DNA comes in many forms

Gene, 135 (1993) 999109 0 1993 Elsevier Scrence Pubhshers

GENE

B.V. Al) rights reserved.

99

0378-l 119/93/$06.00

07516

DNA comes in many forms* (Duplex; triple-stranded DNA; quadruplex DNA; Z-DNA; zuotin; syn and anti conformations; telomers)

Alexander Rich Department of Bwlogy. Massachusetts Recetved by G. Bernardi:

Instrtute of Technology,

1 June 1993; Accepted:

Cambridge,

MA 02139, USA

11 June 1993; Received at pubhshers:

17 August

1993

SUMMARY

Forty years ago, we learned of the major double helical structure adopted by DNA. It combined both elegance and simplicity in its design. Since then we have learned that DNA can also adopt other conformations. We now know that it can exist in a variety of triple-stranded and quadruple-stranded forms, as well as forms that are left-handed. The list of alternative conformations that can be adopted by this molecule is still growing. These conformations represent a major biological challenge to understand their role in biological systems. This type of work represents an active frontier in molecular biology today.

INTRODUCTION

Even before the three-dimensional structure of DNA was understood, it was known that the molecule was polymorphic, and could adopt more than one form. The evidence for this came from the early fiber diffraction studies carried out over 40 years ago by Maurice Wilkins and Rosalind Franklin. A dried, oriented DNA fiber produced the highly crystalline A-form diffraction pattern, but the same fiber, when maintained in a moist form, produced a different B pattern. The B-DNA structure is the familiar double helix with the axis of the helix running through the Watson-Crick base pairs. The DNA conformation producing the A pattern differs from B-DNA and is also adopted by double-stranded (ds) RNA. The base pairs are no longer on the axis of the molecule so there Correspondence to. Dr. A. Rich. Biology MIT, 77 Massachusetts Ave., Cambridge,

Department, Room 16-735, MA 02139-4307. USA. Tel.

(1-617) 253-4715; Fax (l-617) 253-8699. *Presented at the COGENE Symposmm, the Human Genome: 40 Years of Molecular

‘From the Double Helix to Genetics’, UNESCO, Paris,

21-23

April 1993.

Abbrevratrons: ds, double NMR, nuclear magnetic handed DNA.

strand(ed); resonance;

mAb, monoclonal nt, nucleotide(s);

antrbody(ies); Z-DNA, left-

is a hole along the molecular axis. In addition, the base pairs are tilted relative to the helix axis. B-DNA has well defined major and minor grooves, but the minor groove in A-DNA is flat and broad, while the major groove is very deep and narrow. These differences in molecular form become of crucial importance in understanding the manner in which proteins interact and recognize nucleotide sequences in ds DNA in the B form as compared, for example, to ds RNA. The narrow entrance to the deep groove in the A form of ds RNA (or DNA) makes it virtually impossible for amino acid side chains to penetrate into the major groove, thus leading to a different form of protein recognition in RNA duplexes compared to B-DNA. The molecular basis for the distinction between A-DNA and B-DNA is found in the conformational flexibility of the furanose ring of ribose. In B-DNA the sugar adopts a C2’ endo conformation (Fig. l), while in A-DNA the sugar adopts the C3’ endo conformation. Because of the change in sugar pucker, the distance between the adjacent phosphate residues along the chain can vary by more than 1 A. The furanose ring thus represents an extensible or elastic element in the DNA backbone; this permits conformational changes in the backbone of the molecule.

100

C2’ end0

Syn

Anti

POSITION

OF

GUANI NE

Sugur

Pucker

POSITION

OF GUANINE I

C3’ end0

Sugar

Pucker

Fig. 1. Two variables contrtbutmg to conformattonal polymorphrsm in DNA. The S-membered furanose rmg can change Its pucker. In the ‘2’ endo pucker (left), the distance between the successive phosphate groups can be 7 A. The C?’ mdo ring pucker IS found in R-DNA In the C3’ entlo conformatton the phosphate groups are closer together. near 6 A or sometimes less. The C3’ endo conformation ts found m A-DNA as well as m RNA. The other variable

conststs

of rotation

of the base around

SJVIconformatton

IS found in other forms of DNA.

Another

of conformational

element

flexibility

the glycosy)

bond. Only the antt conformatton

1s found in A- and B-DNA.

but the

is found

in the orientation of the planar base relative to the sugar. The base can exist in either SJVXor anti conformations (Fig. 1). Although the nnti conformation is the only one found in the B-DNA double helix, the syn conformation occurs in other DNA conformations. Although many alternative DNA

conformations

are

now known, it is generally very difficult to prove the existence of a conformation in vivo and to uncover its biological role. Some examples are discussed below.

TRIPLE-STRANDED

DNA

In 1957, only 4 years after the formulation

of the DNA

double helix, it was found that synthetic polynucleotides were able to form triple-stranded nucleic acids spontaneously (Felsenfeld et al., 1959). This was first observed in the system in which polyuridylic acid could be added as a third strand to a duplex of polyadenylic acid plus polyuridylic acid. It was interpreted as indicating that the second strand of poIyuridyIic acid would hydrogen bond to the adenine N7 and N” amino group (Fig. 2) a hydrogen bonding that was later seen in a single crystal analysis of 9-methyl adenine and 3-methyl thymine (Hoogsteen, 1963). Triple-stranded nucleic acids have since been found to exist in DNA using a variety of hy-

Fig. 2. Triple-stranded by Feisenfeld et al

structure.

Studtes wtth synthetrc

( 1957) led them to the conclusion

polynl~cleot~des that polyadenyhc

actd would combme with two strands of polyundyhc acid to form a trtple-stranded molecule. The type of hydrogen bondtng between the bases 1s shown in the dragram The sohd ctrcle stands for the sugar: the dotted

circles are nitrogen

atoms

drogen bonding arrangements in the major groove of B-DNA. Trtple-stranded DNA is an intermediate in the action of the E. coli recombination enzyme RecA (Camerini-Otero and Hseih, 1993) although the precise nature of the third strand interactions are not yet under-

101

Fig. 3. Triple-stranded triple-stranded

and quadruple-stranded

or quadruple-stranded

structures.

molecules

The single lines lead to the sugar residues;

hatched

organized

Fiber diffraction

circles are nitrogen

stood. Apparently, any sequence can form a triplestranded complex with RecA. Under the driving force of negative torsional strain, long stretches of homopurine/homopyrimidine DNA duplexes found in DNA undergo a conformational change. A segment of the duplex opens up and the freed homopyrimidine strand hydrogen bonds into a remaining intact segment of homopurine/homopyrimidine duplex (FrankKamenetski, 1990). This has been called H-DNA, and there are numerous studies directed toward its biological role in vivo. A number of biotechnology companies are pursuing the goal of using nucleic acid segments that bind to the DNA duplexes to form triple strands as a way of regulating gene expression.

QUADRUPLE-STRANDED

studies of polyinosinic

with this system of cyclic hydrogen

acid were consistent bonding

between

with the formation

hypoxanthine

of either

bases (Rich,

1958).

atoms.

by guanosine monophosphate and suggested that four guanine residues formed cyclic hydrogen bonds in the center (Fig. 4). They pointed out this bonding is related to that which had been proposed for polyinosinic acid, however, it had much greater stability due to the additional hydrogen bonding afforded by the presence of the guanine 2-amino group. This type of hydrogen bonding was also used to interpret the fiber diffraction patterns produced by polyguanylic acid (Arnott et al., 1974;

DNA AS WELL

The origin of quadruple-stranded nucleic acids goes back to 1958 when the structure of a fiber of polyinosinic acid was studied. The X-ray diffraction fiber pattern indicated that the polynucleotide strands were parallel to each other with a three- or fourfold symmetry axis along the fiber (Rich, 1958). It was suggested that the hypoxanthine residues (guanine minus the C2-amino group) were hydrogen bonded in the center of the molecule in a cyclic fashion (Fig. 3). The diffraction pattern was consistent with either three or four strands. At the time there was some preference for the triple-stranded molecule which had a smaller hole in the center; however, the actual structure is quadruple-stranded (Arnott et al., 1974; Zimmerman et al., 1975). A few years later Gellert et al. ( 1962) studied X-ray diffraction patterns of gels produced

Fig. 4. The guanine quartet. X-ray diffraction study of oriented gels of guanosme monophosphate led Gellert et al. (1962) to suggest that the guanines were held together by this system of cychc hydrogen bonding involving erght hydrogen bonds. The solid black circle represents the sugar and the stippled circles are nitrogen atoms.

102 Zimmermar~ et al.. 1975). In 1978, Miles and Fraser suggested that an axial ion is present in the center of fibers of quadruple-stranded polyinosinic acid and later in polyguanylic acid. The ions were postulated to be located between the successive planes of cyclic bonded bases. so that they could coordinate eight Oh oxygen atoms from the upper and lower base planes. Recently, there has been a considerable revival of interest in this type of hydrogen bonding. This was initiated first by Sen and Gilbert f 1988), who discovered that a part of the immunoglobulin switch region containing several sequential guanine residues formed a quadruplestranded complex. They postulated that complexes of this type may have a role in meiosis and interpreted the quadruple-stranded assembly as being held together by quartets of guanine residues as suggested in earlier work. Strings of guanine residues are found most prominently at the ends of chromosomes in the telomere regions. Telomeres have an unusual role since they help to maintain the length of chromosomes which would otherwise tend to shorten following DNA replication due to the enzymatic mechanism. However, there is a complex telomerase enzyme containing a template RNA which acts to continuously lengthen one strand of the telomere (Blackburn, 1991). Telomeres have one strand that is predominantly G rich. while the other is cytosine rich. Typically, the G-rich strand contains large numbers of repeats such as d(T,G,), found in the ciliated protozoan Os~ltrichitr. These repeats are often many thousand-fold in length, bonded to the complementary strand containing d(C,A,),. In those telomeric ends that have been sequenced, there is an overhang at the 3’ end containing two copies of the G-rich strand. The overhang in the Orytrichiu contains d(T,G,& (Lipps et al., 1982). Furthermore, there is evidence that the telomeric ends of chromosomes are cohering and act in vitro as a locus for connecting two DNA fragments together (Oka and Thomas. 1987). Models were put forward by Williamson et al. (1989) and Sundquist and Klug (1989). proposing that the telomeric ends associate by forming guanine quartet structures in which the 3’-guanine-containing overhang folds back on itself hairpin-style with the thymine residues in the loop and the guanine residues paired to each other. Two of these coming together could form a guanine quartet. These models were proposed on the basis of gel migration and chemical modification experiments which showed that the N7 of guanine was shielded from chemical modification. The guanine N’ is involved in hydrogen bonding in the postulated guanine quartet structure. We have undertaken experiments to define the nature of the telomeric structure through single crystal X-ray diffraction analysis. It was possible to crystallize one and

one-half repeats of the ~.~~,~~j~,~l~f~ tclomerc. The sequtnco d(G,T,G,) was crystallized and solved at 1.5 A resolution (Kang et al., 1992). The crystal structure revealed that two of these molecules folded back on each other to form a hairpin-like structure joined together through guanine quartets in the center. The clcctron density map of one of the quartet planes is shown in Fig. 5. A view of the structure from the side IS shown in Fig. 6 in which the contours of the electron density are due to the presence of an axial potassium ion, as had been postulated many years earlier. In order to form a guanine quartet the two models proposed that one segment of guanine residues was in the trf~ti conformatioil while the folded-back segment of guanine residues was in the SJY~ conformation. These postulated conformational differences were necessary in order to form the G quartet. However, the actual structure revealed that all the guanine segments contain an alternation of .sytl and ~riti conformations (Fig. 6). This conformation produced a more stable interaction within the chains. An NMR study carried out of the same sequence (although with the sodium salt) revealed a similar folded-back arrangement in which the G quartets were found in the center of the molecule and the four thymine residues formed a loop at the end (Smith and Felgon, 1992). Like the X-ray diffraction structure. the NMR structure showed an alternation of .SJV~ and unti guanine residues in the quartet region. However, in the NMR structure, the loop went across the diagonals of the quartet in contrast with the crystal structure in which the thymine loops went across the sides of the quartet structure. This is an illustration of the type of polymorphism which 1s probably going to be characteristic of guanine quartet structures. They are likely to form different stable complexes with subtle variations in the folding theme. There has been an extensive study of the effect of different ions on the stability of guanine quartet structures, and the axial potassium ion is the most stable (Guschlbauer et al., 1990). At present there is no direct evidence showing that the guanine quartet is found in vivo. However, a number of proteins have been identified which have the ability to bind to guanine quartet structures, and it remains to be seen whether these proteins can be unambiguously identified with guanine quartets in vivo. This subject IS one that is actively under investigation, and it is likely that interesting results will be revealed in the near future.

THE DNA DUPLEX CAN BE LEFT-HANDED

AS WELL

In 1979 the crystal structure of a DNA hexamer with the sequence d(CGCGCG) was solved at 0.9 A resolution

103

Fig. 5 Telomere structure. Crystal structure analysis of the Oxytrrchra telomere at 2.5 A resolution revealed a system of cyclic hydrogen bonding invol\ ring four gluanine residues (Kang et al., 1992). The electron density map is shown together with the length of hydrogen bon ds (m A).

(Wang et al., 1979). The structure revealed an interesting variant of the B-DNA double helix. The two strands of the duplex were anti-parallel and connected by WatsonCrick base pairs (Fig. 7); however, the helix was left handed. The backbone had an unusual zig-zag shape, and it was termed Z-DNA. To convert from B-DNA to Z-DNA, the base pairs are flipped upside down. This flipping process involved the rotation of the cytidine residues, both base and sugar, thereby introducing a zig-zag in the backbone. For the guanine residues it involved a rotation from the anti to the syn conformation. Moving along the Z-DNA sugar-phosphate chains, the bases al-

ternate in syn and anti conformations. Thus, the molecule had a dinucleotide repeat in contrast to the mononucleotide repeat found in B-DNA. Together with the change from anti to syn, the guanosine sugars also adopted a C3’ endo conformation (Fig. 1). The alternation along the chain involved glycosyl bond rotation as well as the pucker of the rings, as shown in Fig. 1. This was an unusual conformation, and it had been foreshadowed by earlier circular dichroism studies of Pohl and Jovan (1972), who showed that in a high salt environment poly(dG-dC) had a near inversion of the ultraviolet circular dichroism. This inversion in solution

Fig. 6 The ~tructurc d(G,T,G,)

IS folded

the top and bottom

of the U\~rr~/n~r

t&mere

mto a halrpm

conformatlon

of the complex

are rhown

stereo view, the electron

density

contours

m

a

rc~c~lcd

with

m an X-ray

the four

thymme

III this stereo diagram

,tn,~ly~s

( I(,wg CI xl . 199’7 ), 01x .tnd (WC-halt~cpca~\0111xG-IICII st~antl

residues m the loop. Two The halrpms

the center of the molecule

was later established to be the same as the formation of Z-DNA in the crystal (Thamann et al., 1981 f. The two grooves in B-DNA are transfo~ed in Z-DNA. The minor groove remains as a narrowed, deeper groove in Z-DNA bordered by the zig-zag arrangement of sugar phosphate backbones. The major groove in Z-DNA is no longer a groove but actually forms a convex surface on the outer portion of the molecule. Thus, there has been an interesting transformation of the molecule when it is in the left-handed form. Because pyrimidines have a preference for the arzti conformation (Haschemeyer and Rich, 1967). Z-DNA is formed most readily in sequences with alternations of purine and pyrimidine residues, especially those involving d(CG) sequences. The formation of Z-DNA is not dependent upon an alternation of purines and pyrimidines, but the energy required to form Z-DNA is less when those sequences are present. Energy is required to stabilize Z-DNA. It can be stabilized in vitro by a number of changes in the environment. However, the most important component m vivo for stabilizing Z-DNA is negative supercoiling. The reason for

of these halrpms

are held togther

reveal an aural potassium

by guanme

bound quartets

together :L> shown

with

the loops .tt

In Fig 4 In thl\

IOL

this is that negative supercoiled DNA has an energy of supercoiling which is proportional to the square of the number of negative supercoils. If a turn of DNA goes from right-handed to zero to left-handed, two negative supercoils are removed and the resultant supercoiling energy is used to stabilize the Z conformation. This has been demonstrated in a number of different studies (Rich et al., 1984). As is true for all alternative is a great challenge

conformations

to denlonstrate

of DNA, it

the existence

of the

alternative conformation in vivo and to uncover its biological role. There has been considerable progress in Z-DNA. For prokaryotic cells, a number of studies support the conclusion that Z-DNA exists inside the cell (Rahmouni and Wells, 1989). For eukaryotic cells the situation is made more complex since the DNA is organized into nucleosomes in which the DNA is negatively supercoiled. Any disruption of that structure would release negative supercoiling and form Z-DNA. Thus, it becomes imperative to work in biologically active systems. A method was found which uses the agarose microbead

105

Groove Minor Groove

Z DNA

B DNA

Fig. 7. The structure of left-handed Z-DNA as revealed tn the crystal structure of d(CGCGCG) Z-DNA is bordered by a zig-zag array of phosphate groups and 1s analogous to the minor groove

(Wang et al., 1979). The deep, narrow groove tn of B-DNA. The Z-DNA molecule is slimmer than

B-DNA. The edges of the bases in the major groove of B-DNA form the convex outer surface of Z-DNA.

technique developed by Jackson and Cook (1985). In this technique mammalian cells in culture are encapsulated in agarose microbeads by simply adding low-meltingpoint agarose to the culture solution which is laid over mineral oil. Shaking the solution results in the formation of small spheres of the aqueous phase which, upon cooling, form agarose microbeads containing on the order of a thousand cells. Inside the microbead, these cells carry out normal biological activities. The addition of small amounts of Triton X-100 lyses the cytoplasmic membrane, permeabilizes the nuclear envelope, but leaves the nucleus morphologically intact. Moreover, these nuclei are metabolically active. They replicate DNA at a rate 85% equal to that of the intact cell, and they are active in carrying out transcription (Jackson et al., 1988). Wittig et al. (1989) have introduced biotin-labeled monoclonal antibodies (mAb) against Z-DNA into these metabolically active nuclei. These antibodies have as an epitope the zig-zag backbone of Z-DNA and will bind to

The solid line connects

the phosphorous

atoms.

Z-DNA independent of its nucleotide sequence. A series of quantitative experiments have made it possible to measure the amount of Z-DNA in these nuclei. The amount of Z-DNA as measured by mAb bound to the nucleus is independent of the amount of mAb added, even though the concentration of mAb is changed lOO-fold in the incubation mixture. The level of Z-DNA found in these nuclei is dependent on the torsional strain of DNA. Nicking the DNA results in a rapid loss of Z-DNA; stopping the relaxation of DNA through inhibiting topoisomerase I results in an increase of the level of Z-DNA. These experiments demonstrated that the Z-DNA level is determined by the torsional strain or supercoiling of the DNA. In the absence of DNA or RNA synthesis, the level of Z-DNA declined steadily with incubation. This decline was shown to be due to the continued relaxation of DNA due to topoisomerase I. However, upon the addition of ribonucleoside triphosphates, the level of negative supercoiling rose as the rate

106 of transcript~ol~ increased (~~itti~ et al.. 1991 I. The amount of Z-DNA found in the metabolically active myeloma cell nuclei was measured together with the level of RNA synthesis, as determined by the incorporatron of [r-““P]UTP into RNA strands. The incubations were carried out at different concentr~~t~oils of r~b~~~~uc~eosld~ triphosphates in the incubation media. As the level of transcription increases, so does the level of Z-DNA detected in the nucleus. The mechanism for this increase is undoubtedly that suggested by Liu and Wang ( 1987). They proposed that RNA polymerase does not go along DNA rotating around once every ten base pairs. Due to frrctionaf drag, it plows through the molecule generating positive supercoils downstream and negativTe supercoils upstream behind the RNA polymerase. It seems reasonable to believe that the Z-DNA increase observed with transcriptional activity ( Wittig et al., 1991) is due to the increased amount of negative supercoi~ing associated with RNA polymeras~ activity. The experiments described above were global in nature: the total amount of Z-DNA in the nucleus was measured as a function of transcription. Further experiments were carried out which made it possible to measure the formation of Z-DNA in individual segments of a single gene f Wittig et al.. 19921. For this purpose the technique was modified. ~iotin-labeled monoctonat antibodies were diffused into the agarose-enclosed metabolically active nuclei, and the nuclei were then irradiated with a 266 nm laser for 10 ns. The laser irradiation crosslinked the antibody to the DNA but did not crosslink protein to protein. The proteins can be readily removed from the nucleus and a restriction endonuc~ease illt.tI was diffused into the nudeus. DNA fragments emerged from the microbeads. Most were free Alzrf fragments, but some contained Z-DNA monoclonal antibodies crosslinked to them. The segments with the antibody were fished out by adding magnetobeads covered with streptavidin. The DNA absorbed to the magnetobeads was then freed by adding proteinase K that digested the antibody. This released the A/u1 DNA fragments that initially had antibody attached to them due to Z-DNA formation. The A/z41DNA fragments are separated by electrophoresis. and the material is annealed with a 32P-labeled transcript of a particular individual gene. The human leukemic cell culture U937 was used for these experiments. Three zlftlf DNA fragments were found to have formed Z-DNA through their attachment to the magnetobeads via the biotin-labeled mAb against Z-DNA (Fig. 8). The U937 cell line was used because it has the ability to differentiate into a macrophage when a differentiation agent is added to the cell suspension. When this differentiation process starts, transcription of c-rnyr is

drown-regtli~~ted. This vvas ~~cc~~inp~~~lied hy loss of the Z-DNA component of the c-t?ryc+gene. Thus. Z-DNA formation was faund in the actively transcribing gene, hut when the gene was not transcribing Z-DNA formation disappeared. This experiment thustrates fhc dynamic nature of DNA in the nucteus, changing conformatior~ depending upon transcriptional activity. The ease with which Z-DNA forms in any nt sequence can be determined through a simple calculatton. The energies required to flip different sequences from B to Z have been measured by an extensrve series of experiments. These quar~titative results can bc formlllated mto a computer program which makes it possible to scan the nt sequence and measure the energy required for jnd~vidu~~l segments to form Z-DNA (Ho et al., 1986). When that calculation is carried out on the c-nt)*cgene, three regions are found which have a high potential for forming Z-DNA, These regions are in the same three .ilittl fragments which formed Z-DNA as indicated in Fig. 8. These segments contain about 20-30 nt and have a high concentration of GC base pairs with many alternations of guanine and cytosine (Wittig et al., 1992 1. Thus, the L’Xperimental results revealing the formation of Z-DNA in c-nr~~ agreed well with the calculation regarding the case of formation. Furthermore, it should be noted that the Z-DNA forming segments are largely in the upstream or 5’ portion of the gene rather than the do~~nstre~l~ portion. This is also in agreement with the anatysis by Liu and Wang (1987) which pointed out that the upstream region is most strongly subjected to negative supercoiling whife trallscript~on is proceeding. An extensive compilation has been carried out by Schroth et al. ( 1993 ). A total of 137 human genes that were fully sequenced both in the upstream and downstream regions were subjected to a calculation to determine the positions where the most probable Z-DNA forming elements are found. This distribution revealed a peak of Z-DNA forming elements which are found near the transcription start point in the upstream region of the gene. The resufts with the c-ln_rc*gene are thus in agreement with the general distribution of Z-DNA forming elements in human genes generally, These elements are largely concentrated in the ups&ream region of the gene where they are subject to maximum negative supercoiling due to transcription and thus are likely to result in Z-DNA formation. Another approach to determining Z-DNA functton in biological systems is to isofate and characterize proteins that bind preferentially to Z-DNA. Using a band shift assay in which radiolabeled Z-DNA fragments are used. changes in their mobility are observed in an agarose gel when a protein binds to them. A yeast Z-DNA binding protein has been isolated in this way and cloned (Zhang

107

Fig. 8. The human c-myc gene is shown at the top wtth the promoter region at the left and the four transcrtption start pomts indicated as P,-P,. The cross-hatched segment of the exons represents the c-myc codmg region. The line underneath has vertical marks indicating sites at which the AluI restrictron enzyme digests the DNA. When c-myc is transcribed, three AluI fragments form Z-DNA as indicated by the heavy, black horizontal lines (Wtttig et al., 1992). The number of nt m the AluI fragment is indicated. When the gene is no longer transcrtbed. these fragments no longer exhibit Z-DNA conformation.

al., 1992). The 49-kDa protein called zuotin binds Z-DNA preferentially and has in its sequence homology elements to DnaJ protein as well as histone H-l. A histone H-l analog has not yet been found in yeast. Mammalian histone H-l has been reported to bind Z-DNA preferentially over B-DNA (Mura and Stollar, 1984). The phenotype of a zuotin-less yeast mutant is a very slow-growing organism. Thus, it is not an essential gene, but one that seems to be important for maintaining optimal growth rates. A number of experiments are now being carried out to ascertain whether the Z-DNA binding activity is a central part of the biological role of zuotin. More recently a chicken nuclear protein has been identified that binds to Z-DNA very tightly (Herbert et al., 1993). This protein has a binding dissociation constant which is in the nanomolar range. Its cloning and characterization should lead to more understanding of its biological role. At present a number of suggestions have been made regarding Z-DNA functions. The presence of Z-DNA in the upstream region of a transcribing gene may affect the spacing of RNA polymerase molecules (Wittig et al., 1992). RNA polymerase cannot transcribe through Z-DNA and must await the relaxation of the DNA by topoisomerase. When an RNA polymerase molecule passes by a segment with a high potential for forming Z-DNA, it flips it to the Z-DNA conformation due to the negative supercoiling behind the polymerase. Thus, there is a possibility that the following polymerase is delayed by this Z-DNA element until it is relaxed by topoisomerase I. A system such as this would have the effect of separating RNA polymerases, and in turn separating their RNA transcription products. Since these RNA transcripts must be spliced into active messengers, this increased separation may lead to a decrease in transsplicing, that is, an error in splicing that involves two different messages resulting in abortive transcripts. Experiments are under way to change the nucleotide se-

et

quence of the Z-DNA forming elements of c-myc so that they no longer form Z-DNA and to determine what effect this has on the level of transcription as well as the level of Pans-splicing in vivo. The formation of Z-DNA following transcription may play an important role in the positioning of nucleosomes following the cessation of transcription. Earlier experiments by Garner and Felsenfeld (1987) suggest that nucleosomes form preferentially near Z-DNA forming segments although the Z-DNA is not inco~orated into the nucleosome. In principle this represents a mechanism for nucleosome positioning which could be very important in maintaining chromatin in a configuration that can be subsequently reactivated. This is especially true in the control or promoter regions of the gene where transcription factors are likely to bind during gene activation. Having developed a technique for identifying Z-DNA forming segments in individual genes, we can use mutagenesis to change these sequences so they no longer form Z-DNA and observe what effect if any this has on nucleosome positioning. Likewise, the cloning of Z-DNA binding proteins will make possible further experiments to identify the manner in which they interact with the DNA, both in transcription and in nucleosome positioning.

STILL MORE DNA CONFORMATIONS

In the short listing above, we are far from exhausting the total number of DNA conformations. Another well known conformation is one in which inverted repeat sequences extrude duplexes to make cruciform segments of DNA, using the complementary pairing of the inverted repeats. Inverted repeats are widely found in the eukaryotic genome (Wilson and Thomas, 1974), and it is quite likely that cruciform structures form. It has long been suggested that transiently formed cruciforms may serve in vivo as signals. Although the conditions which favor cruciform formation are not fully known, some proteins

108

have

been detected

which

bind

prcferenti~~lly

forms (Hamada

and Bustin,

1985 1.

A specialized

but related

version

that found during bination, Although

structure

considerable

those nucleotide known

of the cruciform

In homologous

two DNA helices with identical

a crossing-over

lizing

recombination.

about

called

is

recom-

sequences

a Holliday

form

that are important

(Lilley,

1988).

not

the three-dimensional

structural

deal

is

details

of

involve

of proteins

is beginning

structures

of more

change in the DNA.

to accumulate

DNA-protein

with DNA

on these as the

complexes

determined

by either X-ray diffraction

However.

we are still at the very beginning

are being

or NMR

studies.

of determining

the variety of structural changes that occur in DNA when it interacts with proteins. In general the field of DNA conformational changes is still at an early stage of development. As molecular biology progresses, it is likely that even more conformational changes in DNA will be discovered. This is all a reflection of the

enormous

versatility

that

is inherent

in

the

molecule. There is always a constant challenge to relate conformational changes which are discovered in vitro to changes that occur in vivo. The manner in which this is carried out will vary from one problem to another. In general, it will involve a study of the interactions of the altered conformation with protein components of the cell and ultimately a genetic analysis which may lead to a clear characterization of biological functions. This broad field represents an important frontier in developing our understanding

of DNA and the manner

in which it func-

tions. This is a major challenge that will be played out during the next 40 years in the development of molecular genetics.

DNA.J.

Cold

2033

and unu~~sl

Harbor

G.

Nati. Acad

W.. Chantot,

71124

htructurrs

Laboratory

and It>

Press,

EfTect of Z-DNA

Cold

on nucleosome

Biomol

Struct

qomal proteins &chemistry Haschemeyer,

later

from

by gua-

guanosme

nucleic

gels to tclomer

X I 1990) 491 ~51 I

Dynamics M

Htcrarchy

of bmdmg

I and 2 m supercoiled

HMG 23

D.R.: Heir!, formation

SCI. USA 3X (1967) 1013-1014.

J.-F. and Thlele, II.. Four-stranded

25 years

H and Bustm.

analysts

Spring

M N and Da\~es.

Proc

structures

Hamada.

DNA supercoIling

Felbenfeld.

M.. Ltpsett.

acid

ol a three-btranded

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(I9781 8037-8038 ACKNOWLEDGEMENTS

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