and analysis of bromoacetaldehyde-sensitive sites was essentially as describedpreviously (35). Briefly, 2 pg of plasmid in 100 pl of 10 mM Tris.HC1, pH 7.6, 50 mM NaC1,lO mM MgC1, was incubated at 37 "C for 2 hours in the presence of 1% bromoacetaldehyde. The DNA was extracted 3 times with ether and then recovered by precipitation and digested by an appropriate restriction endonuclease. 15 pl of S1 nuclease buffer (40 mM sodium acetate, 50 mM sodium chloride, 1 mM zinc sulfate, pH

HSV-1 DR2 Sequences: Anisomorphic DNA 6409

4.6) containing 1.5 units of S1 nuclease was then added, and incuba- tion was at 37 "C for 15 min. The reaction was terminated by addition of EDTA (pH 7.5) to 50 mM, and the DNA fragments were separated by agarose gel electrophoresis. Chloroacetaldehyde (gift of Dr. Leo Hall, this department) reactions were performed in a similar manner.

Two-dimensional Gel Electrophoresis-Mixtures of topoisomer populations prepared as described above were subjected to gel electrophoresis (36) in 2% agarose at 3 volts/cm in 40 mM Tris.HC1 (pH 8.3), 80 mM sodium acetate, 1 mM EDTA (TAE buffer) with recir- culation of buffer. The gel was then soaked in TAE buffer containing 5 VM chloroquine phosphate for 2 h. Second dimension electrophoresis at 3 volts/cm was carried out at a 90" angle in TAE buffer containing 5 PM chloroquine phosphate. DNA spots were visualized by staining with ethidium bromide. In some cases, topoisomers were analyzed in 89 mM Tris-borate (pH 8.3), 1 mM EDTA (TBE buffer).

RESULTS

Plasmids Containing HSV-1 L-S Junction Fragments-A family of recombinant plasmids containing segments of the a region involved in virus packaging and cleavage as well as segment inversion was prepared and characterized. The inserts containing portions of DR2 and, in some cases, DR4 were cloned into pRW790, which is a 1990-bp derivative of pBR322. The use of this small vector enhances separation of topoisomers during gel electrophoresis, thereby facilitating analysis of potential structural transitions which are induced by negative supercoiling.

Fig. 1B shows the sequences of the recombinant plasmids used in this study. The first three were constructed by insertion of the intact SmaI fragment spanning the L-S junction of HSV-1 (strain F) from pRB601 into pRW790. In some clones, deletions were observed. pRW1201 contains 19 repe- titions of DR2 joined to three repeats of DR4 (of which DR2 is a subset), and pRW1202 and pRW1203 carry 14 and 5 copies of DR2, respectively, but have lost all but part of one of the DR4 repeats. Hence, the length of the SmaI fragment present in pRW1201 is 440 bp, the one in pRW1202 is 265 bp, and the one in pRW1203 is 165 bp. The deletions were found on replication of the plasmids in E. coli thus indicating an inherent instability of this long direct repeat sequence; similar instabilities have been documented for sequences that can adopt left-handed Z structures (37,38) or cruciforms (39). Interestingly, note that integral numbers of the direct repeat sequences are naturally deleted.

The studies described below show that a previously unrec- ognized type of DNA structure is induced by negative supercoiling in the DR2 repeats. Hence, it was important to inves- tigate the influence of the number of repeat units on the properties of the DNA molecules. Accordingly, a second set of plasmids was constructed using synthetic oligodeoxynucleotides as inserts (Fig. 1B). These plasmids contain synthetic DR2 repeats flanked solely by vector sequences. Mul- timers of a synthetic dodecanucleotide containing the DR2 sequence were ligated into the filled-in BamHI site of pRW790. pRW1214 contains four copies of the 12-bp DR2 sequence but has one C to G transversion 4 bp from the 3'- end. pRW1212 contains two copies of the 12-bp DR2 sequence but has one C to T transition in the penultimate position from the 3'-end. Last, pRW1250 contains two perfect copies of the 12-bp DR2 sequence but has an insertion of CGCC in the center. The reasons for our inability to isolate perfect multimeric copies of the 12-bp sequence are unknown but are, no doubt, related to the deletions found for pRW1201-1203. Thus, in general, cloning of these synthetic molecules revealed an unusually high mutation frequency at the 3' end of the pyrimidine-rich strand. In fact, two independently isolated recombinant plasmids containing a dimeric insert in opposing orientations had acquired mutations at the same position

(data not shown). The reasons for this behavior are unknown but may be related to the unorthodox conformation generated by these sequences and the cellular events invoked to cope with it.

SI Nuclease Recognition of DR2 Repeats-S1 nuclease has been used widely for the recognition and specific cleavage of cruciforms (15, 16,40,41); junctions between right-handed B and left-handed Z conformations (13, 29, 41-43), slipped structures (17, 18), and unusual non-B DNA structures adopted by certain homopurine. homopyrimidine sequences (28, 30, 44-48). In general, it is believed that S1 nuclease recognizes an aberration in the duplex DNA structure and not single-strandedness (35):' for the B-Z junctions and for pur. pyr structures.

Fig. 2 shows the results found when the negatively supercoiled recombinant plasmids containing the HSV-1 DR2 repeats (Fig. 1B) were treated with S1 nuclease. The position of the S1 nuclease cleavage sites were then mapped with BglI which cleaves the pRW790 vector once. Treatment of pRW1201 resulted in the appearance of two bands which mapped the nuclease-hypersensitive sites to the DR2 repeats (Fig. 2). The breadth of the bands indicates reaction of the enzyme at multiple sites within the 19-copy insert. This result suggests that the DR2 repeats exist in a non-B DNA structure.

Similar studies (Fig. 2) on the other four plasmids which contain fewer copies of the repeat sequence also showed the presence of S1 nuclease-hypersensitive sites which mapped to the DR2 inserts. However, the resulting bands were substan- tially sharper than for pRW1201 indicating the presence of fewer sites. It, therefore, appears that as few as two copies of the DR2 repeat are sufficient for the presence of a nuclease- sensitive structure and that the presence of a transversion, transition, and insertion in pRW1214, 1212, and 1250, respectively, is tolerated by the unusual conformation. Further- more, similar results were obtained with the parent plasmid pRW601 (not shown) indicating that sensitivity to S1 nuclease attack is not a consequence of vector size. The plasmids used in these experiments had negative superhelical densities in the physiological range, namely 0.04-to 0.06 supercoil/ helix turn.

Interestingly, in the case of pRW1201, a 15-bp alternating purine-pyrimidine stretch, 11 bp of which is perfectly alternating dG-dC, is located close to the DR4 repeats. In pRW1202 and pRW1203 this stretch is present close to the

1201 1202 1203 1214 1212 1250 """

M - + " f - + - + - + - +

1100 - 900 -

FIG. 2. SI nuclease-hypersensitive sites in the plasmids. S1 nuclease treatment of natively supercoiled plasmids (approximately -0.06 supercoil density) was performed as indicated under "Materials and Methods." The number on top of the panel is the plasmid designation as shown in Fig. 1. S1 nuclease-treated plasmids were cleaved with BglI and analyzed on a 1% agarose gel. Lanes marked with minus are controls in which the nuclease treatment was omitted. The first lane is a marker lane (123-bp ladder marker from Bethesda Research Laboratories), and the arrows at the side show the positions of the 900- and 1100-bp fragments, respectively.

' M. J. McLean, J. E. Larson, F. Wohlrab, and R. D. Wells, manuscript in preparation.

6410 HSV-1 DR2 Sequences: Anisomorphic DNA

deletion end point (Fig. 1). These types of sequences have been shown to adopt left-handed Z structures under the influence of negative supercoiling (13). However, this sequence was not recognized by S1 nuclease in any of these three plasmids. This result indicates a hierarchy of S1 nuclease cleavage sites in the plasmids, namely that the unusual conformation adopted by the DR2 sequences is a preferential substrate for S1 nuclease to the putative B-Z junctions (41) or is formed at a lower supercoil density.

The absence of inverted repeat sequences in the region of interest rules out the formation of cruciform structures as an explanation for the S1 nuclease sensitivity.

Effect of pH on Nuclease Recognition-Studies were also conducted with P1 nuclease in place of S1 nuclease as the probe for unusual structures. P1 nuclease was employed previously (49, 50) as a probe a t neutral pH values for other unusual conformations. P1 nuclease reactions were performed on negatively supercoiled pRW1201 and pRW1214 at several pHs (6.0, 7.0, 7.5, and 8.0). Specific cleavage at the DR2 inserts was found in all cases; the cleavage was supercoil dependent. Hence, low pH (4.6) and zinc ions are not required for the new unusual structure. This finding distinguishes the present structure from the conformation described for

At pH values above 5.2, no S1 nuclease specific cleavage was found, even with ten fold excess enzyme. However, the pronounced requirement for low pH by this enzyme may be the explanation for this behavior. Also, when MgC12 was replaced with 1 mM ZnSO, in the P1 nuclease studies, no reaction was found.

In summary, the new unorthodox structure adopted by the DR2 sequences is readily recognized by P1 nuclease a t neutral pH in the presence of MgC12. However, it is uncertain if the conformations are identical under the S1 and P1 nuclease conditions.

Influence of Supercoiling-To explore the nature of the structures present in the insert, the influence of negative supercoiling on S1 nuclease specific cleavage of the DR2 repeat regions was investigated. Families of topoisomers of pRW1214 with different average negative supercoil densities were prepared and assayed for S1 nuclease specific cleavage as described. Fig. 3A shows that under low torsional stress, no nicking was observed. At negative supercoil densities of approximately 0.06 and above, specific cuts are introduced at the DR2 region. At high torsional stress (lunes f and g), a band (1600 bp) corresponding to the major cruciform site in the plasmid vector appears. Control experiments using linearized plasmids as substrates showed the absence of S1 nuclease specific bands (not shown).

Similar studies were performed on four of the other DR2- containing plasmids (Fig. 1B). Fig. 3B demonstrates that the S1 nuclease-hypersensitive structure is formed a t approximately the same superhelical densities for all plasmids studied. This finding is extraordinary with respect to all previously described unusual conformations which are induced by negative supercoiling. A pronounced influence of length and type of DNA sequence on the amount of negative supercoiling required was found for cruciforms (15, 41, 51), left-handed Z- DNA (13, 14, 29, 41-43, 52-55), and pur.pyr sequences (26, 27, 48).

The supercoil energy needed to produce S1 nuclease sensitivity (-0.06 supercoil/turn) is higher than needed to stabilize left-handed Z-DNA in alternating dC-dG sequences. Depend- ing on the length of the oligo(dC-dG) block, the minimum threshold superhelical density for the B-to-Z transitions is -0.03 (for blocks of about 60 bp) to -0.06 (for blocks of about

poly(dG-dA)-poly(dC-dT) (28).

-0- FIG. 3. Influence of negative supercoil density on S1 nu-

clease cleavage. Panel A, pRW1214 topoisomer populations were treated with S1 nuclease followed by digestion with BglI as described under “Materials and Methods.” Lanes a through g represent populations of increasing negative superhelical densities. The median values are: a, 0.00; b, 0.02; c, 0.04; d, 0.055; e, 0.065; f, 0.080; g, 0.088. M , markers of 123-bp ladder (Bethesda Research Laboratories). Panel B, plot of S1 nuclease-specific cleavage of pRW1201, filled square; pRW1202, filled circle; pRW1203, open square; pRW1214, open tri- angle; and pRW1212, open circle versus negative superhelical density. The amount of cleavage is in arbitrary units which approximately corresponds with the percent of DNA cleaved and is not strictly comparable between plasmids.

-

8 bp) supercoil/turn (13,41, 53, 56). The amount of supercoiling needed to extrude cruciforms

from inverted repeats in recombinant plasmids depends on the length and nature of the sequence (57). For the major cruciform in pRW790, the required superhelical density is approximately -0.055 (13). Therefore, in the plasmids studied here, the transition to an S1 nuclease-sensitive structure occurs at supercoil densities comparable to but somewhat lower than those required for the major vector cruciform and, in the case of pRW1201,1202, and 1203, the 14-bp alternating purine-pyrimidine stretch located in the insert. The apparent breadth of the transition (Fig. 3B) presumably stems from the distribution of molecules with different linking numbers in each topoisomer population. In a 2000-bp plasmid, a change in linking number of 1 results in a change of superhelical density of 0.005. Thus, the observed structural transitions appear to be completed within a superhelical density range of 0.01.

For all plasmids assayed, the width of the observed bands does not change with increasing negative superhelical density. This indicates that S1 nuclease cleavage sites do not shift under increasing torsional stress. Thus, the structure appears to undergo an “all-or-none’’ type transition at a critical negative supercoil density. A similar “all-or-none” behavior was described for the formation of cruciforms (40) and Z-DNA (52).


Fine Mapping of SI Nuclease Cleavage Sites-S1 nuclease first recognizes its substrate and introduces a nick into the DNA molecule. Subsequently, the enzyme cuts across from the nick (58) to complete the cleavage reaction as assayed in the experiments described above. To determine the position of the initial nicks on each strand, we subjected the plasmids to the S1 nuclease nicking experiments described under “Ma- terials and Methods.” Typical results obtained for pRW1201 and pRW1214 are shown in Figs. 4 and 5. Fig. 4 shows that the most prominent band on the pyrimidine-rich strand of pRW1201 maps to the center of the array of direct repeats (repeat number 9; lane b) . Densitometric analyses showed

a b c d

4

4

4 i

FIG. 4. Fine mapping of S1 nuclease nicks in pRW1201. pRW1201 was treated with SI nuclease and analyzed as described under “Materials and Methods.” Lanes u and b, pyrimidine-rich strand lunes c and d, purine-rich strand. Lane a, A+G sequencing ladder; lane b, S1 nuclease-treated sample; lune c, C+T sequencing ladder; lane d, S l nuclease-treated sample. None of the SI nuclease- specific bands have mobilities corresponding to the renatured duplex (data not shown), as expected.

a b c d e

I

“k-

f g h i j

Y &

FIG. 5. Fine mapping of S1 nuclease nicks in pRW1214. pRW1214 was treated with S1 nuclease and analyzed as described under “Materials and Methods.” Lanes a-e, pyrimidine-rich strand; lunes f-j, purine-rich strand. Lunes a-d and f-i, Maxam-Gilbert sequencing ladders for G (a, f), G+A (b, g), T+C (c, h), and C (d, i). Lanes e and j , SI nuclease-treated samples.

that this band is at least 50-fold (average of six experiments) stronger than the minor bands described below. Further analysis (not shown) revealed that the nicking site was at the center of a run of 6 C residues. Similarly, as schematically shown in Fig. 6, the major nick in the pyrimidine-rich strand of pRW1202 is situated at an equivalent position in repeat number 7 (not shown). The strongest nicking site is, therefore, located close to the center of the whole array of DR2 repeats for both pRW1201 and 1202.

In addition to these major bands, the pyrimidine-rich strand is also cleaved in a periodic fashion in all but the three 5’- most repeats at position -2 relative to the sole dG residue. The purine-rich strand is cut 5‘ of the sole dC residue (Fig. 4, lane d) , but nicking appears in all repeats.

Fig. 5 shows the nicking profile for pRW1214. On the

6412 HS V- 1 DR2 Sequences: Anisomorphic DNA

. . . . . . . . . . . . . . pRW 1201 . . . . . . . . . . . . . . . . . . .

.... I I I I I pRW 1203 ...

FIG. 6. Schematic representation of the nicking pattern of plasmid inserts by S 1 nuclease. Major cleavage sites are indicated by arrows. Minor sites are shown by dots. The cleavage sites shown above the schematic representation of the repeats are on the pyrimidine-rich strand and those below are on the purine-rich strand. The relative strengths of the cleavage sites can only be rigorously compared within one strand of a given molecule.

pyrimidine-rich strand (lane e ) , the major S1 nuclease-specific nicks map to the TCCT sequences in the center two repeats. Note that the bands obtained by chemical degradation (the Maxam-Gilbert sequencing ladders, lanes a-d) are one base shorter than the corresponding bands in the S1 nuclease- treated samples (lane e). There is no apparent attack by the nuclease on the outside repeats on this strand. On the purine- rich strand (lane j ) the major nicking sites are again at the AGGA sequences in the two centermost repeats.

In the plasmids carrying only 5 (pRW1203), 4 (pRW1214), or 2 (pRW1212) repeat units, the cleavage pattern changes (Fig. 6 ) . The 50-fold preference for the center of the DR2 sequences found in the cases of pRW1201 and -1202 is absent,. While the purine-rich strands display a complex set of SI - specific bands, the principal hits of the enzyme on the other strand are at dCpdT linkages. In pRW1203 and pRW1214, the center repeats are attacked strongest, while only one of the two repeats in pRW1212 was found to be nicked. It is interesting to note that insertion of a 4-bp CGCC sequence between the two repeats of pRW1212 (as present in pRW1250) shifts the major cleavage site on the pyrimidine- rich strand by 8 bp toward the center of the insert region.

For pRW1201-1203, S1 nuclease cleavage sites were found 3’ of the insert in the polylinker region of the vector. The reason for this behavior is uncertain but may be related to prior similar observations on long-range effects (59).

In summary, the results from fine mapping of the S1 nuclease nicking sites show that the two strands in all the plasmids are attacked in an unequal manner. This indicates that the structures on the two strands are not equivalent. In addition, the appearance of a major nicking site in the center of the pyrimidine-rich strand of the high copy number inserts suggests that above a certain length, a new DNA structure dominates the nicking pattern found for the shorter repeats.

Effect of Salt-The results obtained so far indicated that the structure adopted by the DR2 region of HSV-1 is different from the homopurine. homopyrimidine sequences reported previously. To further characterize the alternate structure, we investigated the effect of ionic strength on S1 nuclease cleavage of the repeats. Hentschel (25) reported that S1 nuclease sensitivity of the sea urchin histone gene spacer region containing poly(dA-dG) .poly(dC-dT) was abolished a t NaCl concentrations of 120 mM and higher; these and related results were explained on the basis of strand slippage. Fig. 7 shows the results of cleavage of pRW1201 by S1 nuclease a t NaCl concentrations from 50 to 400 mM and subsequent digestion with BglI. Enzyme activity decreases with increasing ionic

a b c d e M - 2400

- 1400 :- 1000

FIG. 7. Salt dependence of S1 nuclease-hypersensitive sites on pRW1201. S1 nuclease digestions were performed under stand- ard conditions except that different amounts of NaCl were included in the reaction mixtures. The concentrations (in mM) were: a, 50; b, 100; c, 200; d, 300; e, 400. The plasmids were then mapped by digestion with BglI and the products were analyzed on 1% agarose gels. M, markers of 123-bp ladder (Bethesda Research Laboratories).

a b c I

’ - 1400 - 1000

FIG. 8. Reactions of pRW1201 with bromoacetaldehyde and chloroacetaldehyde. Reactions with bromoacetaldehyde and chloroacetaldehyde were performed as described under “Materials and Methods.” Lane a, pRW1201 control (reaction in which bromoacetaldehyde and chloroacetaldehyde were omitted); lane b, pRW1201 treated with 1% bromoacetaldehyde; lane c, pRW1201 treated with 200 mM chloroacetaldehyde. Approximate fragment sizes are indicated at the side of the gel photograph.

strength, but S1 nuclease-specific bands were still observed a t NaCl concentrations up to 400 mM. This suggests that strand slippage alone cannot account for the sensitivity of DR2 to attack by S1 nuclease. Likewise, the pattern of nuclease nicking as well as the high GC content of the DR2 region is not consistent with strand slippage as the mecha- nism.

Bromoacetaldehyde and Chloroacetaldehyde Reactions- The single strand specificity of bromoacetaldehyde has been used previously to detect unpaired loops such as cruciform structures in DNA (35, 51). Bromoacetaldehyde reacts with A and C residues to give imidazole derivatives which cannot base pair (60). The location of these wedged open regions can be mapped with S1 nuclease after linearization of the plasmid. Bromoacetaldehyde does not recognize the junctions between B and Z-DNA (35) or the postulated slipped structures occur- ring a t direct repeats (30) but has been shown to react with nucleotides in the sequences flanking oligo(dC.dG) blocks and vector DNA in recombinant plasmids (59). I t may be noted that recent investigations reveal that the type of DNA structure detected by reaction with bromoacetaldehyde is unusually sensitive to the reaction condition^.^ We used bromoacetaldehyde and chloroacetaldehyde to address the ques- tion of single-strandedness in the alternate structure recognized by S1 and P1 nucleases.

pRW1201 was reacted with bromoacetaldehyde and chloroacetaldehyde, and the position of the modified sites was determined by S1 nuclease digestion of the BglI-linearized plasmid (Fig. 8). In addition of the full-length linear band of 2400 bp, two sets of doublets can be observed. The positions

M. J. McLean, F. Wohlrab, and R. D. Wells, unpublished data.


of these bands map the bromoacetaldehyde and chloroacetaldehyde modifications close to both ends of the DR2 region in the insert. The relative strength of the doublet bands indicates that bromoacetaldehyde and chloroacetaldehyde react a t both sites on the same molecule with approximately equal frequency. Similar results were found with pRW1214 with both reagents. The observed modification pattern is not consistent wit.h the presence of large single-stranded regions within the DR2 repeats but instead supports the notion of a structural anomaly at the interface between the DR2 and the flanking sequences. These results corroborate the P1 nuclease data by showing that the DR2 repeats exist in an altered conformation at neutral pH.

Effect of p H on dG Accessibility-Pulleyblank et al. (28) reported that in the case of poly(&-dG).poly(dC-dT) sequences half of the G residues in the GA strand were protected against alkylation by dimethyl sulfate a t pH 4.5, but not at pH 7.0. These findings were attributed to the protonation of the N3 position of cytosine a t low pH and subsequent formation of Hoogsteen G-CH’ base pairs. Alkylation of the G residues by dimethyl sulfate is then inhibited because of the syn-conformation of the base. We performed a similar exper- iment with pRW1214. The supercoiled plasmid was treated with dimethyl sulfate at pH 4.5, 5.1, 6.0, and 7.0 and the modified bases analyzed by their susceptibility to piperidine cleavage. The results indicate a pH dependence of the methylation pattern. All G residues in both the vector and insert were accessible to dimethyl sulfate a t pH 7.0, whereas a t pH 6.0 some of the insert is methylated more weakly than at pH 7.0 compared to the vector. The alkylation patterns at pHs 4.5 and 5.1 were essentially identical; the level of methylation was reduced approximately 3-fold in the insert compared to the neutral pH results (data not shown). These results suggest that protonation plays a role in the stabilization of the alternate DNA structure adopted by the DR2 repeats. However, we do not know a t present if this unusual structure is identical to the supercoil-induced conformation.

Supercoil-induced Change in the Primary Helix-DNA cruciforms and segments of left-handed Z-DNA are induced by negative supercoiling (15, 29), and the transitions of appropriate sequences from right-handed B structures to these unusual conformations may be assayed by agarose gel electrophoresis (37, 61). Two-dimensional gel electrophoresis is a powerful tool for conveniently monitoring the amount of supercoiling required (free energy) and the extent of relaxation observed (i.e. length and type of alternate conformation) (13, 14, 36, 42, 43, 53, 62, 63).

Mixtures of topoisomer populations of all the plasmids (Fig. 1B) were subjected to two-dimensional agarose gel electrophoresis (43). Fig. 9 shows typical examples of such assays. For pRW1201 (panel a), the relative mobilities of the topoisomers up to a specific linking difference of approximately 11 resemble those of the control vector pRW790 (not shown). Topoisomers with higher specific linking differences have decreased electrophoretic mobilities indicating that a structural transition in the DNA has led to relaxation of superhelical turns. The point a t which this transition is first apparent corresponds to the superhelical density (-0.055) a t which nuclease-specific cleavage was first observed (Fig. 3), thus validating the notion that the formation of the novel conformation generates the nuclease-sensitive sites.

The nature and the extent of the gel retardation are un- precedented with either cruciforms or left-handed Z-DNA. An increase in the specific linking differences seems to produce a gradual increase in supercoil relaxation, so that the mobility of the higher topoisomers does not appreciably

a 2nd dim.

b :I,, ;a ,;:::,

:3

5 2

f Q ‘ A.

0 bo*

+z 0 -2 -4 -6 - 8 -10 - 1 2 - 1 4

Number 01 s~perc01Is

FIG. 9. Two-dimensional agarose gel analysis of DR2-containing plasmids. Topoisomer populations were prepared and analyzed as described under “Materials and Methods.” Panel a, pRW1201. Panel b, relative mobilities of topoisomers of pRW790 and pRW1250. Mobilities were determined relative to the most relaxed topoisomer. Topoisomer mobilities from different experiments were normalized by using the distance between the most relaxed topoisomer and the topoisomer with five superhelical turns as a normaliza- tion factor. Closed circles, pRW790; open triangles, pRW1250.

change. Thus, the extent of the observed transition exceeds greatly the value expected for transitions involving known DNA structures (discussed further below). Also, it should be noted that the gels were run in the presence of EDTA at pH 8.3. Therefore, the structural transition(s) can be observed at slightly alkaline pH in the absence of divalent metal ions.

Similar analyses were conducted on all the plasmids shown in Fig. lB, and in all cases the amount of supercoiling required to cause the gel retardation corresponded to the amount necessary to cause specific nuclease cleavage in the DR2 repeats. However, the extent of relaxation found for the shorter inserts exceeded that expected even if left-handed Z- DNA was generated in the inserts. Fig. 9b shows a plot of relative mobility in the first dimension for each topoisomer of pRW1250 and the control plasmid pRW790. At the limit of resolution of the gel, the extent of apparent relaxation is 7.5 supercoils, and the transition does not appear to be complete at this point. The insert in pRW1250 is 28 bp in length, and the relaxation expected if a Z helix were formed is 5.0 superhelical turns. It is not clear at present if this behavior is due to supercoil relaxation or to abnormal mobility of the higher topoisomers during gel electrophoresis. It should be pointed out that identical results were obtained in three different buffer systems and varying electrophoresis conditions.

A similar graphical treatment of topoisomers of pRW1201 and pRW790 is not feasible because of the large difference in the sizes (and thus the number of topoisomers) of the two plasmids.

Determinations for Bent DNA-Regions of bent DNA (19) have been described in kinetoplast minicircles as well as certain genetic control regions (20-22, 24). Bent DNA has been detected by unusual gel electrophoretic behavior of fragments under various conditions of gel concentration and


temperature, electric dichroism relaxation studies (23, 64), unusual susceptibility to ethanol-induced B- to A-type conformational changes (20, 21), and electron microscopy (65). Two linear DNA fragments (440 and 395 bp from pRW1201) containing the DR2 sequences do not exhibit unusual gel mobilities in polyacrylamide and agarose gels at six different gel concentrations and electrophoresis conditions (data not shown). Also it should be noted that the DR2-containing plasmids (pRW1201 and 1214) do not appear to have unusual properties when examined by electron microscopy (courtesy of Dr. Jack Griffith, University of North Carolina). We con- clude, therefore, that there is no major bend in the DR2 repeat regions and that the negatively supercoiled plasmids (Fig. 1B) do not appear to exist in unorthodox type structures such as toroids.

DISCUSSION

The DR2 sequences of HSV-1 contain cis-acting signals necessary for segment inversion (4, 5) as well as regulatory sequences of a diploid gene encoded by the L component of the viral genome (66). Herein, we describe a novel DNA secondary structure adopted by these sequences which has properties unlike those found for other unusual conformations. This structure is characterized by hypersensitivity to endonucleolytic attack by S1 and P1 nucleases and is induced by negative supercoiling. Supercoiling as a regulatory mech- anism in the viral life cycle is suggested by the fact that HSV DNA circularizes soon after entry into the host cell (67) as well as the implication of topoisomerases in lytic virus infec- tion (68).

The DR2 sequences have some resemblance to the polypurine . polypyrimidine sequences previously identified as sites of preferential attack by S1 nuclease (25, 27, 28, 30, 48), but there are significant differences between the results observed with the two types of sequences. The nature of the alternate structure adopted by pur.pyr sequences is poorly understood, but proposed models include slipped structures at direct repeats (17, 18, 69), non-Z left-handed helices (27), structures containing protonated bases (28), and intramolecular triple- strand formation (47, 70). I t may be noted that early studies on DNA polymers (reviewed in Refs. 71 and 72) by spectro- scopic, physical, and biochemical methods revealed the unorthodox properties and, in some cases, conformations of polypurine. polypyrimidine sequences.

S1 nuclease has the striking and novel preference for nicking at the center of the pyrimidine-rich strand of the higher copy number DR2 inserts and nicks the two complementary strands at approximately 2% the rate in a periodic but unequal fashion. Hence, the two strands are not equivalent. This type of nicking pattern excludes the occurrence of slipped structures in this case. Furthermore, S1 nuclease cleavage, although reduced, still occurs a t NaCl concentrations up to 400 mM. For the slipped structure described by Hentschel (25), a relatively short oligo(dGA) sequence from the sea urchin histone gene repeat, S1 nuclease cleavage was essentially abolished at 120 mM NaCl. Also, this sequence retained its nuclease hypersensitivity in relaxed plasmids, whereas the DR2 repeats are sensitive to nucleolytic cleavage only at negative superhelical densities above 0.05.

Cruciform formation by the DR2 sequences can be excluded on grounds of the nature of the sequences involved, the extent of the observed supercoil relaxation, the S1 nuclease nicking pattern, and the effect of salt. Also, the phage T7 gene 3 product (generous gift of Dr. J. Coleman, Yale University) does not cleave this structure5 whereas prior studies showed

' F. Wohlrab, M. J. McLean, and R. D. Wells, unpublished results. ________

its capacity to cleave cruciforms (73). The large extent of apparent supercoil relaxation observed

in two-dimensional gels indicates an extreme amount of un- denvinding of the DR2 insert DNA. Poly(dG) .poly(dC) tracts have been reported to favor the A form of DNA (74) with a winding angle of down to 16"/base compared to approximately 35"/base for B-DNA, but these values cannot explain the data obtained for the shorter inserts (pRW1212 and 1250). The most extreme case of unwinding known is the transition from B to left-handed Z-DNA (29,38).

For poly(dGA) .poly(dCT) sequences, Cantor and Efstra- tiadis (27) proposed left-handed structures. The extent of supercoil relaxation observed for the plasmids containing the DR2 repeat derivatives appears somewhat larger than can be accounted for by conventional Z-DNA formation, but it is difficult to make definite structural assignments for a tiny DNA segment based on supercoil relaxation data of a several thousand bp plasmid unless the nature of the alternate structure is well defined previously. If the DR2 sequences do exist in a left-handed state, they do not adopt a classical Z-DNA conformation. This is supported by the fact that there is very little, if any, influence of the length of the DR2 insert on the critical negative superhelical density at which the structure becomes nuclease sensitive. In contrast, model Z-DNA form- ing sequences (13, 41, 53) show marked influences of chain length on the required superhelical density.

Although S1 nuclease sensitivity of the DR2 sequences depends strongly on pH and is not observable above pH 5.2, a number of other probes including P1 nuclease, bromoacetaldehyde, and chloroacetaldehyde react at neutral pH values. In addition, the supercoil relaxation observed in two-dimensional gels occurs at pH 8.3. These data suggest the existence of an acid and a neutral form of these structures. Although it is difficult to distinguish between the effect of pH on the probe and on the DNA structure, additional evidence for a pH dependence is provided by the increasing protection of the DR2 sequences from methylation by dimethyl sulfate at decreasingpH.The S1 nuclease nicking data exclude, however, a simple dinucleotide repeat as the one found for oligo (dGA) . oligo(dCT) by Pulleyblank et al. (28). Similarly, most models involving triple-stranded structures require protonation of some of the bases. It might be expected that such structures would be revealed by electron microscopy. However, no evidence was found for loop-backs or toroids under the fixing conditions employed. In summary, it seems probable that both negative supercoiling and low pH stabilize an unusual structure with the DR2 sequence. However, other types of determinations will be required to determine if the structures generated by different perturbants are the same.

Since our results are not consistent with previously characterized structures, we propose the following structure for the DR2 repeats which we refer to as "anisomorphic." The two complementary strands of the double helix have different conformations which are induced by negative supercoiling. The nature of this asymmetry is unclear as yet, but it is possible that the angles between consecutive bases are different on the two strands due to different stacking energies. This will lead to the helix axis describing a curve in space, thereby establishing a periodicity of bases positioned on the outside of the helix. This hypothesis is consistent with the S1 nicking data. The very strong central nick on the pyrimidine-rich strand may be explained by a small structural aberration in the structure which is propagated from both ends toward the center of the insert. Such a gradual structural amplification could for example be due to a different rise/residue on the two strands. The purine-rich strand would remain stacked,


but the pyrimidine-rich strand would be unstacked as a consequence of the unequal structure. A sufficient length of DR2 sequences (pRW1201 and -1202) would be required to provide a sufficient amplification of the structural aberration to elicit the pronounced nicking on the pyrimidine-rich strand. Long range interactions in DNA were reviewed (72).

In addition to the asymmetry of the two helix strands, we propose that the DR2 repeats are relatively stiff and inflexible compared to the rest of the plasmid. This resistance to bending will manifest itself upon supercoiling (Fig. 10, center) and lead to an unequal distribution of supercoil density along the plasmid. As a consequence, torsional stress will be exerted on the ends of the DR2 repeats, leading to a distorted vector- insert interface region (Fig. 10, right side). In addition, above a certain length of insert, this bilateral stress will tend to physically generate a structural discontinuity at the center as schematically indicated in Fig. 10, right side. This physical effect may act in concert with the unstacking of the pyrimidine-rich strand in the center of the DR2 repeats described above to generate the observed S1 nuclease nicking patterns.

It has been proposed that polypurine.polypyrimidine sequences are resistant to bending (75-78). The first evidence for this fact came from the observation that pur.pyr sequences do not easily form nucleosomes (79, 80). It is interesting to note that HSV-1 DNA seems to be devoid of nucleosomal organization in vivo (81-83). As expected, fragments containing DR2 sequences do not appear to be bent. This model is consistent with the pattern of S1 nuclease nicking and the lack of insert length on the critical superhelical density required.

Interpretation of two-dimensional gel relaxation data usu- ally assumes a simple correlation between writhe and twist of a DNA (29) and its electrophoretic mobility. Specifically, the nature of a secondary structure present in a plasmid cannot influence per se the mobility of a certain topoisomer. This assumption has been valid thus far for cruciforms and Z-DNA segments (11). However, the large supercoil relaxation observed in the DR2-containing plasmids could be due to a direct influence of the anisomorphic DNA structure on electrophoretic behavior. Thus, it is not rigorously possible to deduce if the DR2 repeats exist in a right- or left-handed helix at present. Alternatively, it is possible that flanking vector sequences are influenced by the DR2 structure and/or that the new conformation causes even more unwinding than left- handed Z-DNA. One possible reason for this could be a supercoil-induced bend as indicated on Fig. 10, fur right side. It should be pointed out that the types of analyses performed in this study can only provide inferential structure information. Other determinations (x-ray, NMR, etc.) will be required to define conformational details. Since the observed phenomenon is supercoil dependent, substantial work will be neces-

FIG. 10. Schematic representation of supercoil-induced anisomorphic DNA. Left side, relaxed plasmid with insert (open box) in right-handed B structure. Center, moderately supercoiled plasmid, below the structural transition. Right side, plasmid at supercoil densities above the transition which induces the conformational aberra- tions ( 3 arrows).

sary to resolve the structure at the nucleotide level. For poly(dG) .poly(dC) sequences present in recombinant

plasmids, Kohwi-Shigematsu and Kohwi (59) proposed radial asymmetry as first postulated by Arnott et al. (84) as a cause for bromoacetaldehyde reactivity of neighboring sequences. The type of nuclease nicking pattern described in this work also demonstrates polarity of S1 cleavage. It is possible that the mutation frequency observed at the 3' end of the pyrimidine-rich strand is related to this phenomenon. However, the supercoil-dependent reactions of the vector-insert junctions with both bromoacetaldehyde and chloroacetaldehyde could equally be a consequence of the rigidity of the DR2 sequences, as shown in Fig. 10.

The term anisomorphic DNA refers to both the dissimilar structures of the insert and the vector and their mutual forces on each other as well as the dissimilarities of the complementary strands of the DR2 regions. The flanking vector sequences invoke a physical force causing a deformation or crack at the center of the tandem repeats; also, a feature of the nonidentical complementary strands generates unpairing at the center, thus enhancing the lability of the center region. In contrast, the term heteronomous DNA (84) was coined to describe the structure of dA,.dT, deduced from fiber diffrac- tion studies where each of the strands had a different conformation, one A-like and one B-like.

Anisomorphic DNA differs markedly from certain other unusual conformations since it may contain a partially unstacked pyrimidine-rich region. Thus, this segment is ideally suited for multiple and specific contacts with regulatory pro- teins. Some eukaryotic protein factors have been shown to bind to DNA regions containing polypurine-polypyrimidine motifs (i.e. the spl protein) (85). It is possible that structural transitions in the DR2 region regulate protein binding to this and flanking sequences. In this context it is noteworthy and unexpected (13) that a neighboring 15-bp alternating purine- pyrimidine sequence does not appear to undergo a B-to-Z DNA transition even at high negative supercoil densities. While the a sequences containing the DR2 repeats are essen- tial regions of the viral genome (I), the effect of DR2 copy number on biological functions is unclear but is under inves- tigation.

Acknowledgments-We thank Dr. Bernard Roizman (University of Chicago) for plasmids containing segments of HSV-1 and Drs. Wolf- gang Zacharias and Stephen C. Harvey (this department) for helpful discussions.

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