enzyme-mediated dna looping_halford, welsh, szczelkun_2004

30 Apr 2004 15:49 AR AR214-BB33-01.tex AR214-BB33-01.sgm LaTeX2e(2002/01/18)P1: FHD10.1146/annurev.biophys.33.110502.132711

Annu. Rev. Biophys. Biomol. Struct. 2004. 33:1–24doi: 10.1146/annurev.biophys.33.110502.132711

Copyright c© 2004 by Annual Reviews. All rights reservedFirst published online as a Review in Advance on December 12, 2003

ENZYME-MEDIATED DNA LOOPING

Stephen E. Halford, Abigail J. Welsh,and Mark D. SzczelkunDepartment of Biochemistry, School of Medical Sciences, University of Bristol,University walk, Bristol BS8 1TD, United Kingdom; email: [email protected];[email protected]; [email protected]

Key Words restriction endonuclease, DNA structure, DNA-protein interaction,protein-protein interaction, molecular motor

■ Abstract Most reactions on DNA are carried out by multimeric protein com-plexes that interact with two or more sites in the DNA and thus loop out the DNAbetween the sites. The enzymes that catalyze these reactions usually have no activityuntil they interact with both sites. This review examines the mechanisms for the as-sembly of protein complexes spanning two DNA sites and the resultant triggering ofenzyme activity. There are two main routes for bringing together distant DNA sites inan enzyme complex: either the proteins bind concurrently to both sites and capture theintervening DNA in a loop, or they translocate the DNA between one site and anotherinto an expanding loop, by an energy-dependent translocation mechanism. Both captureand translocation mechanisms are discussed here, with reference to the various types ofrestriction endonuclease that interact with two recognition sites before cleaving DNA.

CONTENTS

INTRODUCTION: ENZYME REACTIONS AT TWO DNA SITES . . . . . . . . . . . . . 1LOOPING BY RESTRICTION ENZYMES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2LOOP CAPTURE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Preassembled Protein. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5Assembly on DNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Assembly on Protein. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

LOOP TRANSLOCATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11The Tracking Model for Loop Expansion and Contraction. . . . . . . . . . . . . . . . . . . 12Topological Constraints of the Tracking Model. . . . . . . . . . . . . . . . . . . . . . . . . . . . 14Alternative Ways to Distribute Topology into Loops. . . . . . . . . . . . . . . . . . . . . . . . 15

FUNCTIONS OF ENZYME LOOPS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

INTRODUCTION: ENZYME REACTIONS AT TWO DNA SITES

Some reactions on DNA are carried out by single proteins acting at solitary sites.For example, certain restriction endonucleases, such as EcoRI, EcoRV, and BamHI,catalyze separate reactions at each copy of their recognition sequence (35). These

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30 Apr 2004 15:49 AR AR214-BB33-01.tex AR214-BB33-01.sgm LaTeX2e(2002/01/18)P1: FHD

2 HALFORD ¥ WELSH ¥ SZCZELKUN

enzymes are dimers of identical protein subunits that interact symmetrically withpalindromic nucleotide sequences. They position the active site from one subunitagainst one strand of the DNA and likewise that from the other subunit on thecomplementary strand (68). Consequently, they cut a DNA with two copies of thesequence first at one site and then, in an entirely separate reaction, at the othersite (10, 30). They can, however, act processively, cutting two sites in successionwithout leaving the “domain” of the DNA molecule (83, 91).

Nevertheless, the vast majority of reactions on DNA are mediated not by indi-vidual enzymes acting at solitary sites but rather by multimeric proteins interactingwith multiple sites that are often distant from each other along the DNA (23, 100).Examples of this behavior occur in DNA replication and repair (3, 6), transcription(54, 64), and genome rearrangements by site-specific recombination and transpo-sition (17). In many of these situations the sites are specific DNA sequences, oftentwo copies of the same (or a similar) palindromic sequence. These are commonlybridged by a tetrameric protein composed of identical subunits: Two of the subunitsinteract with one copy of the palindrome (in the same way as a dimeric restrictionenzyme at its recognition site), and the other two subunits interact with the secondcopy (15, 19, 94). Alternatively, the two targets may be unrelated sequences that arerecognized by distinct subunits in a heterologous protein assembly, for example,transcription factors activating RNA polymerase (64). In other situations, one ormore of the subunits may be a “molecular motor,” capable of coupling the hydrol-ysis of nucleoside triphosphates to the processive translocation of DNA. In thesecases, motion along DNA results in the bridging interaction spanning specific andnonspecific sites, or even two nonspecific sites (36).

Two main kinds of pathway can lead to communications between distant DNAsites (1). In one kind, the sites become juxtaposed in three-dimensional spaceas a result of the dynamic flexibility of the DNA chain, and the protein(s) bindsconcurrently to both sites. This captures the intervening DNA in a loop. The loophas a fixed size, which corresponds to the length of DNA between the sites. Inthe other kind, energy-dependent translocation of one or more subunits along theone-dimensional DNA contour relative to the other subunits results in DNA beingpumped into or out of a sequestered loop. In this case, loop size is not fixed buteither increases or decreases with time, depending on the polarity of the motor. Inmany instances these two pathways can be distinguished if the bridging interactioncan occur on a catenane containing two interlinked rings of DNA, with one targetsite in each ring (1). Such a catenane allows for action by systems that bridgethe sites through three-dimensional space but not by systems that follow the one-dimensional contour from one site to the other (88, 89).

LOOPING BY RESTRICTION ENZYMES

A restriction enzyme that cleaves each recognition site in a separate reaction willinitially cut a DNA with two copies of its site at just one site. This reaction proceedsat the same rate as that on a DNA with one copy of the site (for example, Figure 1a),

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RESTRICTION ENZYMES LOOPING DNA 3

Figure 1 Enzyme reactions on DNA with one and two target sites. The reactionscontained a Type IIS restriction enzyme, (a) BsaI or (b) FokI, and a plasmid with eitherone or two copies of the relevant recognition sequence. For each reaction the amountof the plasmid substrate was measured as a fraction of the total DNA at the time pointsindicated: black circles, one-site plasmid; white squares, two-site plasmid.

although flanking sequences may modulate the rates. However, many restrictionenzymes have either no activity or only weak activity against DNA with a singletarget sequence. The DNA with one site is then cleaved either slowly, often withthe liberation of nicked intermediates, or not at all, whereas DNA with two or moresites is cleaved rapidly and efficiently (Figure 1b). The simplest explanation forthis behavior is that these enzymes interact with two recognition sites. Loopinginteractions through three-dimensional space normally occur more readily withsites incis (in the same molecule of DNA) than with sites intrans (in separateDNA molecules), because the effective concentration of one DNA site in thevicinity of another is usually higher for sites incis than intrans(70, 71). However,looping reactions that follow the one-dimensional DNA contour can only operatewith sites in the same chain (88).

Many nucleases that need multiple sites act concertedly on DNA with two sites,generating directly the final product cut at both sites, without liberating en routethe DNA cut at one site (63, 104). For concerted action, the enzyme must interactsimultaneously with both sites. However, some enzymes rapidly cleave just one ofthe two sites: in several cases, the residual site is then cleaved slowly, at the samerate as a DNA with one site (25); in other cases, the residual DNA is resistant tofurther cleavage despite the presence of an intact site (67). Hence, even thoughcertain restriction enzymes can catalyze independent reactions at individual sites,a larger number adhere to the common requirement for enzyme reactions on DNA;namely, the need to interact with two sites in the DNA before initiating the catalyticprocess (35).

The restriction enzymes that interact with two sites in the DNA include all of theendonucleases from the Type I and the Type III restriction-modification systems(56). These cleave DNA some distance away from their recognition sites becauseof an energy-dependent translocation of the adjacent nonspecific DNA, driven by

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nucleoside triphosphate hydrolysis (13, 21). Both types recognize asymmetric sitesthat, unlike palindromic sites, possess an orientation within the DNA. While theType III enzymes demand DNA substrates with two sites in inverted orientation(13, 67), the Type I enzymes can cleave circular DNA with a single site (20, 41, 88).Only on linear DNA do two sites become essential for the Type I enzymes: The sitescan then be in either inverted or repeated orientations. Three-dimensional loopingbetween pairs of sites has also been observed with EcoKI (7). In addition, the GTP-dependent systems that restrict methylated DNA operate via two sites (66, 84).

The Type II restriction enzymes cleave DNA at fixed locations relative to theirrecognition sites, in reactions that normally require only Mg2+ ions as a cofactor,but many of these also need two copies of the site (35). The latter include examplesfrom several subsets of the Type II systems (73). Among these are the Type IIEand the Type IIF enzymes, which mostly recognize palindromic sites. The TypeIIE enzymes, such as EcoRII (50), NaeI (39), and Sau3AI (29), are usually (but notalways) dimers. The dimer has two separate DNA-binding clefts, at the top and atthe bottom of the subunit interface. Both clefts can hold the relevant recognitionsequence, but only one possesses the catalytic functions for phosphodiester hydrol-ysis (39). The catalytic cleft is, however, inactive unless the other (allosteric) cleftis also filled with cognate DNA. On the other hand, the Type IIF enzymes, such asBse634I (34), Cfr10I (80), NgoMIV (19), SfiI (104), SgrAI (10), and many others(D. Gowers & A. Welsh, unpublished data), are generally tetramers. Two subunitsinteract symmetrically with one copy of the palindromic recognition sequence, inthe style of a dimeric restriction enzyme at an individual site, and likewise theother two subunits with the second copy. Yet these tetramers are fully active onlywhen both clefts are filled. They then cleave both sites in both strands within thelifetime of the complex (63).

Interactions with two sites are also common among the Type IIS enzymes,which cleave DNA at fixed positions downstream of asymmetric recognition sites,for example, FokI (5), BsgI (5), BspMI (31), MboII (82), and BfiI (52). BecauseType IIS enzymes recognize asymmetric sequences, they have the potential to beaffected by the relative orientation of their sites. Indeed, given two sites 700 bpapart in supercoiled DNA, BspMI has a∼20-fold-higher affinity for the repeatedorientation over the inverted orientation, but strikingly, this preference is abolishedby linearizing the DNA (46). Supercoiling may allow only repeated sites to becomealigned appropriately for the catalytic reaction, whereas no such constraint appliesto linear DNA (1, 46).

In addition, almost all of the Type IIB enzymes, which cleave DNA both up-stream and downstream of their recognition sites, need two copies of their targetsite. This was shown first with BcgI (49) but it also applies to AloI, BaeI, BsaXI,and PpiI (J. Marshall & D. Gowers, unpublished data). The Type IIB enzymesthus cleave eight phosphodiester bonds per turnover—both strands on both sidesof both sites.

The frequencies with which these various types of restriction-modification sys-tems appear in the sequenced bacterial genomes (48, 74) suggest that maybe 75%of the restriction endonucleases present in nature need to interact with two DNA

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sites in order to cut DNA. Indeed, Type I, Type III, and methyl-dependent systemsprobably constitute by themselves∼50% of the restriction-modification systemsin nature, and Type IIS and Type IIB systems may comprise another 10%–15%. Inaddition, whereas genome sequence analysis cannot reveal whether a conventionalType II enzyme that recognizes a palindromic sequence needs to interact with twosites, biochemical assays have already shown that a significant fraction of suchenzymes definitely require two sites (35). Historically, many restriction enzymeshave been assayed only on DNA substrates with multiple recognition sites, andit had not been noted before just how ineffectual they often are on DNA with asingle site: FokI (Figure 1b) and Sau3AI are just two examples (5, 29).

Even though the restriction enzymes that are often considered as the archetypesof this group, such as EcoRI, EcoRV, and BamHI, act at individual sites, these nowseem to be in the minority. The reason why some enzymes that act at solitary sitesare better known than those needing two sites is most likely because they are muchmore convenient as tools for DNA manipulations in vitro. EcoRI and its ilk have, ofcourse, vast applications as molecular biology reagents. In contrast, the nucleasesacting at two sites are difficult, or impossible, to use as tools in recombinant DNAtechnology, because their activities can depend on the number and the positions ofrecognition sites in the DNA and many of them cleave DNA considerable distancesaway from their recognition sites. However, SfiI, a restriction enzyme that needstwo sites, yielded a novel application as a sensor to measure the compaction ofDNA (79). Moreover, the many restriction enzymes that interact with two sites canbe used to illustrate the range of mechanisms of enzyme reactions across distantDNA sites, as this review will reveal.

LOOP CAPTURE

The capture of a DNA loop of fixed length by the concurrent binding of a protein(s)to two separate sites in the DNA can be accounted for by least three differentschemes. In the first, the protein exists in an aggregation state that enables it tobind directly to both DNA sequences (78), i.e., the native protein possesses twoseparate surfaces for binding DNA (Figure 2a). In the second, separate moleculesof the protein bind to the individual sites and then associate to a higher-orderassembly via protein-protein interactions (Figure 2b). In some such cases, thedimeric form of the protein binds initially to each site and the DNA-bound dimersthen associate to a tetramer (38). In the third, the protein binds first to the DNAand then to another molecule of either the same or a different protein. The secondprotein binds to another site in the DNA and so captures the loop. All three routescan be illustrated by Type II restriction enzymes.

Preassembled Protein

An enzyme with two separate DNA-binding surfaces can capture a loop on aDNA with two target sequences by binding first to one of the targets and thento the other target incis (Figure 2a). Many of these enzymes have virtually no

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Figure 2 Loop capture by (a) preassembled protein and (b) assembly on DNA.(a) The tetrameric protein (four gray subunits) binds first to one copy of its palindromicrecognition sequence (head-to-head arrows) via two protein subunits (the flat edgesmark the DNA-binding surface), and then to the second copy via its other subunits: Thiscauses a conformation change in all four subunits (denoted in black). Alternatively,the DNA carrying a tetramer at one site is prevented from looping by binding a secondtetramer at the vacant site. (b) A dimeric protein (two gray subunits) binds to one copyof a palindromic recognition sequence; another dimer binds to the other copy. The twoDNA-bound dimers then associate to form a tetramer, with a new conformation in allfour subunits.KD andKL are defined in the text. The unequal arrows for the loopingequilibrium indicate the thermodynamically favorable direction.

activity when bound to one site and become active only when bound to both sites.This scheme thus poses three questions: First, how is the enzyme barred from itsreaction when bound to a single site? Second, how quickly and by what route doesthe enzyme bound to one site contact the second site? Third, how stable is thecomplex of a single molecule of the enzyme spanning two sites incis relative tothe alternatives, separate molecules of the enzyme bound to each site (Figure 2a)or a single molecule spanning sites intrans(not shown)?

Activity at a single site can be averted by the protein subunits bound to one site,positioning their catalytic functions not on that segment of DNA but rather on theother segment. For example, in the synaptic complex of the MuA transposase withboth ends of the transposon, the active sites from the subunits bound to one end

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of the transposon are positioned against the other end of the DNA (2). However,this scheme cannot apply to the Type II restriction enzymes noted above. In thetetrameric enzymes such as SfiI and NgoMIV (19, 102), the two subunits thatinteract with one copy of the recognition sequence contain the catalytic functionsfor cutting that copy, as do the other two subunits with second copy. The two DNA-binding clefts are on the opposite sides of the protein, separated by about 60A.Nevertheless, SfiI binds two cognate duplexes that contain its recognition sitewith a high degree of cooperativity (26). But SfiI cannot bind concurrently twononcognate duplexes that differ from the cognate site by 1 bp; instead, it binds onlyone such duplex. In addition, the complex with one noncognate sequence cannotbind the cognate sequence at its vacant cleft (26). Moreover, in a complex withtwo specific duplexes, a phosphorothioate in place of the target phosphodiester inone duplex prevents SfiI from cleaving not only that duplex but also the bona fideduplex (106). Hence, the nature of the DNA at one cleft is transmitted to the othercleft on the opposite side of the protein. By initiating its catalytic reaction onlywhen both clefts contain scissile DNA, SfiI is largely precluded from cleavingDNA at any sequence other than its recognition site.

If a preassembled protein with two DNA-binding surfaces is to bind concur-rently to two sites incis, the intrinsic flexibility of the DNA chain needs to bringthe protein bound to one site into close proximity with the second site. Browniandynamic simulations of supercoiled DNA indicate that the first juxtaposition ofthe sites is likely to occur within a few milliseconds (for sites∼400 bp apart in a∼3-kb DNA), although it takes much longer in relaxed DNA (40, 44, 47). Hence,the rate-limiting step in forming a looped complex by a preassembled protein ismore likely to be the initial binding to the first site than the capture of the secondsite, at least in supercoiled DNA.

The protein at the initial site is, however, unlikely to encounter straightawaythe free target site but will instead collide first with some random nonspecificsite. DNA-binding proteins have long been thought to locate their target sites byfirst binding to the chain at random and then “sliding” along the DNA by one-dimensional diffusion (97). However, sliding is unlikely to play any significantrole in the location of the second DNA sequence. If one DNA-binding surfaceof the protein follows the helical path as it diffuses along the nonspecific DNA(45) while the other surface retains its contacts with the initial specific site, theintervening DNA will then soon become twisted severely out of its minimal free-energy state (see below). Hence, it is much more likely that the protein bound toone specific site finds the second site through a series of dissociation/reassociationswith the nonspecific DNA before encountering the specific sequence. This nowseems the predominant pathway for target-site location by DNA-binding proteins(32, 83). Indeed, many restriction enzymes that act at two sites operate efficientlyon DNA catenanes with one site in each ring (25, 89), where the protein at one sitecannot reach the site in the other ring by sliding alone.

A preassembled protein bound to one site in a DNA with two target sites willalmost always bind more tightly to another site in the same chain than to a site in

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another DNA, because the effective concentration of the second site in the vicinityof DNA-bound protein is usually higher for a site incis than for one intrans(71). Moreover, the preference is even larger in supercoiled DNA, as supercoilingenhancescis interactions while impedingtrans interactions. The probability ofjuxtaposition of two DNA sites incisdecreases as their separation increases above0.5 kb, but at all separations the probability on supercoiled DNA is much higherthan that on relaxed DNA (96). On the other hand, an interaction between sitesin separate DNA molecules requires one molecule to penetrate the domain of theother molecule, a process that occurs more readily if at least one of the moleculesis linear (104). The linear DNA can presumably worm its way end-first through thevolume occupied by the other DNA. SfiI follows the above principles: On a 7-kbDNA with two sites 1 kb apart, it forms its most stable bridging interactions on thesupercoiled circle; its next most stable on a linear form with sites 1 kb apart; thenthe alternative linear form with sites 6 kb apart; after that, two linear fragmentswith isolated sites; finally, two supercoiled DNA with solitary sites (62).

On DNA molecules with two closely spaced SfiI sites≤200 bp apart, the sta-bility of the loop trapped by SfiI varies cyclically with the length of the DNAbetween the sites, with a periodicity corresponding to the helical repeat of DNA(103), as observed previously with many other looping systems (78). The loopinginteraction requires both sites to present the requisite face of the DNA, usuallythe major groove, to the corresponding DNA-binding surfaces in the protein, butaltered separations of the sites cause the position of one face relative to the other torotate around the DNA once every helical repeat. Hence, inappropriate separationsrequire the intervening DNA to be twisted out of its minimal free-energy state toalign the two faces on the protein. However, for SfiI, the maxima in the periodicresponse of loop stability against intersite spacing occurs not when the sites areseparated by an integral number (n) of helical turns, but rather at spacings ofn ±1/2 turns (102). The additional half-turn arises from the geometry of the synapseby SfiI (Figure 3). Because one DNA segment crosses over the other, the curvatureof the intervening DNA becomes analogous to a writhe of either+1/2 or −1/2,

Figure 3 Geometries of loops trapped by SfiI. The binding of two segments of DNAon the opposite sides of the SfiI tetramer traps either (a) a negative node (4), as innegatively supercoiled DNA, or (b) a positive node, as in positively supercoiled DNA.

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as in DNA that has been, respectively, over- or underwound by 180◦. Hence, SfiIloops are most stable when the intervening DNA is either over- or undertwisted bythis amount: Unwinding results in the structure shown in Figure 3a, overwindingthat in Figure 3b.

Assembly on DNA

Instead of a protein with two DNA-binding surfaces associating first with onesite and then with the second (Figure 2a), many looping reactions proceed by theproteins binding individually to the separate sites and then associating with eachother via protein-protein interactions (Figure 2b). Two restriction enzymes thatfollow this route, albeit in different ways, are Sau3AI (29) and SgrAI (18). Sau3AIcleaves both strands at a palindromic recognition sequence but is a monomer insolution. However, the monomer may possess two DNA-binding domains but onlyone active site, and the minimal structure for making one double-strand break seemsto be two monomers spanning two copies of the recognition site. This enzyme thuscleaves DNA with two Sau3AI sites faster than DNA with one site, but the two-site DNA is cut at just one site, in the manner of a Type IIE enzyme (29). SgrAIalso cleaves DNA with two copies of its recognition site faster than DNA withone copy, but it cleaves the DNA with two sites concertedly at all four scissilephosphodiester bonds, giving rise directly to DNA cut in both strands at both sites,similar to a Type IIF enzyme (10). Yet unlike the tetrameric Type IIF enzymes(104), SgrAI is a dimer with presumably two catalytic centers (18). It binds to itsrecognition site as a dimer to give a complex with low activity, which cleaves DNAmuch more slowly than enzymes such as EcoRV. But on a DNA with two SgrAIsites, the dimers at the separate sites associate to a tetramer that rapidly cleavesthe DNA at both sites (Figure 2b).

The equilibrium constant for the association of two protomers bound to separatesites in the same chain of DNA is usually much larger than that for the associationof the same two protomers in free solution (38), due to the local concentrationeffect noted above (71). The maximal distance between DNA-bound protomersis the length of the intervening DNA, whereas no such limit applies to the freeprotomers in solution.

The aspects of DNA structure (supercoiling, site separation, and so forth) thatinfluence loop stability by a preassembled protein (Figure 2a) pertain equally tolooping by protein assembly on the DNA (Figure 2b). However, altered proteinconcentrations affect the two sorts of loop differently (Figure 4). Looping by apreassembled protein with two DNA-binding surfaces is inevitably blocked athigh concentrations of the protein (89) because the DNA must then bind a separatemolecule of the protein at each target site (Figure 2a). Both the maximum in theyield of looped DNA and the decline in the yield at excess protein concentrationsare mutual functions ofKD, the equilibrium dissociation constant for the bindingof the protein to an individual target, andKL, the equilibrium constant betweenthe looped DNA and the singly bound DNA (57). Hence,KL can be evaluated by

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Figure 4 Loop stability. The percentage of the DNA in its looped state at varied ratiosof protein subunits per molecule of DNA was calculated for the scheme in Figure 2a,a tetramer with two DNA-binding surfaces (solid line), and the scheme in Figure 2b,the association of two dimers bound to separate sites in the DNA (dashed line). Thecalculation was made by numerical integration (as in Reference 57) using the followingparameters: DNA concentration= 10 nM;KD = 1 nM; KL = 10.

measuring the modulation of looping with protein concentration (57). In contrast,if the protein has a single DNA-binding surface (Figure 2b), looping by assemblyon the DNA is not blocked by excess protein concentrations (Figure 4). Indeed,this pathway operates best when the protein is at higher concentration than theDNA-binding sites. At lower protein concentrations, only a fraction of the DNAmolecules will carry the protein at both sites and only these produce the loop.

The disruption of the loop by an excess of a preassembled protein may be oflittle consequence for an enzyme reaction. The dynamic equilibria ensure that allof the individual DNA molecules in the reaction will repeatedly visit the loopedstate and, provided that the half-time for the disassembly of the loop is not verymuch shorter than that of the catalytic reaction, all of the DNA molecules willsooner or later be converted to product. The disruption by excess protein may,however, have severe consequences for a regulatory system, for example, wherethe level of gene expression is determined by the equilibrium distribution betweenlooped and unlooped states (78).

Assembly on Protein

A further possibility for DNA looping systems is that a protein first binds to onesite in the DNA and then recruits a second protein from free solution, which in turnbinds to a second site in the DNA (Figure 5). This route is common among transcrip-tion factors, which often have, by themselves, relatively low affinities for DNA,but higher affinities when tethered to another protein already on the DNA (64).

This scheme is perhaps illustrated most clearly by the FokI endonuclease. FokIis a Type IIS restriction enzyme that recognizes an asymmetric sequence andcleaves both strands of the DNA at specified positions on one side of the sequence

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Figure 5 Loop capture by assembly on protein. The scheme shown here is for the FokIendonuclease. The protein is shown as a large DNA recognition domain connected bya flexible linker to a small catalytic domain. To cut both strands, the monomer boundto its asymmetric recognition sequence (arrow) associates with a second monomerthrough its catalytic domain, but the resultant dimer normally disassembles back tomonomers before it can cleave the DNA. On a DNA with a second site, the dimer canbind to that site through its second recognition domain.

(73). It exists in solution as a monomer with two domains, one of which contactsthe entire recognition sequence while the other possesses the catalytic functionsfor cleaving a single phosphodiester bond (99). Some Type IIS enzymes use asingle active site to act successively on the two strands of the DNA (77), but FokIuses a dimer created by the association of the catalytic domain in one monomerwith a second catalytic domain (12). The interface of the two catalytic domains hasa much smaller surface area than is common for protein-protein interactions (98),so the dimer is probably unstable. Hence, the most likely reason why this dimercleaves DNA with a single FokI site at a slow rate (Figure 1b) is that it usuallydissociates back to monomers before carrying out the catalytic reaction. On theother hand, if the DNA possesses a second FokI site, the DNA recognition domainof the second monomer can bind to the vacant site (Figure 5). The tethering ofboth monomers to the DNA, via their DNA recognition domains, stabilizes theassociation of the catalytic domains. The active form of FokI clearly involves thedimer bound to two copies of the recognition sequence (93). Hence, the reason whythis dimer cleaves DNA with two FokI sites at a relatively rapid rate (Figure 1b)is that the catalytic reaction is no longer impeded by the dissociation back tomonomers (5).

LOOP TRANSLOCATION

Although less well documented, loop formation by one-dimensional translocationis likely to be as common as loop capture through three-dimensional space. En-zymes that translocate loops include the mismatch repair enzymes (3), the RAD54

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Figure 6 Generation of loops by a motor enzyme. The model shown is based onbidirectional translocation of a Type I restriction enzyme. An asymmetric recognitionsequence (arrow) is bound by a single HsdS subunit (rectangle) that aligns two HsdMsubunits (ovals) over the site and two HsdR subunits (gray) to the nonspecific DNA oneither side. ATP hydrolysis by the HsdR subunits pumps the DNA ahead of the motorstoward the HsdS subunit, which remains attached to its site throughout. Consequently,the DNA on either side of the enzyme is translocated independently into two expandingloops of variable size.

recombinase (72), and the chromatin remodeling enzymes (37, 105). The TypeI and Type III restriction endonucleases are large multimeric proteins that havefour enzyme functions within the same complex (13, 21): a methyltransferase, anendonuclease, an ATPase, and a translocase. Both the HsdR subunits of the TypeI enzymes and the Res subunits of the Type III enzymes carry helicase motifs thatare capable of coupling the last two functions. The Type I endonucleases wereamong the first enzymes demonstrated to form loops (27, 75, 107). A model basedon the biochemical evidence (13) is shown in Figure 6. In short, an enzyme remainsbound to its recognition site while translocating adjacent nonspecific DNA pastthe stationary complex. The DNA ahead of the motor is the contracting domain(or loop, on circular DNA), and the translocated DNA forms the expanding loop.Translocation can occur bidirectionally so that two expanding loops are extruded(with EcoKI, EcoR124I, and EcoAI), or it can occur unidirectionally from one sideof the asymmetric site so that a single loop is extruded (with EcoBI). An equiva-lent unidirectional mechanism has been suggested for the Type III enzymes (56).For a bidirectional scheme, the translocating motors appear to act independentlyso that the expanding loops can differ in size (87). DNA cleavage is stimulatedwhen an HsdR or Res subunit is stalled by collision with a related motor subunittranslocating from the opposite direction.

The Tracking Model for Loop Expansion and Contraction

From DNA structures observed by electron microscopy during translocation byEcoBI and EcoKI, it was realized that loop formation might be accompanied bychanges in DNA twisting or wrapping (27, 107). If a motor enzyme moves alongDNA one base pair at a time, the protein must track the helical path of the DNAstrands. If the protein is prevented from rotating around the DNA, for instance, by

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being part of a larger complex, the DNA must rotate around its axis, causing anincrease in DNA twist ahead of the complex and a decrease behind it (53, 65, 92,101). This tracking model is illustrated in Figure 7a.

Of particular relevance to the Type I and Type III enzymes are the experimentscarried out with a chimera of Gal4 repressor and T7 RNA polymerase (65). On arelaxed, circular DNA carrying Gal4 binding sites and a T7 promoter, binding ofthe chimera trapped a figure-of-eight structure. Subsequent transcription caused

Figure 7 Alternative mechanisms for partitioning DNA twist between loops. The motordomain of an enzyme is shown (black oval) attached to the rest of a complex (gray shading).Translocation of the motor from left to right causes the DNA ahead of the motor to betranslocated from right to left, from the contracting domain into the expanding loop. Theresulting topological penalties are indicated to the right of each scheme. (a) The trackingmodel. The motor follows a helical pathway, causing the DNA to rotate around its helix, asindicated. (b) The relaxation model. The motor translocates the DNA as in (a), except that ittransiently dissociates to allow the induced rotation to freely reverse. (c) The walking model.The motor steps forward along one surface of the DNA; to remain in binding register, theDNA rotates around its axis in a left-handed direction (as viewed from the expanding loop;upper pathway) or a right-handed direction (lower pathway). The DNA is pulled back into theexpanding loop, the topology of which depends on the handedness of the trapped rotations.In (b) and (c) backtracking of the DNA out of the expanding loop must be prevented.

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DNA from one loop to be translocated into the other. Because the two domains weretopologically constrained, the resultant DNA twisting was manifested as negativesupercoiling in the expanding loop and positive supercoiling in the contractingloop (Figure 7a). Similarly, a restriction-deficient mutant of EcoAI was shownto partition a circular DNA into positive and negative domains; the supercoilingincreased with time and the effect was specific to ATP-driven translocation (42).Intriguingly, when the same approach was applied to a Type III endonuclease, nochanges in supercoiling were detected (43); one-dimensional DNA translocationhas yet to be proven unambiguously for the Type III enzymes.

Topological Constraints of the Tracking Model

DNA topology can be described by its linking difference (1Lk), which is the sumof two geometric terms for the changes in DNA twist (1Tw) and writhe (1Wr)relative to the unconstrained configuration (4). One way to define the additionalchange in twist (11Tw) induced by translocation is by a “twisting step size”(StepTw); this is the number of base pairs translocated before a unitary changein 1Tw is constrained in the loop(s) (i.e.,11Tw = ± 1). Equally, StepTw is thedistance translocated before a 360◦ rotation of the DNA is trapped. Thus, the changein DNA twist accompanying translocation is described by11Tw = d/StepTw (or−d/StepTw), whered is the distance translocated. The sign of this equation dependson the domain and on the model (see below). For the tracking model (Figure 7a),StepTw corresponds toh, the number of base pairs per helical turn, and11Tw isnegative in the expanding loop and positive in the contracting domain.

A fundamental concern with the tracking model is that relatively large changesin11Tw per base pair translocated must be accommodated in the loops. For loopsmany kilobases in length, this is less of a problem. Correspondingly, for translo-cation on linear DNA the contracting domain is freely rotating. However, for TypeI enzymes the problem of accommodating twist is particularly acute with the ex-panding loop. The size of this domain prior to the start of translocation is suggestedto be 10–20 bp (69). Assuming a tracking model withStepTw = ∼10 bp (Figure7a), then the specific linking difference (σ ) of the expanding loop drops rapidly tohypersupercoiled levels, even after relatively short distances (Figure 8a). Becausethe free energy of supercoiling varies with the first power of DNA length andwith the second power ofσ (4), translocation over longer distances would requirean energetic input far in excess of that available from coupled ATP hydrolysis.Moreover, the torque required far outstrips the stall forces typical of DNA-basedmotors (28). If the loop could expand sufficiently,11Tw would distribute morefreely andσ would plateau, but this steady-state level is energetically unobtainablewith these twisting parameters. Even if the restriction enzymes were to start witha larger expanding loop (e.g., by initially wrapping DNA around the complex), animpassable topological barrier would still rapidly accumulate (Figure 8a).

A further concern is how11Tw distributes through small DNA loops. Forsmaller domains, torsional DNA rigidity disfavors bending and1Wr→ 0 such

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Figure 8 The effect of translocation on expanding loop topology.σ was calculatedfollowing translocation ofdbp of DNA withh = 10.5 bp/turn andStepTw, as indicated,into an expanding loop of initial sizeL. σ = −0.06 at the start of the reactions.(a) Varying expanding loop size;StepTw = 10 bp andL = 10 bp (black circles),20 bp (black squares), 50 bp (black triangles), or 250 bp (inverted black triangles).(b) Varying StepTw; L = 10 bp andStepTw = 10 bp [black circles; data from (a)],50 bp (triangles), 70 bp (squares), 100 bp (inverted triangles), or 150 bp (diamonds).Note the differences in scale between the axes in (a) and (b).

that1Lk is mostly1Tw (4). For small expanding or contracting loops, rapidlyaccumulating11Tw could not partition into writhe. Regular B-form duplex DNAwould not be able to accommodate these changes entirely as1Tw and the DNAwould deform (76).

It is commonly argued that these may be moot problems, as the cellular activityof topoisomerases would counteract any changes induced by translocation. Al-though this is true for larger loops formed by, for instance, RNA polymerases (55),it seems less likely where small DNA loops, by virtue of their size, may preventprotein access; where enzyme activity is prevented by1Lk being partitioned al-most exclusively into1Tw, with little1Wr (14, 85); or where translocation causesa gradient of11Tw to accumulate (61, 101).

Alternative Ways to Distribute Topology into Loops

Although it has been suggested that stalling of an endonuclease by topology couldbe a trigger for DNA cleavage (88), it now appears that this is unlikely (42).Instead, the kinetics of cleavage by Type I enzymes of circular substrates withone site appears to correlate with the collision of the translocating HsdR subunitsfrom a single enzyme complex when they have each traveled, on average, half thelength of the circle (S. McClelland, unpublished data). Because this is a distancegenerally measured in kilobases, it becomes difficult to reconcile a tracking modelin which 360◦ of twist is introduced for every 10.5 bp translocated. In fact, forthe kinetics of EcoAI at least, the peak in induced DNA supercoiling coincided

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with the appearance of cleavage product resulting from translocation of the HsdRsubunits over half the length of the 2.8 kb plasmid (42). IfStepTw were∼10 bp,the supercoiling should have peaked much earlier in the reaction. Furthermore, themajority of the DNA loops observed by either electron microscopy (27, 75, 107)or atomic force microscopy (24) were relaxed or only moderately twisted; highlytwisted expanding loops were only rarely observed. Clearly, the Type I enzymescan avoid hypersupercoiling their expanding loops early in the reaction.

One way to avoid overtwisting is to release the strain before11Tw can ac-cumulate. Speculations for how this may occur have included topoisomerase-likenick-closure activity (107) or an initial nicking of the expanding loops to allowfree rotation of the DNA (88). However, translocation by nuclease-deficient mu-tants indicates that neither approach is likely to be used by Type I enzymes (42;S. McClelland, unpublished data). Alternatively, any mechanism that allows forStepTw to increase will also allow11Tw to be introduced more slowly into the ex-panding loop (Figure 8b). Consequently, supercoiling can reach a plateau at a morerealistic level and translocation can proceed over longer distances, particularly ifStepTw > 100 bp (Figure 8b). Two models that could achieve this are illustrated inFigure 7b,c.

In the first scheme (Figure 7b), the motor still tracks the DNA helix but dissoci-ates transiently to allow11Tw to repartition between the topological domains byDNA swiveling (42). This may occur in a regular fashion (e.g., after every 10 bptranslocated) or may fluctuate as a function of strain (e.g., more relaxation stepswould be required when the loops were small). In either case,StepTw would begreater thanh and would represent the11Tw trapped on average after multiplerelaxation steps. The distribution of DNA topoisomers formed by EcoAI appearsbroader than expected from a simple Poisson relationship. This supports a swivel-ing model in which the DNA rotations are unconstrained and variable11Tw istrapped. Further support comes from the apparentKm for ATP determined from thesupercoiling assays (42). This constant was higher than that estimated by ATPaseassays, indicating that more11Tw is constrained as the ATP concentration isincreased. Because the processivity of translocation (the probability of the proteinremaining bound to the DNA) is likely to be a function of ATP concentration,11Tw can only remain trapped if the nucleotide concentration is sufficiently highto prevent motor domain dissociation and DNA swiveling.

In the alternative scheme (Figure 7c), the motor does not track the helix butinstead “walks” along the DNA surface, similar to kinesin along a microtubule(51). Although some enzymes, such as polymerases, must take single base pairsteps and thus conform to a helical pathway (53, 92), there is no formal reasonwhy every DNA-based motor should act in the same way. Indeed, translocation onintact duplex DNA and with translocation step sizesÀ1 bp have been suggestedfor some DNA helicases (8, 81). For a walking model,StepTw is defined by thearchitecture of the translocation complex. For example, a motor domain may movea significant distance (tens of base pairs) forward along one face of the DNA. Forsteps sizes other than integral turns of the helix, axial DNA rotation is required to

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bring the protein into contact with the correct face of the DNA. These adjustmentsare equivalent to then ± 1/2 turns seen in some three-dimensional looping reactions(102) (Figure 3). When the motor domain returns to its starting position, drawingthe DNA into the expanding loop, any additional DNA rotation will also be trapped.As the number of steps accumulate, the number of rotations will also accumulateuntil 11Tw = ± 1. As a result,StepTw could be tens, or even hundreds, of basepairs. It is possible that this model also introduces variable11Tw at each step.The other key feature of a walking model is that the DNA rotations may, likethose in Figure 3, be either left- or right-handed (Figure 7c); accordingly,11Twin any given loop may be negative, positive, or, by rotations alternating betweeneither handedness, canceled out to zero. The supercoiling assays used with bothType I and Type III enzymes (42, 43) assume the formation of an undertwistedexpanding loop and an overtwisted contracting loop (65, 92). However, we cannotformally rule out a stepping model in which it is actually the expanding loopthat becomes positively supercoiled. To determine unambiguously, loop topologyrequires alternative assays that utilize enzyme reactions that are activated only byparticular DNA topologies (37, 101).

FUNCTIONS OF ENZYME LOOPS

Some of the enzymes that act on DNA have clear-cut reasons for interacting withtwo sites before initiating their reactions. For instance, many recombinases andtransposases mediate “cut-and-paste” reactions, in which the DNA is first cut attwo separate sites and the termini from each site are then pasted onto those fromthe other site (17). In these cases, the enzyme needs to be prevented from carryingout the “cut” stage unless it can complete the “paste” stage (2). If the enzyme isactive when bound to a solitary site, instead of becoming active only after bridgingtwo sites, it may cleave the DNA at the individual site without being able to jointhe resultant termini to the sister site. Such cleavages may be lethal for the cell(9). On the other hand, it is less obvious why many restriction enzymes need tointeract with two sites, particularly when enzymes such as EcoRI and EcoRV showthat endonucleases acting at individual sites can accomplish the biological processof restriction in vivo. Indeed, SfiI is ineffective in vivo at restricting DNA witha single SfiI site, though it restricts DNA with two and three SfiI with the sameefficiencies as EcoRI on DNA with one and two EcoRI sites, respectively (11).

It has been proposed that the Type II restriction enzymes that bridge two sitesmay be related through evolution to the recombinases that catalyze cut-and-pastereactions at two specific sites (9, 60, 104). There is, however, no current justifica-tion for this view. In the structures of the Cre and the Flp recombinases bound totheir recombination sites, the two DNA duplexes lie next to each other so that thestrand transfer steps can occur without any gross rearrangements of the complex(15, 94). In marked contrast, in the crystal structures of the NgoMIV and the NaeIrestriction enzymes bound to two copies of their recognition sequence, the two

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DNA duplexes are located on the opposite sides of the protein, thus excludingany possibility of transferring termini between the sites (19, 39). Moreover, thereactions of the cut-and-paste enzymes usually conserve the energy of the phos-phodiester bond by employing a covalent enzyme-DNA intermediate where theterminal phosphate is attached transiently to a tyrosine residue. Such reactions re-tain the stereochemical configuration at the phosphate (58). Conversely, the directhydrolysis of phosphodiester bonds by the EcoRI and EcoRV restriction enzymesinverts the stereochemistry (16, 33). DNA cleavage by SfiI proceeds with stereo-chemical inversion, thus excluding a covalent intermediate (59). The Type IIrestriction enzymes that interact with two DNA sites are thus unrelated to thesite-specific recombination enzymes in terms of both protein structure and chem-ical mechanism.

At present, perhaps the most plausible rationale for why many Type II re-striction enzymes need two sites is that these enzymes effectively double-checkthe DNA sequence to which they are bound before cleaving the DNA (26). Thepivotal requirement for a restriction enzyme is not to cleave DNA at its recogni-tion sequence but rather to avoid cleaving DNA at any sequence other than theunmodified recognition site. In vivo, the modification methyltransferase protectsevery copy of the recognition sequence in the host chromosome, but not everypossible sequence that differs from the recognition site by one or two base pairs(90). Hence, if the restriction endonuclease cleaves these alternative sequences, thechromosome will soon be degraded. Even though EcoRI and EcoRV achieve suf-ficient levels of fidelity while recognizing individual sites (35, 68), an alternativeroute to this end is that the enzyme bridges two sites and that it initiates its DNAcleavage reaction only when both sites possess the cognate sequence. In this way,a relatively low level of discrimination against alternative sequences at one site ismultiplied by the same (relatively low) level at the second site to obtain an over-all level of discrimination that is much higher than that at either site alone (23).The need to interact with two unmodified sites may also prevent the restrictionendonuclease from cleaving its host chromosome when the latter is incompletelymethylated.

It is likely that the Type I and Type III enzymes have evolved to benefit fromsimilar enhanced specificity, except that a one-dimensional linear search is used tocommunicate between sites instead of a three-dimensional search. However, whyemploy such baroque protein architectures to spool loops (Figure 6)? For somelooping enzymes, such as those involved in mismatch repair (3), a linear searchis the only way to maintain strand polarity over long distances. For the Type IIIenzymes at least, one-dimensional tracking prevents the formation of site pairingswith inappropriate geometries (as is possible with three-dimensional looping, e.g.,Figure 3) such that cleavage occurs exclusively at pairs of sites in inverted repeat(56, 67). However, the most plausible reason for why Type I enzymes translocateDNA may lie in the modular adaptability of the complexes. By translocating andcleaving nonspecific DNA through discrete HsdR subunits, the HsdS subunits canreadily adapt to new specificities without the need to also adapt a catalytic site (22).

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In comparison, to change the specificity of the majority of Type II enzymes wouldrequire both an endonuclease and a methyltransferase to converge separately onthe same, new sequence (48).

Notwithstanding the energetic exertion required to trap11Tw in loops, theresulting changes in DNA topology may be key to driving otherwise unfavor-able processes. For example, it has been suggested that formation of a negativelysupercoiled expanding loop by RAD54 facilitates strand invasion by a RAD51nucleoprotein complex (95). Alternatively, the generation of torque in small con-tracting or expanding loops can be used to destabilize nucleoprotein complexesby altering DNA twist to a level unacceptable for protein association. Examplesinclude chromatin remodeling (37, 105) and displacement of stalled polymerasesby transcription coupled repair factors (86). Because high levels of torque need tobe generated in these cases,StepTw may be∼10 bp and translocation over tens ofbase pairs is sufficient (105). Although Type I enzymes undoubtedly form topo-logically constrained DNA loops (42), translocation over thousands of base pairsrequiresStepTw to beÀ10 bp. Nonetheless, the more moderate torque introducedmay still affect the action of other proteins on a target bacteriophage genome. Al-ternatively, since collision with Type I enzymes can displace Lac repressor fromits operator (20), translocation may be required simply to displace proteins fromthe DNA while maintaining contact with the specific site via a DNA loop.

ACKNOWLEDGMENTS

We apologize to those colleagues whose work we have not cited due to spacelimitations. We thank other members of The Bristol DNA-Protein InteractionsGroup, past and present, for input and discussion. Our research is supported by theWellcome Trust and by the BBSRC. M.D.S is a Wellcome Trust Senior Fellow inBasic Biomedical Sciences.

The Annual Review of Biophysics and Biomolecular Structureis online athttp://biophys.annualreviews.org

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P1: FDS

April 12, 2004 13:39 Annual Reviews AR214-FM

Annual Review of Biophysics and Biomolecular StructureVolume 33, 2004

CONTENTS

ENZYME-MEDIATED DNA LOOPING, Stephen E. Halford, Abigail J. Welsh,and Mark D. Szczelkun 1

DISEASE-RELATED MISASSEMBLY OF MEMBRANE PROTEINS,Charles R. Sanders and Jeffrey K. Myers 25

CONFORMATIONAL SPREAD: THE PROPAGATION OF ALLOSTERICSTATES IN LARGE MULTIPROTEIN COMPLEXES, Dennis Brayand Thomas Duke 53

A FUNCTION-BASED FRAMEWORK FOR UNDERSTANDINGBIOLOGICAL SYSTEMS, Jeffrey D. Thomas, Taesik Lee, and Nam P. Suh 75

STRUCTURE, MOLECULAR MECHANISMS, AND EVOLUTIONARYRELATIONSHIPS IN DNA TOPOISOMERASES, Kevin D. Corbettand James M. Berger 95

STRUCTURE, DYNAMICS, AND CATALYTIC FUNCTION OFDIHYDROFOLATE REDUCTASE, Jason R. Schnell, H. Jane Dyson, andPeter E. Wright 119

THREE-DIMENSIONAL ELECTRON MICROSCOPY AT MOLECULARRESOLUTION, Sriram Subramaniam and Jacqueline L.S. Milne 141

TAKING X-RAY DIFFRACTION TO THE LIMIT: MACROMOLECULARSTRUCTURES FROM FEMTOSECOND X-RAY PULSES ANDDIFFRACTION MICROSCOPY OF CELLS WITH SYNCHROTRONRADIATION, Jianwei Miao, Henry N. Chapman, Janos Kirz,David Sayre, and Keith O. Hodgson 157

MOLECULES OF THE BACTERIAL CYTOSKELETON, Jan Lowe,Fusinita van den Ent, and Linda A. Amos 177

TETHERING: FRAGMENT-BASED DRUG DISCOVERY, Daniel A. Erlanson,James A. Wells, and Andrew C. Braisted 199

THE USE OF IN VITRO PEPTIDE-LIBRARY SCREENS IN THEANALYSIS OF PHOSPHOSERINE/THREONINE-BINDING DOMAINSTRUCTURE AND FUNCTION, Michael B. Yaffe and Stephen J. Smerdon 225

ROTATION OF F1-ATPASE: HOW AN ATP-DRIVEN MOLECULARMACHINE MAY WORK, Kazuhiko Kinosita, Jr., Kengo Adachi, andHiroyasu Itoh 245

ix

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April 12, 2004 13:39 Annual Reviews AR214-FM

x CONTENTS

MODEL SYSTEMS, LIPID RAFTS, AND CELL MEMBRANES,Kai Simons and Winchil L.C. Vaz 269

MASS SPECTRAL ANALYSIS IN PROTEOMICS, John R. Yates, III 297

INFORMATION CONTENT AND COMPLEXITY IN THE HIGH-ORDERORGANIZATION OF DNA, Abraham Minsky 317

THE ROLE OF WATER IN PROTEIN-DNA RECOGNITION, B. Jayaramand Tarun Jain 343

FORCE AS A USEFUL VARIABLE IN REACTIONS: UNFOLDING RNA,Ignacio Tinoco, Jr. 363

RESIDUAL DIPOLAR COUPLINGS IN NMR STRUCTURE ANALYSIS,Rebecca S. Lipsitz and Nico Tjandra 387

THE THERMODYNAMICS OF DNA STRUCTURAL MOTIFS,John SantaLucia, Jr. and Donald Hicks 415

SPIN DISTRIBUTION AND THE LOCATION OF PROTONS INPARAMAGNETIC PROTEINS, D. Goldfarb and D. Arieli 441

INDEXESSubject Index 469Cumulative Index of Contributing Authors, Volumes 29–33 495Cumulative Index of Chapter Titles, Volumes 29–33 498

ERRATAAn online log of corrections to Annual Review of Biophysicsand Biomolecular Structure chapters may be found athttp://biophys.annualreviews.org/errata.shtml

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enzyme-mediated dna looping_halford, welsh, szczelkun_2004

Documents

dnaprotein interaction

dna structure

dna sitessome reactions

capture theintervening

enzyme reactions

recognition sites

proteinprotein interaction

solitary sites