Mechanisms of DNA bending

688

Mechanisms

of DNA bending

L James Maher III Genome Recent involved notably

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in DNA bending

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Addresses Department of Biochemistry and Molecular Biology, Mayo Foundation, GUI 6, 200 First Street SW, Rochester, MN 55905, USA; e-mail: [email protected] Current Opinion in Chemical

Biology

Current Opinion in Chemical

Biology

1998, 2:688-694

http://biomednet.com/elecref/1367593100200688

Examples of two classes of DNA bending proteins (red). (a) Class 1 proteins, such as mammalian TBP bind bent DNA (blue) on its convex

0 Current Biology Ltd ISSN 1367-5931

surface, bending the DNA away from the protein. (b) In contrast, class 2 proteins, such as the Escherichia co/i CAP protein, bend DNA toward the protein-DNA

Abbreviations bZlP basic leucine zipper CAP TBP

catabolite activator protein TATA box binding protein

Introduction Genomes consist of long, double-helical DNA molecules. These double helices are right-handed and are characterized by major and minor grooves due to Watson-Crick base pairing. The persistence length (A) of DNA refers to the distance over which a segment of the molecule tends to remain in a linear trajectory. This parameter is estimated to be -50 nm (-150 bp) for duplex DNA. For very long DNA molecules (many thousands of base pairs in length), polymer flexibility and Brownian motion reduce the average end-to-end distance to (AL)“.5, where L is the molecular contour length [l]. In contrast, over the shorter lengths commonly bound by proteins, naked (i.e. uncomplexed) duplex DNA behaves as a stiff, worm-like coil. Because of this inherent stiffness, the biologically relevant bending of DNA into compact structures such as nucleosomes and transcription complexes requires energy. DNA-bending energy is provided by favorable protein-DNA interactions involving van der Waals contacts, burial of hydrophobic surfaces, formation of hydrogen bonds, and Coulombic interactions that release condensed counterions. Simplification of an expression for the free energy of DNA bending [Z] leads to Equation 1.

(kcal/mol)

AGbend = 0.0135

(1)

Lbp DNA bending free energy at room temperature (AGbend) is expressed in kcal/mol, assuming a DNA persistence

interface.

length of 150 bp, where DNA is bent by 0 degrees over a contour length of Lb>. Physiological thermal energy is predicted to allow a random root mean square fluctuation of -7” bending per base pair. Because this motion is random, however, its time-average produces little coherent local curvature. Sites of induced DNA bending may be of biological importance because they can contribute significantly toward the overall cost of DNA bending in higherorder nucleoprotein structures. For example, the cost of bending 100 bp of DNA by 90” is calculated to be -1.1 kcal/mol, whereas the presence of a pre-existing bend of 20” reduces the required additional bend angle by -22% while reducing the required additional bending energy by -40% (to 0.66 kcal/mol). High-resolution structural studies have provided evidence for at least two motifs in nucleoprotein complexes involving bent DNA. These two motifs suggest different bending mechanisms. The first important class of DNA-bending proteins (here termed class 1) contact bent DNA on its convex surface, such that the double helix curves away from the bound protein (Figure la). Examples include the TATA box binding protein (TBP), which interacts with the TATA box in the promoter region of genes in order to initiate transcription, high mobility group (HMG) box proteins such as the human male sex determining factor and the human lymphoid enhancer binding protein 1, and other molecules that are often classified as ‘architectural’ binding proteins [3,4]. The biological role of such proteins appears to primarily derive from their ability to strongly bend DNA, thus modifying the local nucleoprotein geometry. The mechanism of DNA-bending by class 1 DNA-bending proteins appears to involve intercalation of hydrophobic amino acids between adjacent base pairs in the minor groove of

Mechanisms

DNA, thus dramatically enlarging altering the helix axis [3].

this groove, and thereby

A second key class of DNA-bending proteins (here termed class 2) contact DNA on its concave surface, curving the double helix toward the bound protein. Many class 2 DNA bending proteins have been described. These include the E. coli catabolite activator protein (CAP; Figure lb) [S] that regulates transcription of certain bacterial genes and the eukaryotic histone octamer, which is responsible for packaging DNA in chromatin by wrapping -150 bp of DNA by almost 720”. Histones form disc-like structures around which eukaryotic DNA is wound, creating a repeating structural unit called a nucleosome. The engaged surfaces of class 2 proteins often contain cationic amino acids, suggesting an important role for electrostatic interactions. In this review, I will discuss two classes of DNA bending proteins, together with hypothetical bending mechanisms.

Figure

of DNA bending Maher

689

2

(4

-

Variable linker Reference bend

Crystal structures involving bent DNA Perhaps the most spectacular recent advance in our understanding of induced DNA bending comes from the highresolution structure of the mammalian nucleosome [6”]. This work required the independent expression and purification of recombinant histones and their reconstitution with uniform DNA fragments into this massive complex prior to crystallization. This structure reveals in detail how 146 bp of DNA are arranged in 1.7 turns of left-handed DNA superhelix. With respect to the mechanism of DNA bending in this class 2 complex, the DNA is contacted mainly at 10 bp intervals, corresponding to sites where the minor groove faces the histone octamer core. At each of these sites, many favorable protein-DNA interactions appear to stabilize the bent DNA configuration. The authors note five modes of protein contact with the deoxyribose phosphate backbone of DNA, which are sequence-independent (as expected because the nucleosome serves a genetic DNA packaging function that is not strongly influenced by DNA sequence): the amino termini of a-helical segments face specific DNA phosphate groups, presumably generating a favorable Coulombic interaction based on the a helix dipole (arising because of the alignment of partial positive and negative charges inherent in each polarized peptide bond); mainchain amide nitrogen atoms of the histone proteins form hydrogen bonds to DNA phosphate groups; arginine residue sidechains often approach the DNA; nonpolar contacts are made with the deoxyribose sugars; and hydrogen bonds and salt bridges are observed between certain DNA phosphate groups and protein sidechains. Thus, this remarkable crystal structure provides a wealth of information for designing experiments to discriminate between the roles of various forces in DNA bending. Three other recent X-ray structures of class 2 proteins complexes are also worthy of mention. A re-examination and refinement of the structure of the E. coli CAP protein bound to DNA has been presented [7]. This structure

Current Opinion in Chemical

Biology

Two phasing assays for DNA bending by proteins. (a) Depiction of a family of electrophoretic phasing probes, which are made up of several hundred base pairs of duplex DNA (cylinders) flanking a phased array of As_, tracts (5 to 6 consecutive adenosine residues on the same DNA strand recurring every 10 base pairs) as a reference bend, a short phasing linker of variable length, and an unknown bend, typically induced by protein binding. The range of overall probe shapes as a function of phaser linker length is indicated by the oval with arrowheads. Electrophoretic mobilities therefore depend on the phasing of the unknown and reference bends. Shorter

end-to-end

molecular distances correspond to lower gel mobility. (b) In minicircle competition binding assays, DNA probes contain protein-binding sites (filled ovals) at various distances from phased As_, tracts (heavy lines), either constrained in minicircles (above) or in linear fragments (below). Relative to linear forms of the binding site, proteins that bend DNA should bind more tightly to the minicircle form of the probe whose protein-binding site is pre-bent in the appropriate direction.

largely agrees with that previously published [S], while clarifying aspects of the sharply kinked DNA conformation that results in an overall DNA bend of -90”. The more subtle example of DNA bending caused by the PU.l ETS domain has also been recently analyzed [8]. PU.l is a transcription factor in the Ets family of DNA-binding proteins, and functions in the differentiation of human hematopoetic cells. PU.l contacts DNA over 10 bp, inducing a gentle 8” bend in the helix axis. DNA sequence recognition is reminiscent of the prototypic helix-turn-helix motif seen

690

Biopolymers

Figure

3 Proposed

(4

Intercalating protein

roles of electrostatic effects in DNA

bending by class 1 and class 2 proteins. (a) Schematic depicting enhanced interphosphate

repulsions

(arrow) by a

reduced local dielectric environment in the DNA groove as an intercalating protein approaches. (b) Models for DNA bending by class 2 proteins. In the traditional ‘attraction’ model, DNA bending results only from favorable protein-DNA interactions. In the ‘collapse’ model, some of the DNA bending is induced by unbalanced interphosphate repulsions (shown as horizontal arrows) on the DNA face away from the cationic surface of the protein. 0 represents face of the DNA duplex.

‘Attraction’

c\

n

the neutralized

‘Collapse’

+ljlj~2p~ Current Opinion in Chemical

Biology

in bacterial DNA-binding proteins such as CAP. The authors note that salt bridges to DNA phosphate groups occur on the helical face that bends toward the protein. Finally, the structure of the yeast Mat&-MCMl-DNA ternary complex has been obtained. This complex is involved in establishing the mating type in haploid a cells and repressing haploid functions in diploid Saccharoqces cermisiaecells. a2 protein is expressed in cells of the a mating type and interacts with the ubiquitous MCMl protein, binding to DNA and repressing genes specific for the alternative a mating type. The Mat&-MCMl-DNA complex shows the DNA to be bent by -70” [9]. DNA bending appears to maximize hydrophobic and hydrogen bonding interactions in the major groove, and hydrogen bonding with the phosphate backbone.

by positive base-pair roll at single sites (if one imagines walking up ‘stairs’ formed by the base pairs of the double helix, roll corresponds to the tilting of a step towards the walker). Examples of such kinks are seen in DNA complexes with CAP and the E. coli integration host factor (IHF). Second, writhe (superhelical twisting of the double helix) results when positive roll occurs at a series of adjacent base pairs (e.g. TBP). Third, some protein-DNA complexes cause a continuous, smooth curvature of the double helix axis. 28 of the 86 examples constitute complexes with obvious induced DNA bends. By focusing only on the structure of DNA in these protein-DNA complexes, this analysis permits an appreciation of how inherent DNA properties are exploited by proteins to induce DNA bending.

A related and noteworthy recent contribution by Dickerson [10”] analyzes and catalogs DNA bending in 86 published examples of protein-DNA complexes deposited in the Nucleic Acid Database. This comprehensive study draws several interesting conclusions. Three well-defined types of bending are observed. First, kinks are produced

The basic leucine zipper controversy DNA bending by the basic leucine zipper of transcription factors (including Fos-Jun Jun-Jun and GCN4 homodimers and their been actively investigated. These proteins 7 bp AP-1 recognition site 5’-TGACTCA in

(bZIP) family heterodimers, relatives) has bind to the duplex DNA.

Mechanisms

Recent developments in this area feature an ongoing debate between the laboratories of Kerppola and Crothers as to whether any of these proteins actually cause significant DNA bending. The disagreement has been described in two recent reviews [11,12’]. At issue is the interpretation of experimental results obtained using different assays for DNA bending. On one hand, certain conventional electrophoretic circular permutation and phasing experiments provide evidence that some bZIP proteins (e.g. Jun homodimers) but not others (e.g. S. cereuisiae GCN4 homodimers) induce DNA bending in the range of 10-30” [13,14’,15’,16]. On the other hand, published crystal structures and electrophoretic phasing experiments with other probe geometries fail to show DNA bending by full-length Fos-Jun complexes and smaller derivatives [17]. In addition, the results of ringclosure experiments and minicircle binding competition assays in solution fail to support the bending interpretation [17,18’]. The kinds of probes used for electrophoretic phasing assays and minicircle binding competition assays are shown schematically in Figure 2. Much attention has focused on the reliability of the various bending assays in use [ll,lZ’]. For example, methods that depend upon mobility anomalies in polyacrylamide gels cannot be interpreted from first principles, as a detailed theory of polyacrylamide-macromolecule interactions is lacking. On the other hand, effects of imprecise binding-site phasing in minicircle competition binding assays are uncertain, as is the expected effect if a protein under study introduces a site of nonisotropic DNA flexibility (rather than a static bend); however, an equally important additional issue that must be addressed is the exact protein complex under study. For example, a recent report from the Crothers’ laboratory described evidence that a truncated Fos-Jun complex [Fos(ll%?l l)-Jun( 199-334)], which had been shown in gel phasing experiments to bend DNA by less than So, does not bend DNA ([18’]; see [15’]). Thus, the particular protein complex selected by the Crothers’ laboratory for detailed study was not an example the Kerppola laboratory would have predicted to cause significant DNA bending. In any case, it seems that resolution of this dispute will require further comprehensive studies, combining several techniques and a full panel of bZIP derivatives.

Electrostatic effects With regard to possible mechanisms of DNA bending, several recent studies have suggested roles for electrostatic effects in both class 1 and class 2 proteins. In the case of class 1 proteins such as TBP (Figure l), an interesting molecular modeling simulation was performed [19] to explore the prediction that protein occupation of a DNA groove could reduce the apparent local dielectric constant [ZO]. This simulation used a ‘virtual protein’ whose only property was a specified degree of reduced dielectric character. Electrostatic effects were evaluated using the Poisson-Boltzmann equation. As this virtual protein was brought near the modeled DNA, the double helix was

Figure

of DNA bending Maher

691

4

Oligonucleotide

Restraining

(domain 1)

tether

Oligonucleotide (domain 2)

Current ODinion in Chemical Biolow

Schematic illustration of a two-domain oligonucleotide designed to capture a bent conformation of a duplex DNA target sequence through the formation of two oligonucleotide-directed triple helices separated by a restraining tether.

observed to become highly deformed at the protein-DNA interface, mimicking in some ways the conformation of DNA in TBP-DNA structures. This DNA deformation was attributed entirely to increased interphosphate repulsions along the helical face contacted by the simulated protein. Thus, the case was made for a plausible electrostatic component in DNA bending caused by of class 1 proteins (Figure 3a). The suggestion that protein binding can induce DNA bending by ‘revealing’ latent forces within the double helix also figures prominently in the asymmetric phosphate neutralization hypothesis that was originally proposed by Rich and co-workers (reviewed in [Zl]). This hypothesis applies to class 2 proteins, suggesting that salt bridges on one helical face neutralize residual phosphate charges on that face, leaving an unbalanced set of interphosphate repulsions along the helix axis. The double helix is thought to relax toward the neutralized face (Figure 3b). Electrophoretic experiments with chemicallyneutralized DNA molecules tend to support this conclusion [Z?] and have recently extended the result to other sequences [23’] and neutralization patterns [24’]. Mathematical [ZS] and computational simulations [26] are in agreement with these experimental data. When patterns of phosphate neutralization are created to mimic salt bridge contacts with transcription factors, DNA bending of approximately the correct magnitude and direction is

692

Biopolymers

observed [27’,28”,29’]. Charge patterns on certain bZIP proteins appear in electrophoretic phasing experiments to induce DNA bending in the direction predicted by the neutralization phosphate model asymmetric [28”,30,31’,32,33]. On the other hand, chemical substitution of methylphosphonate analogs is an imperfect approach to charge neutralization [24’], introducing effects on DNA shape that may not be electrostatic in origin; however, additional experiments wherein excess cations are tethered to one DNA face also suggest that detectable DNA bending can be observed when unbalanced electrostatic forces are present along the double helix, though the induced bending is not as dramatic [34,35].

CAP-DNA, TBP-DNA) and recombination (e.g. IHF-DNA). Two general classes of DNA-bending proteins can be discerned. For class 1 DNA-bending proteins, intercalation of hydrophobic amino acids between base pairs is a major bending mechanism, perhaps augmented by the electrostatic effect of reduced local dielectric constant. For class 2 proteins, favorable protein-DNA interactions and unbalanced phosphate neutralization may be important factors in DNA bending. Important questions for future study involve better understanding of the origin of bending forces applied to DNA by proteins, and to what extent the physical stiffness of DNA constrains protein-protein interactions in viva. in the context of chromatin.

Ionic effects

Acknowledgements

In a discussion of protein-induced DNA bending, it is important to recall that ionic conditions in solution can themselves alter the bending properties of DNA. For example, the persistence length of DNA has been measured in the presence of low concentrations of multivalent ions (e.g. metals such as Mg2+, cobalt[III] hexaammine3+ and polyamines such as spermidinex+ and spermine4+) using a variety of techniques. Under these conditions, the persistence length of DNA is observed0 to drop dramatically0 below the conventional value of 500 A to as little as 1.50 A. An interesting recent experimental approach involves studies of this phenomenon using single molecules of h phage DNA stretched by laser tweezers [36’]. In related work, theoretical analysis has led to a proposal in which this decrease in persistence length is caused by the purely electrostatic effects of transient, groove-bound multivalent cations causing transient local DNA bending [37’]. The proposed mechanism involves a combination of phosphate attraction to each transiently-bound cation, and repulsion of other solvent cations that would otherwise tend to screen phosphate charges.

Artificial DNA-bending

agents

An ingenious series of recent experiments has investigated the interesting possibility that artificial DNA-bending agents can be designed [3841]. This approach is based on oligonucleotide-directed triple helix formation using two oligonucleotides linked by a restraining tether. Because of this tether, simultaneous occupation of both triplex target sites requires DNA bending (Figure 4). It was found that highly bent target DNA configurations could be ‘captured’ using such oligonucleotides [38,40]. Thermodynamic approaches have been applied in an attempt to deduce bending energies from these experiments [39,41]. The development of sequence-specific DNA-bending proteins, perhaps by the combination of independent DNA binding and bending protein domains, represents an exciting prospect for molecular nanotechnology.

Conclusions Over tens to hundreds of base pairs, duplex DNA behaves as a relatively stiff, worm-like coil. The shape of DNA is more radically altered in nucleoprotein complexes essential for DNA packaging (e.g. histone-DNA), transcription (e.g.

Experiments Foundation, of Health. particularly

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References and recommended

reading

Papers of particular interest, published within the annual period of review, have been highlighted as: l l

1.

of special interest * of outstanding interest Trun NJ, Marko JF: Architecture of a bacterial chromosome. Sot Microbial News 1998, 64:276-283.

Am

2.

Liu-Johnson H-N, Gartenberg M, Crothers DM: The DNA binding domain and bending angle of E. co/iCAP protein. Cell 1986, 47:995-l 005.

3.

Werner MH, Groneborn AM, Clore GM: Intercalation, DNA kinking and the control of transcription. Science 1996, 271:778-784.

4.

Werner MH, Burley SK: Architectural transcription that remodel DNA. Cell 1997, 881733.736.

5.

Schultz SC, Shields GC, Steitz TA: Crystal structure of a CAP-DNA complex: the DNA is bent by 900. Science 1991, 253:1001-l 007.

factors: proteins

6. ..

Luger K, Mader AW, Richmond RK, Sargent DF, Richmond TJ: Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 1997, 389:251-260. This paper has been accurately described as a ‘tour de force’ of biochemistry, involving full reconstitution of the histone octamer from recombinant proteins. The unprecedented resolution of this nucleosome structure permits a full accounting of protein-DNA contacts, and makes predictions about the kinds of forces that give rise to the dramatic DNA bending observed. 7.

Parkinson G, Wilson C, Gunasekera A, Ebright VW, Ebrighi RE, Berman HM: Structure of the CAP-DNA complex at 2.5 A resolution: a complete picture of the protein-DNA interface. J MO/ Bioll996, 260:395-408.

8.

Pio F, Kodandapani R, Ni CZ, Shepard W, Klemsz M, McKercher SR, Maki RA, Ely KR: New insights on DNA recognition by ets proteins from the crystal structure of the PU.l ETS domain DNA complex. J Biol Chem 1996, 271123329.23337.

9.

Tan S, Richmond TJ: Crystal structure of the yeast MATu2/MCMl /DNA ternary complex. Nature 1998,

391:660-666.

10. ..

Dickerson RE: DNA bending: the prevalence of kinkiness and the virtues of normality. Nucleic Acids Res 1998, 26:1906-l 926. This paper provides a comprehensive survey of bent DNA structures extracted from 86 complexes involving sequence-specific proteins. Several useful summary statistics and diagrammatic representations are introduced. Three categories of DNA bending are discerned, with the interesting observation that base pair tilt is never a significant component of DNA bending. This summary is uniquely comprehensive. 11.

Hagerman PJ: Do basic region-leucine zipper proteins bend their DNA targets ... does it matter? froc Nat/Acad SC; USA 1996, 93:9993-9996.

Mechanisms

12. .

McGill G, Fisher D: DNA bending and the curious case of Fos/Jun. Chem Bioll998, 5:29-38. This brief review paper is particularly helpful in its diagrammatic summaries of various DNA bending assays. The authors provide useful background and a description of the ongoing debate concerning DNA bending by basicleucine zipper proteins. 13.

Kerppola TK: Fos and Jun bend the AP-1 site: effects of probe geometry on the detection of protein-induced DNA bending. froc 0122. Nat/ Acad SC; USA 1996,93:10117-l

14. .

Kerppola T: Comparison of DNA bending by Fos-Jun and phased A tracts by multifactorial phasing analysis. Biochemistry 1997, 36:10872-l 0884. The author presents a comprehensive analysis of variables influencing electrophoretic phasing experiments. Among the contributions of this work are the introduction of a damped harmonic oscillation function for curvefitting of phasing data. In the context of the debate concerning DNA bending by basic-leucine zipper (bZIP) proteins, the author uses intrinsically curved A tracts to argue for three criteria to establish actual DNA bending, and presents evidence that the phasing properties of bZlP proteins meet these criteria. Kerppola TK, Curran T: The transcription activation domains of Fos and Jun induce DNA bending through electrostatic interactions. EM60 J 1997, 16:2907-2916. Using electrophoretic phasing experiments, the authors explore an impressive number of combinations of protein domains from Jun and Fos proteins to explore which protein domains contribute to phasing anomalies consistent with DNA bending. It is argued that, although they are distant from the DNA binding domains, the anionic transcription activation domains of these basic-leucine zipper proteins can also influence DNA bending through electrostatic effects. These bending effects are argued to deflect the DNA gently away from the transcription activation domain of the bound protein. The authors suggest that the opposite directions of apparent DNA bending by Fos and Jun are related to the opposite disposition of the acidic transcription activation domains in these proteins. 15. .

16.

Leonard D, Rajaram N, Kerppola T: Structural basis of DNA bending and oriented heterodimer binding by the basic leucine zipper domains of Fos and Jun. froc Nat/ Acad Sci USA 1997, 94:4913-4918.

17.

Sitlani A, Crothers DM: Fos and Jun do not bend the AP-1 recognition site. froc Nat/ Acad Sci USA 1996, 93:3248-3252.

18. .

Sitlani A, Crothers DM: DNA-binding domains of Fos and Jun do not induce DNA curvature: An investigation with solution and gel methods. froc Nat/ Acad Sci USA 1998, 95:1404-l 409. This paper presents experiments using DNA minicircle competition assays and DNA cyclization kinetics measurements. Supporting the contention of this group that Fos-Jun heterodimers do not, in fact, bend DNA, the data are interpreted to show that DNA bending must be less than 5’. The authors suggest that electrophoretic mobility anomalies are associated with the relative positions of the leucine zipper of the bound protein and the local region of curved DNA. It must be noted that the particular protein combination studied in this paper [Fos(ll8-21 l)-Jun(l99-334)] has been assigned an induced DNA bend angle of only 4’ in electrophoretic phasing experiments in the Kerppola laboratory (see [15’]), so there is, in fact, no disagreement between the groups on this point. It will therefore be important to extend the minicircle competition assays to other basic-leucine zipper combinations thought to bend DNA more dramatically. 19.

Elcock AH, McCammon JA: The low dielectric interior of proteins is sufficient to cause major structural changes in DNA on association. J Am Chem Sot 1996, 118:3787-3788.

20.

Travers AA: Reading the minor groove. 2:615-618.

21.

Crothers DM: Upsetting 1994, 266:1819-l 820.

22.

Strauss JK, Maher LJ: DNA bending by asymmetric neutralization. Science 1994, 266:1829-l 834.

23. .

the balance

Nat Struct

of forces

Bioll995,

in DNA. Science

phosphate

Strauss-Soukup JK, Rodrigues PD, Maher LJ: Effect of base composition on DNA bending by phosphate neutralization. Siopbys Cbem 1998, in press. This paper employs electrophoretic assays of DNA duplexes ligated end-toend to explore how DNA base composition affects DNA bending by asymmetric phosphate neutralization. Patterns of six phosphate groups are neutralized, three consecutive phosphate groups on each side of one minor groove of DNA, and DNA bending is monitored in GC- versus AT-rich contexts. Perhaps surprisingly, induced DNA bending (-20’) was comparable in both cases, suggesting little difference in the intrinsic minor groove compressrbrlrtres of the two sequences studied.

of DNA bending

Maher

693

24. .

Strauss-Soukup JK, Maher LJ: Effects of neutralization pattern and stereochemistry on DNA bending by methylphosphonate Biochemistry 1997, 36:8692-8698. substitutions. The authors explore how the pattern and stereochemistry of laterally asymmetric methylphosphonate substitutions in DNA affect induced bending. It was first determined that an alternating pattern of six phosphate neutralizations (three neutralizations alternating with charged phosphate diester linkages on each side of one minor groove) caused -13’ of DNA bending, compared with -20’ for consecutive neutralizations. These DNAs with diastereomeric mixtures (RP and Sp) of chiral methylphosphonates were then compared to duplexes wherein the methylphosphonate substitutions were only the Rp stereoisomer. Induced DNA bending was -go, about 30% less than for the racemic methylphosphonates (-13’). These results suggest that methylphosphonate analogs (particularly S, stereoisomers) distort DNA structure to a small extent by nonelectrostatic effects, but that electrostatic effects remain a plausible explanation for induced DNA bending by asymmetric methylphosphonate substitutions. 25.

Manning GS, Ebralidse KK, Mirzabekov AD, Rich A: An estimate of the extent of folding of nucleosomal DNA by laterally asymmetric neutralization of phosphate groups. J Biomol Struct Dyn 1989, 6:877-889.

26.

Sanghani SR, Zakrzewska K, Lavery R: Modeling DNA bending induced by phosphate neutralisation. In Ninth Conversation in Biomolecular Stereodynamics. Edited by Sarma RH, Sarma MH. Schenectady: Adenine Press;1 996: 267-278.

27. .

Strauss-Soukup JK, Maher LJ: Role of asymmetric phosphate neutralization in DNA bending by PU.l. J Biol Cbem 1997, 272:31570-31575. This paper applies the predictions of asymmetric phosphate neutralization to the case of an actual DNA-protein complex for which high-resolution structural data are known. In the X-ray crystal structure, PI-t.1 protein gently bends DNA (-8’) as it contacts one face of the double helix, making asymmetric salt bridges to DNA phosphates. The shape of the unbound DNA site was determined by electrophoretic analysis before and after neutralization of the relevant phosphate groups by methylphosphonate substitution. DNA bending induced by charge neutralization (-28’) is larger than observed for protein binding, and is in a similar direction. 28. ..

Strauss-Soukup J, Maher L: Electrostatic effects in DNA bending by GCN4 mutants. Biochemistry 1998, 37:1060-l 066. Electrophoretic phasing analyses are used to explore the consequences of modifying the basic-leucine zipper region of the DNA binding protein GCN4 (which does not itself appear to bend DNA) to include various numbers of anionic or cationic amino acid residues just amino-terminal to the basic region that contacts DNA. The amplitude of the electrophoretic anomaly in all cases is proportional to the net charge of the amino terminus of the protein. Moreover, the direction of apparent DNA bending is as might be expected from simple electrostatic considerations-toward the cationic domain, and away from the anionic domain. Alternative explanations for these data include the possibility that local amino acid charges cause phase-dependent changes in the interaction of the complex with the gel matrix, or in the rigidity of the complex. 29. .

Tomky L, Strauss-Soukup J, Maher L: Effects of phosphate neutralization on the shape of the AP-1 transcription factor binding site in duplex DNA. Nucleic Acids Res 1998, 26:2298-2305. Following from [28”], the authors have chemically neutralized phosphates near sites of possible contact with cationic amino acids of GCN4 derivatives in the AP-1 DNA sequence thought to be bent upon protein binding. The extent of phosphate neutralization correlates well in both quantitative and qualitative terms with the results of electrophoretic phasing experiments involving binding of GCN4 derivatives with various numbers of amino-terminal cationic amino acid residues. 30.

Kerppola TK, Curran T: Selective DNA bending proteins. MO/ Cell Bioll993, 13:5479-5489.

by a variety

of bZlP

31. .

Paolella D, Liu Y, Fabian M, Schepartz A: Electrostatic mechanism for DNA bending by bZlP proteins. Biochemistry 1997, 36:10033-l 0038. Electrophoretic phasing analyses and chemical phosphate neutralization experiments are used to demonstrate that, consistent with an asymmetric phosphate neutralization model for DNA bending, basic-leucine zipper (bZIP) peptide derivatives with cationic amino acid substitutions just aminoterminal to the basic region influence the apparent shape of DNA bound at the CREB responsive element (CRE) sequence. Intrinsically curved CRE DNA is straightened by certain bZlP proteins, and neutralization of two symmetryrelated phosphate groups flanking the DNA minor groove (on the DNA face opposite to the leucine zipper of the protein) is shown to be correlated to this effect. 32.

Strauss-Soukup JK, Maher LJ: DNA bending by GCN4 mutants bearing cationic residues. Biochemistry 1997, 36:10026-l 0032.

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33.

Metallo S, Paolella D, Schepartz A: The role of a basic amino acid cluster in target site selection and non-specific binding of bZlP peptides to DNA. Nucleic Acids Res 1997, 25:2967-2972.

34.

Strauss JK, Roberts C, Nelson MG, Switzer C, Maher LJ: DNA bending by hexamethylene-tethered ammonium ions. froc Nat/ Acad SC; USA 1996,93:9515-9520.

35.

Strauss JK, Prakash TP, Roberts C, Switzer C, Maher LJ: DNA bending by a phantom protein. Chem Bioll996, 3:671-678.

Bauman C, Smith S, Bloomfield V, Bustamante C: Ionic effects on the elasticity of single DNA molecules. froc Nat/ Acad SC; USA 1997,94:6185-6190. The authors used laser tweezers to measure the elasticity of individual phage h DNA molecules under a variety of ionic conditions, including the presence of various multivalent ions such as Mg2+, Co(NHs)ss+, putrescines+, spermidines+ and spermineb+. Using this interesting approach, the authors demonstrated that the DNA persistence length is in the expected range of$50-500A in monovalent salt, but is substantially reduced to 250-300 A by multivalent cations. The authors make a convincing case for the utility of single-molecule methods for studying physical properties of DNA. 36. .

37. .

Rouzina I, Bloomfield A: DNA bending by small, mobile multivalent cations. Siopbys J 1998, 74:3152-3164. In this theoretical paper related to the experiments described in [36’], the authors approach the problem of how multivalent cations reduce the

persistence length of duplex DNA below that achieved under high monovalent salt conditions. An electrostatic mechanism is proposed in which multivalent cations transiently occupy sites within the DNA major groove, electrostatically repelling sodium counterions from neighboring phosphate groups. According to this mechanism, the differential unscreening of local phosphate groups would lead them to be highly attracted to the multivalent cation, generating a transient bend toward of the major groove of -20-40’. The number of multivalent cations positioned to induce such bends is thought to be small, because only cations with particularly strong electrostatic interactions will persist at the major groove site with a lifetime comparable to the time required for DNA bending. 38.

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