THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 3, Issue of January 25, pp. 1352-1359,199O Printed in U.S.A.

Left-handed Z-DNA and Intramolecular Triplex Formation at the Site of an Unequal Sister Chromatid Exchange*

(Received for publication, July 25, 1989)

Ari WeinrebSQ, David A. Colliern, Barbara K. BirshteinSlj , and Robert D. Wellsq From the $Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461 and the ITDepartment of Biochemistry, Schools of Medicine/Dentistry, University of Alabama, Birmingham, Alabama 35294

An unequal sister chromatid exchange (USCE) in the mouse myeloma cell line MPG11 between 3’ regions of the Cy2a and Crab heavy chain genes results in duplication of the Cr2a heavy chain gene and genera- tion of a novel recombination joint. The USCE occurs between (TC), tracts adjacent to alternating purine- pyrimidine tracts. We have investigated the capacity of both the donor regions and the recombinant product involved in this event to adopt left-handed Z-DNA and intramolecular triplexes. The results of chemical prob- ing with diethylpyrocarbonate and osmium tetroxide at the base pair level demonstrate that under the influ- ence of negative supercoiling the alternating purine- pyrimidine regions of these plasmids can adopt Z-DNA at neutral pH, and the oligopurine. oligopyrimidine (pur . pyr) regions of these regions can adopt intramo- lecular triplexes at low pH (or pH 6.0). At intermediate pH values, mixtures of both structures are present. Increasing the negative superhelical density of the plasmid does not increase the amount of triplex present at neutral pH indicating that the presence of long Z- DNA segments adjacent to pur . pyr tract prevents in- tramolecular triplex formation. In summary, we con- clude that the sequences involved in the USCE can form either an intramolecular triplex in the (TC), tract or Z-DNA in the alternating purine-pyrimidine tract and that Z-DNA will predominate under physiological conditions. The presence of segments which adopt Z- DNA at a site of USCE suggests that formation of this structure may enhance recombination between adja- cent pur. pyr tracts.

The mouse myeloma cell line MPC-11 (IgG2b, K) carries a tandem duplication of the Cy2a gene together with a deletion of a portion of the CySa-associated tandem repeats on the expressed chromosome (1). This duplication has been shown to be the result of an unequal sister chromatid exchange (USCE)’ (1). The DNA sequences 3’ of the CrPb gene and 3’

* This work was supported by National Institutes of Health Grants GM30822 and NC1 CA-13148 (to R. D. W.) and AI13509, A110702, and P30 CA13330 (to B. K. B.), National Science Foundation Grant 86-07785 (to R. D. W.), and by a grant from the Irvington House Institute for Medical Research (to B. K. B.). 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 solelv to indicate this fact.

!j Supported by Training Grant TS 2GMf288 from the National Institutes of Health.

11 Established Investigator of the American Heart Association. To whom correspondence should be addressed.

‘The abbreviations used are: USCE, unequal sister chromatid exchange; bp, base pair(s); pur .pyr, oligopurine oligopyrimidine, homopurine in one strand and homopyrimidine in the complementary strand; DEPC, diethyl pyrocarbonate; pur-pyr, purine-pyrimidine, strictly alternating purine-pyrimidine sequence in both complemen- tary strands.

of the Cy2a gene that are involved in this crossover are nearly identical simple repeats of (TC),, which are followed by stretches of alternating purine-pyrimidine bases comprised mostly of (TG), (2). Sequencing of the USCE joint has revealed that both recombination breakpoints occur within the (TC), tracts, giving a product containing segments of both (TC), tracts plus an insertion of 64 bp containing additional (TC), nucleotides and two shorter segments of 6 and 8 bp (3). The mechanism of this insertion is unknown.

Because of the localization of the crossover point to within the (TC), tracts and adjacent to (TG), tracts, we explored the possibility that these segments may adopt unusual DNA structures and that such structures may have contributed to the observed recombination event. Recent studies have dem- onstrated the ability of short oligopurine . oligopyrimidine (pur.pyr) stretches such as (TC), to adopt intramolecular triplexes ((4-6) reviewed in Ref. 7), and stretches of (TG), are known to adopt left-handed Z-DNA (Ref. 8 and references therein). It has recently been shown that alternating purine- pyrimidine sequences can adopt a left-handed Z-DNA confor- mation in uiuo (9).

To date, there are several studies which suggest that Z- DNA plays a role in recombination (10-12). The role of pur . pyr stretches in recombination is just beginning to be exam- ined. For example, the HSV-1 inversion segment contains sequences which adopt an anisomorphic DNA structure (13), and a rat LINE mobile genetic element has been shown to contain a guanine-rich homopurine stretch with unusual structural properties (14). In addition the DR2 repeat (AG- GAG)2, of the mouse IgA immunogiobulin switch region, which is involved in heavy chain class switch recombination, is capable of adopting a non-B-DNA structure which may be an intramolecular triplex (5).

In this study we have found that the (TC), and (TG), tracts that lie 3’ of the Crab and Cy2a genes which are involved in the USCE event are hyperreactive to a variety of chemical probes in negatively supercoiled plasmids. The pattern of modification of bases by these chemical probes indicates that the (TC), tracts are able to form triplexes at low pH (5 pH 6.0) and the (TG), tracts are able to form Z-DNA at pH values closer to neutral (2 pH 6.0). Under all conditions studied, the triplex structure predominated at low pH and Z- DNA predominated at neutral pH. These resuIts suggest that intramolecular triplex formation is not likely to be directly involved in in uiuo recombination in this system but instead that Z-DNA may contribute to recombination within the flanking (TC), tracts.

MATERIALS AND METHODS

Plasmids-All plasmids were cloned and prepared using standard procedures (15) and the sequences confirmed by the Maxam and Gilbert chemical degradation method (16). pAW8.12 is a 3929-bp

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pBR322 derivative containing the 99-hp HindIII-SphI fragment lo- cated 3’ of the CyPa gene in MPC-11. pAW7A3 is a 3968-bp pBR322 derivative containing the 138-bp XbaI-SphI fragment located 3’ of the Cr2b gene is MPG11 (12). pNJ41 contains a 2.5-kilobase Hind111 fragment cloned in pBR322 from the USCE crossover site in MPC- 11 (3). pNJ25 is identical to pNJ41 except that it has a deletion of 62 bp within the TC tracts, which occurred during cloning procedures. pNJ25.5 contains a 208-bp HindIII-SspI fragment which was derived by truncation of the insert present in pNJ25. The fragments sub- cloned into these plasmids are shown in Fig. 1, upper and lower panels. The pur.pyr and alternating pur-pyr sequences present in these plasmids which were analyzed are shown in Fig. 2.

A.

Base Pair Resolution Mapping of Chemical Modification Sites- Base pair resolution mapping of osmium tetroxide (0~04) and diethyl pyrocarbonate (DEPC) was essentially as described by Collier et al. (5). Briefly, 3 c(g of DNA was incubated with each of the reagents in the appropriate buffer at pH values ranging from 5.0 to 7.6. After the reactions were stopped, the DNA was recovered by precipitation, digested with the appropriate restriction endonucleases, and labeled using either the Klenow fragment of DNA polymerase I or T4 polynucleotide kinase (United States Biochemical Corp.). After iso- lation from a polyacrylamide gel, the DNA fragments were treated with 1 M piperidine for 20 min at 90 “C, lyophilized, and applied to a denaturing polyacrylamide gel in parallel with sequencing markers generated by the chemical degradation method.

Generation of Topoisomers-Topoisomers of plasmids were pre- pared as described previously (17). Calf thymus topoisomerase was a kind gift of J. E. Larson, University of Alabama at Birmingham.

RESULTS

Plasmids Containing Segments of Regions Flanking the Cy2b and Cy2a Immunoglobulin Constant Region Genes-In order to characterize the sequences involved in the USCE in the MPC-11 heavy chain locus, we made a series of constructs containing segments of DNA from the donor regions and from the product of this rearrangement. The sizes and compositions of these constructs are described under “Materials and Meth- ods.”

All of the inserts contained in the plasmids used in these studies contain a (TC), tract adjacent to an alternating pur- pyr tract, which is predominantly (TG),. The regions sub- cloned into these plasmids are shown in Fig. 1, and details of the sequences are shown in Fig. 2. The complete sequences of these fragments are shown in Figs. 4 and 7.

The Inserts of pA W8.12 (y2a-3’) and pA W7A3 (-y2b-3’) Can Adopt Z-DNA and Intramolecular Triplex Structures in Vi- tro-OsO1 and DEPC were used as conformational probes because of their ability to recognize bases in double-stranded DNA which are in an alternate conformation such as those in cruciform loops, intramolecular triplexes, and B-Z junc- tions (reviewed in Refs. 7 and 8). 0~0~ adds to the C-5, C-6 double bond of pyrimidines to form osmate esters and is substantially more reactive to single-stranded than to double- stranded DNA. DEPC carboxyethylates purines at the N-7 position, which results in opening of the imidazole ring. DEPC is weakly reactive toward B-DNA, but its reactivity is en- hanced when bases are single-stranded or in a syn conforma- tion, as in Z-DNA (7). Therefore, we used these reagents to characterize the conformations adopted by the plasmids in this study.

The results of probing the pyrimidine-rich strand ((TC&- C-(TG)16) of pAW8.12 (y2a-3’) with OsOl are shown in Fig. 3, lanes 1-4, and the results of DEPC probing of the purine- rich strand ((AG)13-G-(AC)16) of pAW8.12 (y2a-3’) are shown in Fig. 3, lanes 5-8. DNA linearized prior to chemical modi- fication showed only background reactivity in all cases; there- fore, modification and hence structure formation were totally dependent on supercoiling (Fig. 3, lanes 2,4, 6, and 8).

At pH 7.6,0s04 is strongly reactive with T residues in both the 5’ and 3’ regions flanking the (TG)16 tract and shows

C 2b

c -1 m1 &dIII L&I

DAW7A3 PAW8.17

B. C 2b

Y

UdIII &I Hind111

pNJ41

FIG. 1. Inserts in plasmids. Panel A, the upper line depicts the region of the germline murine heavy chain immunoglobulin locus containing the C$b, CyPa, and Cr constant region genes. The lower line shows the regions flanking the Cr2b and Cy2a genes from which the inserts of pAW7A3 and pAW8.12 were isolated. Panel B, the upper line depicts the MPC-11 heavy chain immunoglobulin gene locus with the duplicated y2a gene. The lower line shows the USCE- joint clone pNJ41.

PAW8.17 (@a-3'):

[ (i-C) ,,I = { (Pyr-Pur) 20 I

m7A3 (yZb-3'1 :

[ (TC)&C(TC)e] RGTGTCTCTT{ * (PYr-k,,)

pNJ25.5 (AUSCE):

[ (TC) 22]~~~~~~~~~~{ * (Pyr-Pur) ,,}

FIG. 2. Juxtaposed purine. pyrimidine and alternating pur- ine-pyrimidine tracts analyzed by chemical modification. The pur.pyr and alternating pur-pyr sequences present in each construct are shown. The pur .pyr tracts are indicated by 11, and the alternating pur-pyr tracts are indicated by {). The * indicates imperfect alterna- ting pur-pyr tracts with occasional breaks in the alternating sequence. The complete sequences are shown in Figs. 4 and 7.

weaker reactivity with some T residues within the (TG),, tract (Fig. 3, lane 1). At pH 7.6, DEPC strongly modifies the A residues of the (AC)16 tract and an adjacent A residue in the 5’ portion of the (AG)13 tract; a weaker reactivity is observed with the other A residues of the (AC),, tract (Fig. 3, lane 5). A summary of the modifications of pAW8.12 (y2a-

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Z-DNA and Triplexes at an USCE Site 1354

OS04 1234

AC GT

GC =

f -_

(TG), 6

C L L

-m 4

T- VC)13 -

I- TT

GT

DEPC 5678

TG I:

1 AA - ~-

CA ir -

FIG. 3. Base pair level mapping of the sites of modification of pAW8.12. Lanes l-4, pAW8.12 (y2a-3’) was treated with 0~0, as described under “Materials and Methods.” Plasmid DNA was cleaved with Hind111 and labeled at the 5’ terminus with T4 polynu- cleotide kinase. After cleavage with a second restriction endonuclease, the fragments were treated with piperidine and applied to a denatur- ing polyacrylamide gel adjacent to a sequencing ladder. Lane I shows supercoiled pAW8.12 at pH 7.6, lane 2 shows linear plasmid at pH 7.6, lane 3 shows supercoiled plasmid at pH 5.0, and lane 4 shows linear plasmid at pH 5.0; lanes 5-8, pAW8.12 (r2a-3’) DNA was treated with DEPC as described under “Materials and Methods.” Plasmid was cleaved with Hind111 and Sal1 and the Hind111 site labeled with Klenow fragment of DNA polymerase I. After treatment with piperidine the DNA was applied to a denaturing polyacrylamide gel adjacent to a sequencing ladder. Lane 5 shows supercoiled plasmid at pH 7.6, he 6 shows linear plasmid at pH 7.6, lane 7 shows supercoiled plasmid at pH 5.0, and lane R shows linear plasmid at pH 5.0.

3’) with 0~0~ and DEPC at pH 7.6 is shown in panel A of Fig. 4.

The strong 0~0, reactivity at the T residues flanking the (TG)16 tract at pH 7.6 is diagnostic of the presence of B-Z junctions (18), and the strong reactivity of the DEPC with the A residues in the (AC)l, tract is consistent with these bases adopting a syn conformation (19,20). Therefore we can conclude that, the (TG)ls stretch of pAW8.12 (y2a-3’) adopts a left-handed Z-DNA conformation at pH 7.6 in the super- coiled plasmid.

Panel B of Fig. 4 gives a summary of 0~0~ and DEPC modification of pAW7A3 (-y2b-3’) at pH 7.6 where 0~0, reacted strongly with almost all T residues in the alternating pur-pyr tract of the (AC), strand (lower strand in Fig. 4B) and many of the T residues on the (TG), strand (upper strand in Fig. 4B). The modification pattern of the alternating pur- pyr region of pAW7A3 (-y2b-3’) differs from that of pAW8.12

(y2a-3’) since the sequence of the alternating pur-pyr stretch of this plasmid is somewhat more complex. The reactivity of the T residues in both strands of the alternating pur-pyr tract to OsO., is a characteristic feature of Z-DNA tracts, since thymidine residues in left-handed DNA are hyperreactive to this reagent (21).

When the chemical modification of pAW8.12 (y2a-3’) was conducted at pH 5.0, strong OsOl reactivity was observed at the center T residues of the (TC)1,3 tract and the 5’ T residues of the adjacent (TG)16 tract (Fig. 3, lane 3). At pH 5.0, the reactivity of the (AC)1G tract to DEPC was diminished but still detectable, and a strong reactivity of the 5’ half of the (AG),% tract was observed (Fig. 3, lane 7). A summary of the modifications of pAW8.12 (y2a-3’) with 0~0, and DEPC at pH 5.0 is shown in panel A of Fig. 4.

These modification patterns are diagnostic of intramolec- ular triplex formation (5). The strong 0~0~ reactivity in the center of the (TC),, tract results from modification of the central triplex loop ((5) reviewed in Ref. 7), while 0~0, reactivity at the 3’ end of the (TC),s tract indicates the single- stranded region at the point before the pyrimidine and the purine strands rejoin to form duplex DNA. The strong DEPC reactivity of the 5’ half of the (AG),., strand is characteristic of the unpaired 5’ half of the purine strand. The weak reac- tivity of the (AC)1G stretch with DEPC at pH 5.0 suggests that there may still be some Z-DNA present at this lower pH. Fig. 5 illustrates the structure and the sites of chemical modification of the intramolecular triplex adopted by the (TC),., tract of pAW8.12 at pH 5.0.

Panel B of Fig. 4 gives a summary of 0~0, and DEPC modification of pAW7A3 (r2b-3’) at pH 5.0. Strong OsO., modification is observed at the T residues in the center and 3’ of the (TC), tract. In addition, strong 0~0, modification is observed on a T residue immediately 5’ of the (AC), tract while strong DEPC modification is observed in the 5’ half of the (AG),, tract.

Although generally similar to that observed for pAW8.12 (y2a-3’), pAW7A3 (y2b-3’) has some additional reactivities which result from an interruption in the TC repeat (an A residue in the 3’ half of the (AG), tract (lower strand, Fig. 4B) has been replaced by a G residue). Because of this inter- ruption, the mirror repeat required the formation of the triplex stem is imperfect and a Hoogsteen hydrogen bond cannot form at this position (6, 7). Two additional T residues become reactive, one 5’ and one 3’ of the modification in the center of the (TC), stretch. A similar phenomenon has been observed for short (TC), tracts containing interruptions in the mirror repeat pattern (6). Thus the pattern of reactivity of the (AC), tract of pAW7A3 (y2b-3’) is also consistent with the formation of an intramolecular triplex.

In summary, the (TG), tracts of these plasmids are predom- inantly Z-DNA at pH 7.6 and the (TC), tracts are predomi- nantly intramolecular triplex at 5.0.

The Inserts of pNJ41 (USCE) and pNJ25.5 (A USCE) Adopt pur.pyr Structures and Z-DNA in Vitro-The studies de- scribed above for pAW8.12 (r2a-3’) and pAW7A3 (+y2b-3’) were repeated for the recombination product sequence of pNJ41 (USCE) and pNJ25.5 (A USCE). The results of 0~0~ and DEPC chemical modification of the purine strand of the insert of pNJ41 (USCE) molecule are shown in Fig. 6. As for pAW8.12 (r2a-3’) and pAW7A3 (y2b-3’), in all cases DNA linearized prior to chemical modification showed only back- ground reactivity, again indicating that structure formation was totally dependent on supercoiling (Fig. 6, lanes 1, 3, 5, and 7).

At pH 7.6 for pNJ41, 0~0, reacted strongly with all of the

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A. pAW8.12 (Y2a-3’)

Z-DNA and Triplexes at an USCE Site 1355

0~04 pH 7.6 :

oso4; pH 5.0 I

DEE, pH 5.0

DEPC, pti 7.6

B. pAW7A3 (Y2b-3’)

FIG. 4. Schematic diagram of chemical modification studies on pAW8.12 and pAW7A3. Panel A, chemical reactivity of supercoiled pAW8.12 (y2a-3’). Panel B, chemical reactivity of supercoiled pAW7A3 (r2b- 3’). The relative extent of modification is indicated by the height of the bar corresponding to each base. Data were obtained by densitometric scanning of autoradiograms, and thus the height of bars is plotted in arbitrary units and is only comparable between each probe on the same strand.

FIG. 5. Schematic of intramolecular triplex structure adopted by the pur’pyr tract of pAW8.12 at pH 5.0. The 3’ half of the (TC)u tract is stabilized in the major groove of the adjacent double helix through Hoogsteen base pairs. The 5’ half of the (AG),, tract is unpaired. The symbols are as follows: ., Watson-Crick base- pairs; +, Hoogsteen base pair formed between a protonated cytidine residue and a guanosine residue; +, Hoogsteen base pair formed between a thymidine residue and an adenosine residue; 0, sites mod- ified by osmium tetroxide; A, sites modified by diethyl pyrocarbonate. Stronger reactivity is indicated by the larger size of the symbol.

T residues (Fig. 6, lane 4), and DEPC reacted with some of the A residues (in the central region) of the alternating pur- pyr tract of the (AG),(AC), strand (Fig. 6, lane 8). In addition,

DEPC exhibited weak reactivity with the A residues at the center of and at the 5’ end of the (AC), tract at pH 7.6 (Fig. 6, lane 8).

At pH 7.6 for pNJ25.5 (A USCE) (Fig. 7, panel B), 0~0~ reacts with almost all of the T residues of both strands within the alternating pur-pyr tract.

The pattern of modification at pH 7.6 of pNJ41 (USCE) and pNJ25.5 (A USCE) within the alternating pur-pyr tract is summarized in Fig. 7, panels A and B. Chemical modifica- tion is similar to the pattern observed for pAW7A3 (y2b-3’), which has an identical alternating pur-pyr tract. As for pAW7A3 (y2b-3’), the pattern of modification is consistent with these regions adopting Z-DNA under these conditions.

When the chemical modification of pNJ41 (USCE) was conducted at pH 5.0, 0~0~ exhibited strong reactivity with two T residues in the 5’ region of the (AC), tract (Fig. 6, lane 2). In the (TC), strand at pH 5.0, 0~0~ modified the T residues of the 5’ third of the segment, the two T residues of the central (TTTCTG) N6 segment, the two 5’ residues of the central (TTGCTCAC) N8 segment, and the T residues of the 3’ third of the segment (Fig. 7, panel A). At pH 5.0 on the (AC),, strand, DEPC modified the A residues in the 5’ half of the first (AC), segment (closest to the (AC),, blocks), the two

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1356 Z-DNA and Triplexes at an USCE Site

T PUFVPYR 76-bp

W), 7 TG

GAG - AAC -

W,

GAC d-

AAA l-

W)24

1234 5678

i

AAC AAA

FIG. 6. Base pair level mapping of sites of chemical reactiv- ity on DNJ~~ WSCE). Chemicallv modified ulasmids were cleaved with Hind111 and SspI and end-labeled with the Klenow fragment of DNA polymerase I. After treatment with piperidine, the fragments were applied t.o a denaturing polyacrylamide gel adjacent to a sequenc- ing ladder. Lanes l-4, pNJ41 (USCE) treated with 0~0~ as described under “Materials and Methods.” Lane I, linear plasmid at pH 5.0; lane 2, supercoiled plasmid at pH 5.0; lane 3, linear plasmid at pH 7.6; lane 4; supercoiled plasmidat pH 7.6. Lanes 5-8, pNJ41 (USCE) treated with DEPC as described under “Materials and Methods.” Lane .5, linear DNA at pH 5.0; lane 6, supercoiled DNA at pH 5.0; lane 7, linear DNA at pH 7.6; lane 8, supercoiled DNA at pH 7.6.

central A residues of the (AACGAGTG) segment adjacent to this stretch, and almost all of the A residues in the remaining purine run (Fig. 6, lane 6). All of these modification patterns are summarized in Fig. 7, panel A. Note that at pH 5.0, almost all of the pyrimidines on the 5’ side are reactive to both DEPC and 0~0~.

At pH 5.0 for pNJ25.5 (II USCE), 0~0~ modifies the T residues at the 5’ segment of the (TC), tract and a T residue at the 5’ end of the (AC),, tract (Fig. 7, panel B). DEPC modifies the A residues in the central and 5’ ends of the (AG), tract and the A residue at the 3’ end of the (TC), tract opposite the reactive T residue at the 5’ end of the (AG), tract.

The reaction pattern at pH 5.0 within the (TC), tracts of pNJ41 (USCE) and pNJ25.5 (A USCE) differs from the reactivity pattern observed for the shorter (TC), tracts of pAW8.12 (y2a-3’) and pAW7A3 (y2b-3’) in that reactivity to

these chemical probes has now spread to almost the entire insert. This transition to a different pattern of reactivity from that typical of a triplex conformation as the length of the pur. pyr tract is increased has been observed for several other longer pur.pyr tracts.’ This may represent a mixture of dif- ferent triplex isomers, which gives rise to this complex pattern of chemical modification. Interestingly, the shorter (AC), tract closest to the (TG), of the pNJ41 (USCE) block appears to adopt a separate short intramolecular triplex of its own, as characteristic reactivity is obtained within this segment (Fig. 7). This indicates that the break in the run of purines (TGAGC) at the boundary of this segment is enough to isolate triplex formation at this site from the rest of the (AC), stretch.

pH Titration of Triplex and Z-DNA Structures of pA W8.12 (y2a-3’) and pNJ41 (USCE)-In order to determine if a mixture of triplex and Z-DNA existed at pH values interme- diate between 5.0 and 7.3, we performed chemical modifica- tion studies on the inserts pNJ41 (USCE) and pAW8.12 (y2a- 3’) at a variety of pH values using 0~0~ as a probe. Fig. 8 shows the result of decreasing pH on the pattern of 0~0~ modification on these plasmids and demonstrates that for both plasmids there is a gradual change in the equilibrium between intramolecular triplex and Z-DNA as the pH is lowered. For pAW8.12 (y2a-3’), triplex begins to form below pH 6.5 with a concomitant decrease in Z-DNA-associated reactivity. At -pH 6.0, strong modifications associated with both triplex and Z-DNA are present, indicating that a mixture of both structures is present. However, because the modifi- cations associated with triplex and Z-DNA structures are overlapping, it is likely that these structures form in different rather than in the same plasmid molecules. At pH 5.5, triplex reactivity has reached a maximum, with the concomitant loss of modification associated with Z-DNA.

For pNJ41 (USCE), reactivity associated with triplex for- mation is present at all pH values and begins to increase at pH 6.0, with a concomitant decrease in reactivity associated with Z-DNA. At -pH 6.0, strong modification associated with both triplex and Z-DNA is present, indicating that a mixture of structures is present. However, as modification patterns for the two structures do not overlap, it cannot be determined whether they exist together in the same molecule or in differ- ent molecules. At pH 5.0, triplex reactivity has reached a maximum, and Z-DNA reactivity has reached its minimum.

Effect of Increased Superhelical Density-The studies on natively supercoiled plasmids (-a = -0.06) described above indicate that the intramolecular triplex is the predominant structure at low pH and Z-DNA the predominant structure at neutral pH. Accordingly, we attempted to shift the triplex/ Z-DNA equilibrium to determine if triplexes can form at neutral pH, since it has been demonstrated that intramolec- ular triplexes can exist at neutral pH under high levels of negative supercoiling (22). We generated topoisomer popula- tions of these plasmids with a decreasing linking number so that an average negative superhelical density of -0.09 was obtained for pAW7A3 (r2b-3’) and pNJ41 (USCE). For pAW7A3 (Tab-3’) and NJ41 (USCE), increasing the negative superhelical density did not increase the amount of triplex present at pH 7.6, as determined by OsOI reactivity; at pH 6.0 equal amounts of Z-DNA and triplex were present. At pH 5.0, triplex remained the predominant structure in these topoisomer populations (data not shown). Therefore, in- creased negative supercoiling did not alter the ratio of Z-DNA to triplex from pH 7.6 to pH 5.0. Thus of the two variables, negative supercoiling or pH, the pH is the primary determi- nant of the type of structure adopted by the inserts in these

’ D. A. Collier and R. D. Wells, submitted for publication.

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A. pNJ41

Z-DNA and Triplexes at an USCE Site 1357

B. pNJ25.5

FIG. 7. Schematic diagram of chemical modification studies on pNJ41 (USCE) and pNJ25.5 (A USCE). Panel A, chemical reactivity of supercoiled pNJ41 (USCE). Panel B, chemical reactivity of supercoiled pNJ25.5 (1 USCE). The relative extent of modification is indicated by the height of the bar corresponding to each base. Data were obtained in the same manner as for Fig. 3.

pAW8.12

flanking (TG), tracts are not uncommon in eukaryotic DNA (23-25).

DISCUSSION

USCE is the result of unequal pairing, followed by a cross- over event, between homologous regions of sister chromatids

c se- .* -.-- * of a replicated chromosome. This results in a duplication of a

.-w region of the chromosome on one sister chromatid with a

2

-I i

reciprocal deletion of that region on the other chromatid. If a USCE occurs in the vicinity of a gene, copy number variation

L of that gene may result which can be detected by molecular x genetic techniques.

There are several known examples of the involvement of USCEs in genetic regulation, including the maintenance of homogeneity of yeast rDNA repeats (26) and the magnifica- tion of ribosomal RNA genes in Drosophila bobbed mutants (27). Indeed, USCE may contribute to the development of multigene families from a single ancestral gene (28). However, the mechanisms which govern or enhance USCE events are not yet known.

FIG. 8. 0~0~ modification of pAW8.12 (y2a-3’) and pNJ41 (USCE) as a function of pH. Supercoiled plasmid DNA was treated with OsOl at the appropriate pH as described under “Materials and Methods.” Left panel, chemically modified pAW8.12 was cleaved with Hind111 and Sal1 and labeled with T4 polynucleotide kinase. Right panel, chemically modified pNJ41 was cleaved with Hind111 and SspI and labeled with Klenow fragment of DNA polymerase I. After treatment with piperidine, DNA fragments were applied to a dena- turing polyacrylamide gel.

DNJ~~ Studies on the immunoglobulin heavy chain locus of MPC-

11 have identified a site of USCE, which resulted in the duplication of the Cy2a constant region genes (1). Sequencing of the regions involved and USCE joint has shown tracts of (TC), on both recombining chromatids at the crossover site with tracts of alternating purine/pyrimidine bases, predomi- nantly (TG),, adjacent to these (TC), tracts (2,3). Since these sequences have the potential to adopt intramolecular triplexes and Z-DNA, respectively, we explored the possibility that non-B-DNA structures were indeed adopted by these se- quences.

plasmids. This result means that significant amounts of in- tramolecular triplex could not be stabilized in these sequences at neutral pH. Since longer pur .pyr sequences can form triplexes at neutral pH when adjacent to nonrepeating DNA,’ the Z-DNA flanking sequence must be destabilizing triplex formation. This is an important observation since (TC), tracts

Analysis of plasmids containing sequences from the regions 3’ of the mouse germline Cy2a and Cr2b heavy chain genes and the USCE joint from the MPC-11 tandem duplication of the Cy2a gene reveals that in negatively supercoiled plasmids these segments are hypersensitive to the chemical probes DEPC and Os04 in a pH-dependent manner. Base pair reso- lution of the sites of chemical modification for the inserts of

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Z-DNA and Triplexes at an USCE Site

the donor sequences for the USCE, pAW8.12 (yZa-3’), and pAW7A3 (-yZb-3’) shows that hyperreactivity to OsOl and DEPC falls within the alternating purine-pyrimidine stretches at neutral pH, indicating that Z-DNA is forming under these conditions. At pH 5.0, the adjacent (TC), tracts adopt an intramolecular triplex, and reactivity in the alter- nating purine-pyrimidine segment is lost. This phenomenon is also seen for pNJ41 (USCE) and pAW25.5 (A USCE). At an intermediate pH (6.0), the inserts of pAW8.12 (yZa-3’), pNJ41 (US&E), and pAW7A3 (y2b-3’) exist as similar amounts of both structures.

This pH dependence can be readily explained; intramolec- ular triplex formation is dependent on low pH because of the requirement of protonated cytosines for the third Hoogsteen- bonded strand of the triplex stem (7). Z-DNA formation is less sensitive to pH (20). Since the formation of both these structures is driven by negative supercoiling, the structure most favored at a given pH will form first, leaving insufficient negative supercoil energy for the formation of the second structure. At pH 6.0 both structures are equally stable, and a mixture of intramolecular triplex and Z-DNA will be present. It is not possible to say whether both structures exist side by side in the same molecule at pH 6.0. However, this seems unlikely, and in the case of pAW8.12 (yZa-3’), the segments which form intramolecular triplex and Z-DNA overlap, prob- ably precluding their simultaneous existence. In the case of pNJ41 (USCE), the TC and TG segments do not overlap, but the level of negative supercoiling required to drive the for- mation of both structures would be very high.

Increasing the level of negative supercoiling in these plas- mids should stabilize the formation of both structures at neutral pH. Intramolecular triplexes at short pur.pyr seg- ments have been demonstrated to form at neutral pH at extreme negative superhelical densities (ZZ), and for a suffi- ciently long pur. pyr tract the structure will form at a negative superhelical density of less than 0.05.’ However, chemical probing of highly supercoiled topoisomer populations (-c = -0.09) of pNJ41 (USCE) and pAW7A3 (y2b-3’) at pH 7.6 did not reveal any increase in intramolecular triplex forma- tion. Therefore, the large relaxation of negative superhelical turns which occurs on the transition to Z-DNA has not been sufficiently compensated for by further decreasing the linking number. It is possible that greater increases in negative su- perhelical density will eventually stabilize the triplex at neu- tral pH. However, this level of underwinding of the helix may be well outside the level of negative supercoiling currently believed to exist in uivo. The failure to observe significant amounts of the intramolecular triplex at neutral pH with increased levels of negative supercoiling in these segments suggests that the in viuo formation of the triplex in such sequences may not be possible by simply increasing the local negative superhelical density. Although it is possible that as yet unknown cellular factors may exist which preferentially stabilize intramolecular triplex formation, it seems that the presence of sequences which can adopt left-handed Z-DNA adjacent to pur pyr segments destabilizes the formation of intramolecular triplexes at neutral pH.

In addition, a significant increase in the amount of Z-DNA at low pH with increased levels of negative supercoiling was not seen. This can be attributed to the fact that intramolecular triplex formation is more favorable than Z-DNA formation at low pH; once formed, the triplex may destabilize adjacent left-handed Z-DNA segments.

Thus, the segments involved in the USCE event are able to adopt Z-DNA at neutral pH. Similar sequences in the inter- vening segments of human fetal globin gene have also been

demonstrated to adopt Z-DNA in supercoiled plasmids (29). These sequences are “hot spots” for recombination and also contain alternating purine-pyrimidine stretches comprised mainly of (TG), of 40-60 bp. Negative superhelical densities of approximately 0.045 were required for the B-Z transition of these segments. The longer alternating purine-pyrimidine segments of pNJ41 (USCE) and pAW7A3 ($L!b-3’) are likely to adopt left-handed helices at comparable or lower negative superhelical densities and the shorter (TG), segment of pAW8.12 (y2a-3’) at higher negative superhelical densities. The in vivo superhelicaI density in eukaryotic cells is not known, although it may reach fairly high levels because of superhelical domains generated by transcription (30).

Although we have not shown a direct relationship between Z-DNA formation and the MPC-11 USCE, it is likely that non-B-DNA structure formation at this site will enhance recombination. Z-DNA has been demonstrated to exist in uivo in Eschmichia coli (9, 32, 37) and is known to have biological consequences in the cell since sequences which adopt Z-DNA undergo frequent deletions (31, 32). Recombination may be enhanced by either the recognition of the Z-helix or B-Z junctions by cellular proteins such as repair or recombination enzymes or by denuding the DNA helix of nucleosomes to allow interaction of two separate DNA helices (38). The ability of pur-pyr stretches to interact with each other through the formation of multistranded structures such as intermolecular triplexes (33-35) or tetraplexes (36) may fur- ther increase the propensity for these sequences to recombine.

Acknowledgment-We would like to thank Dr. J. Maio for his critical reading of this manuscript.

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Left-handed Z-DNA and intramolecular triplex formation at the site of an unequal

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