giant proteins that move dna: bullies of the genomic playground

© 2006 Nature Publishing Group

The information content in a given length of DNA is limited by the simplicity of the quaternary genetic code. Consequently, the nucleic-acid blueprint of even the simplest organism is vastly greater in length than the organism itself. The DNA-packaging problem has been well appreciated for some time1,2. Equally important is the task of moving DNA during replication and repair, during the process of simplifying the DNA topology and during chromosome segregation. This review dis-cusses the highly specialized proteins that move DNA, often in defined directions with great speed and accuracy. These include DNA replicases, helicases, translocases, topo isomerases and recombinases. The growth of genomes, in keeping pace with increasing cellular complex ity, is bound to have driven the evolution of the proteins that are discussed in this review. For example, the requirement for type II topoisomerases that cut and rejoin DNA duplexes to allow others to pass3,4 was needed to resolve rapidly the topological entanglements of large chromosomes.

We will consider four motions relative to the double-helical axis of DNA: rotation about the axis, translation along the axis, translation lateral to the axis and separa-tion of the DNA strands. Rotation twists the DNA helix and leads to over- or under-winding. The DNA trans-locases that we discuss move along the DNA axis. Lateral motions are needed to align DNA and promote repair by homologous recombination, and are used by type II topoisomerases, the actions of which allow the segregation of chromosomes to proceed despite the replication-induced entanglements of newly synthesized chromosomes. Last, the complete conversion of double-stranded DNA to single-stranded DNA is required to duplicate any genome, which is accomplished by the replicative helicases.

Five important questions will guide our discus-sion. First, what is it that actually moves, the DNA or the proteins? Second, why are enzymes that move and organize DNA generally large and/or multimeric? Third, how do enzymes use energy to maintain the genomic order? Fourth, why is the movement of DNA molecules entrusted to processes that are often stochastic given that the consequences of mislocalization are so dire? Fifth, why do proteins that move and organize DNA cause changes in DNA topology?

The theory of non-relativity: DNA movesFrame-of-reference questions are commonplace in physics, but they are also important in biology. A protein that moves on a DNA molecule can also be thought of as a DNA mover. Until recently, it has been assumed that enzymes migrate longitudinally over tens of thousands of base pairs (bp) across DNA that is mostly stationary. For example, a recent time-lapse fluorescence-microscopy study has revealed that the DisA protein (or a protein in complex with it), which is involved in the Bacillus subtilis DNA-damage response, is a nonspecific DNA-binding protein that forms a single focus in vivo and seems to scan the chromosome rapidly, pausing at sites of DNA damage5. Other enzymes have been suggested to use a similar mechanism to scan the DNA template6.

Surprisingly, in several other cases, protein complexes do not move on DNA but rather DNA is moved through them. In these cases, proteins that move DNA can be thought of primarily as DNA pumps rather than DNA-tracking enzymes. This distinction has implications for their mechanisms of action, but how do we know that it is the DNA that is moving and why is this knowledge important?

16 Barker Hall, Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720-3204, USA.* in memoriamCorrespondence to G.J.C. e-mail: [email protected]:10.1038/nrm1982

Giant proteins that move DNA: bullies of the genomic playgroundNicholas R. Cozzarelli*, Gregory J. Cost, Marcelo Nöllmann, Thierry Viard and James E. Stray

Abstract | As genetic material DNA is wonderful , but as a macromolecule it is unruly, voluminous and fragile. Without the action of DNA replicases, topoisomerases, helicases, translocases and recombinases, the genome would collapse into a topologically entangled random coil that would be useless to the cell. We discuss the organization, movement and energetics of these proteins that are crucial to the preservation of a molecule that has such beautiful biological but challenging physical properties.

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DNA motorsDNA-binding enzymes that use energy to move a segment of DNA processively from one point to another or to track along DNA.

SeptumThe protein ring at the mid-point of a bacterial cell that demarcates the site of future cell division.

ReplisomeA multiprotein complex that is involved in DNA replication.

Depending on what moves, the consequences are dramatically different. For example, the relevant frame of reference in cell biology is the cell. The creation of a new cell boundary is the fundamental and irrevocable act of cell division. Movement of DNA within the cell has to be regulated and must be accurate with respect to this event. Large DNA motors have been shown to be anchored to the cell membrane, as in the case of the prokaryotic DNA translocases FtsK and SpoIIIE7–13. This anchoring fixes their position relative to the cell boundary and ensures that the DNA moves relative to it. These processes involve the rapid and directional translocation of large amounts of DNA (~106 bp in Escherichia coli) over long distances and the movement of protein complexes that are small in comparison to the DNA displacement. SpoIIIE is such a translocase. Its role is to package DNA into the forespore during B. subtilis sporulation. SpoIIIE was shown to form stationary foci at the sporulation septum in vivo and to pump DNA from the mother cell into the forespore14.

RNA polymerase (RNAP) has been shown to move DNA when fixed to a solid surface15. To move DNA in vivo, RNAP would need to be stationary, and this possibility has been raised16,17. Results from experiments based on the inhibition of transcription in B. subtilis are also consistent with an immobile RNAP18. More recently, an actin-like cytoskeletal protein (MreB) was shown to

interact with RNAP in vivo and in vitro, raising the pos-sibility of an interaction that will enforce a physical con-straint to RNAP movement. These experiments would support a model in which RNAP moves DNA rather than moving itself along DNA.

In a similar manner, the E. coli replisome, a ~106-Da multiprotein complex that is responsible for DNA repli-cation, has long been assumed to propel itself along DNA. Recently however, the replisome was shown by immuno-fluorescence microscopy to move only small distances with respect to the length of the replicated DNA19–21. It should be remembered that the replisome contains two kinds of DNA motors, a DNA polymerase and a DNA helicase. It has been argued that because DNA synthesis is ~10 times faster than RNA synthesis, the replisome should be at least as powerful as RNA polymerase22, which can exert forces in excess of ~25 pN15. So, the contribution of two motors — one to unwind DNA and one to polymerize it — have been coupled. It has been speculated that an additional benefit of a stationary replisome would be the capacity to separate the newly replicated sister chromatids rapidly22. Stationary replicons have also been proposed to exist in higher eukaryotes23. When assembling large and complicated protein complexes, it seems that the cell prefers to run DNA through the complex rather than drag the complex through the cellular milieu.

Table 1 | General properties of DNA-moving and DNA-binding proteins

Name Type Function Organism Size (kDa) Subunit composition ATPase activity

Active, DNA-moving proteins

FtsK DNA translocase Couples replication to cell division

E. coli 1,788 (α6) or (α6)2Yes

Rho Helicase Transcription-termination factor

E. coli 300 Hexamer Yes

Bacterial replisome

DNA replicase DNA synthesis E. coli 1,328 26 protomers Yes

Yeast replisome DNA replicase DNA synthesis S. cerevisiae 2,038 28 protomers Yes

Swi/Snf Chromatin remodelling Moves nucleosomes S. cerevisiae 1,150 17 protomers Yes

Condensin SMC complex Condenses chromosomes

S. cerevisiae 631 5 protomers Yes

Cohesin SMC complex Pairs chromosomes S. cerevisiae 625 (702*) 5 (6*) protomers Yes

DNA gyrase Type II DNA topoisomerase (–) supercoiling E. coli 374 α2β2 Yes

Topoisomerase IV Type II DNA topoisomerase Decatenase E. coli 308 α2β2Yes

Topoisomerase II Type II DNA topoisomerase Decatenase S. cerevisiae 328 Dimer Yes

Passive, DNA-binding proteins

Topoisomerase I Type I DNA topoisomerase Relaxes (–) supercoils E. coli 97 Monomer No

Topoisomerase I Type I DNA topoisomerase Relaxes (+/–) supercoils H. sapiens 91 Monomer No

Fis DNA-binding protein Nucleoid-associated protein or transcriptional regulator

E. coli 22 Dimer No

H-NS DNA-binding protein Nucleoid-associated protein or transcriptional regulator

E. coli 31 Dimer No

BAF DNA-binding protein Chromatin-associated/ transcriptional regulator

H. sapiens 20 Dimer No

* With Rec8 (another cohesin subunit that is exclusively expressed during meiosis). BAF, barrier-to-autointegration factor; E. coli, Escherichia coli; Fis, factor-for-inversion stimulation; H. sapiens, Homo sapiens; S. cerevisiae, Saccharomyces cerevisiae; SMC, structural maintenance of chromosomes.

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G segmentATPasedomain

T segment C gate

+2 ATP

1 2 3 4

Ground state/transition stateThe most stable/least stable (lowest-energy/highest-energy) state of a chemical reaction.

800-kD gorillas: DNA movers are largeTABLE 1 compares and contrasts the properties of enzymes that actively transfer and order DNA with proteins that have a more static and/or structural role. Note the differ-ence in size between the two classes — many DNA-moving proteins have a total molecular mass of over a million Da, whereas more passive DNA-binding proteins such as the transcriptional regulators Fis, H-NS and BAF are uniformly small. These passive DNA-binding proteins might still have roles in DNA compaction when they are multimerized, but they seem to assemble in an unordered fashion24–27. Why are DNA-moving proteins so large? Intuitively, large binding surfaces would seem to provide an advantage when moving large stretches of DNA. Such surfaces can be provided by large subunits and/or by the combination of many smaller subunits (see TABLE 1).

Why are DNA-moving proteins not only large but also often multimeric? To cope with the relatively large size of even the smallest genomes, DNA-moving and DNA-metabolizing enzymes need to be fast. The on rate of many of these enzymes is slow relative to their rate of catalysis. For example, the ordered assembly of repli-some subunits is time consuming compared with the rate of DNA synthesis28. Multimerization presumably helps to convert events that would otherwise lead to disassociation (and require slow re-assembly) into mere pauses. The binding energy of individual monomers to DNA or to each other might be small, allowing for easy association and dissociation. By contrast, the energy of association of the multimeric complex will be much larger, making it stable but also dynamic. So, in contrast to static DNA-binding proteins, which might multimer-ize along DNA in both directions, the geometry of the association of DNA-moving proteins seems to be crucial for completing DNA synthesis, DNA reorganization or DNA movement in a timely manner.

Many enzymes that impel DNA have DNA-motor activity and an additional function. Replicases couple DNA-strand-separation (helicase) activity with the poly-merization of mononucleotides. The translocases FtsK and SpoIIIE couple a membrane-anchored flexible linker region with their translocase domain. DNA translocation by the helicase RecBCD is coupled to its nuclease activity.

Finally, topoisomerases couple DNA breakage and ligation with the lateral translation of a single or double strand of DNA through the body of the enzyme. The generally large size of these ‘gorilla-like’ DNA movers results both from their complex organization and is an evolutionary con-sequence of their being forged from multiple functional domains into indispensable cellular tools.

Avoiding equilibrium by using energyIn his classic text What is Life?, the physicist Erwin Schrödinger describes life as a localized violation of the second law of thermodynamics29. Cells are obliged to devote vast amounts of energy to copy, order and partition DNA. But there is more to energy utilization than meets the eye. William Jencks was among the first to understand that energy has an expanded and com-plex role30. In enunciating the so-called Circe effect of enzymes, he posited that the strong attractive force of binding would counterbalance the repelling forces that are experienced by the portions of the substrate that are being modified. So, more than just effecting impres-sively tight substrate association, the free energy of binding entices substrates into the highly destabilizing active-site environments, thereby leading to either ground-state destabilization or transition-state stabiliza-tion30. In a similar way, for enzymes that move DNA, nucleotide binding and release is often as important in luring enzymes into catalytically proficient states as ATP hydrolysis itself. ATP contributes enormously not only to force generation, but also to the processivity and direc-tionality of enzyme-mediated movement. Ultimately, it is the coupling of a propelling force with continuous irreversible and directional turnover that places DNA in the right place at the right time — usually far from its topological equilibrium. Below we discuss some relevant examples that illuminate the energetic distinctions that are described above.

Energy consumption determines the reversibility of the reaction, but it can also affect the balance of reactants and products. The energetics of DNA move-ment during disentanglement is of particular interest, especially with respect to the role of DNA topoisomer-ases. Topoisomerases solve all the topological problems

Figure 1 | Conformational changes during type II topoisomerase action. When two DNA duplexes — the gate (G; dark blue) segment and the transported (T; light blue) segment — are bound to the type II topoisomerase enzyme (red and yellow) (step 1), two ATP molecules are hydrolysed and the N-terminal ATPase domains (yellow) close (step 2). The G segment is then cleaved and the T segment is transported from the first cavity to the second one through the G segment (step 3). Last, the T segment exits via the C gate (step 4). Although poorly understood, the conformational changes that occur during the transport of the T segment from the first to the second cavity are thought to be driven by ATP hydrolysis74,75.

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b

a (–) crossing (+) crossing

d

c

B-form DNAA right-handed double-helical conformation of DNA that is the most common form seen in solution.

Decatenase/catenaseAn enzyme that removes/adds catenanes.

Endergonic reactionA reaction that consumes or traps energy.

Exergonic reactionA reaction that produces or releases energy.

that are related to the physical structure of the DNA double helix by transiently introducing single-strand breaks (via type I enzymes) or double-strand breaks (via type II enzymes) into DNA and by promoting the passage of DNA through the breach created31 (FIG. 1). The E. coli gyrase, for example, introduces (–) supercoils into (+) and (–) supercoiled DNA substrates. This reaction involves the inversion of a DNA crossing with a specific sign (see BOXES 1,2)32 and it requires energy to do the mechanical work against the torque (rotational force) in (–) supercoiled DNA. The process of ATP hydrolysis involves the irreversible conversion of chemical energy into entropy. So, ATP consumption not only provides energy for a reaction, but it also assures its thermo-dynamic irreversibility. DNA motors often use the bind-ing of ATP to produce specific structural changes in the enzyme and to promote reaction directionality. In the

case of gyrase, ATP binding captures a DNA crossing with a specific geometry, and ensures the introduction (rather than the relaxation) of (–) supercoils.

Relaxation of DNA by topoisomerases reduces mechanical strain and is therefore energetically favourable. Indeed, all the type I topoisomerases (with the excep-tion of reverse gyrase) use the topological stress of their substrate as energy to carry out the relaxation reaction31. Topoisomerase IV (topo IV) can preferentially relax (+) over (–) DNA crossings and is the main decatenase in E. coli. Interestingly, topo IV requires ATP to achieve relaxation of (+) supercoiled DNA. Why? The energy from ATP is needed to attain large coordinated confor-mational changes in the protein–DNA complex and also to reset the enzyme in preparation for a new catalytic cycle33–35.

The consequence of energy consumption is also funda mental for the reaction in systems where the enzyme requires composite activities. It is not well known that Ham Smith discovered DNA helicases. He did so by appreciating the need for ATP in DNA movement. High-energy phosphate bonds of deoxynu-cleoside triphosphates (dNTPs) are needed for synthetic reactions, such as DNA polymerization, as energy is needed for the endergonic bond-formation reaction. But, it was puzzling that some degradative reactions such as that of RecBCD required ATP even though DNA depo-lymerization is exergonic. Smith postulated that enzymes, such as RecBCD, used the energy of ATP to denature the DNA strands before nuclease digestion. He proved this mechanism in 1976, and many enzymes have since been shown to denature their substrate before making a covalent change36. In such cases, the energy of ATP is essential for producing DNA conformational changes that are necessary for generating the final product.

Because of the passive enzymatic mechanism of ATP-independent type I topoisomerases, they only relax DNA supercoiling to its value at thermodynamic equilibrium. By contrast, the ATP-dependent type II topoisomerases catalyse topological changes in DNA that result in non-equilibrium distributions of topoisomers. For example, topo IV in prokaryotes and topo II in eukaryotes sim-plify DNA topology by reducing the number of knots and catenanes well below their equilibrium levels37 (see BOX 2). The selective advantage is obvious because even a single catenane link that remains at the end of replication is lethal38,39. Similarly, in bacteria with circular chromo-somes, gyrase drives DNA into non-equilibrium levels of high (–) supercoiling. This necessary under-winding of the DNA both compacts DNA and also facilitates the melting of the DNA strands that is required for transcription and repair.

Movement via directed stochasticityCounter-intuitively, DNA-moving enzymes often func-tion stochastically rather than in a deterministic fash-ion. Their action at a given instant is not always in the direction of the overall process. All that is needed is a fail-safe, non-reversible conclusion to the process for eventual success. This principle applies across the diverse phenomena that are discussed below.

Box 1 | DNA topology for beginners

Crossings in DNA can be (+) or (–) (figure, part a). The arrows indicate the orientation of two crossing DNA segments. The sign of the crossing is determined by the direction in which the upper segment needs to be rotated (by < 180°) so that it points in the same direction as the lower segment: (–) for clockwise rotation and (+) for counter-clockwise rotation.

For topologically closed DNA circles, the linking number (Lk) is the sum of two geometric properties, twist (Tw) and writhe (Wr). Lk is obtained by adding up all the signed strand crossings and dividing this number by two. The mathematical properties of Lk have two important implications for DNA topology. First, Lk remains unchanged under any continuous deformation of the DNA strands (that is, as long as no break is made in them) and it is independent of the viewpoint of the observer. Second, Lk always has a non-negative integer value. A topological description of closed circular B-form DNA can be made by breaking down the topological quantity Lk into the geometric quantities Wr and Tw. Tw describes how the individual DNA strands coil around the axis of the DNA helix, whereas Wr describes how the path of the DNA-helix axis coils (that is, supercoils) in three dimensions. Although Wr and Tw need not be integers, their sum must be.

For closed circular duplex DNA, any change in Wr is accompanied by an exact change in Tw such that Lk = Wr + Tw. Therefore, any change in Lk due to enzymatic activity manifests geometrically as a change in Tw and Wr, so that ∆Lk = ∆Tw + ∆Wr. In part b of the figure, most of the supercoiling is condensed into writhe (Wr ~–3; Tw ~0). As the ends of the DNA are pulled apart (but not twisted) the absolute value of Wr decreases and that of Tw increases (figure, part c). Last, in part d, the supercoiling has been completely converted into twist so that the DNA helix no longer coils in three dimensions and Tw = –3 and Wr = 0. Vertical marks represent registration marks. Adapted with permission from REF. 56 © (1990) Cold Spring Harbor Laboratory Press.

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Supercoils Catenanes Knots

18

12

6

dif

6

12

18

0 10 20 240 250 260 270

Time (s)

FtsK

pos

itio

n (k

b)

CatenaneA topologically linked circular molecule.

difA 28-nucleotide sequence near the terminus of DNA replication in E. coli that is the recognition site for the XerC and XerD recombinases.

AAA+ family of ATPases(ATPases with associated activities). A large family of ATP hydrolases that is typified by a highly conserved catalytic motif. The members of the family greatly vary in both form and function.

KinetochoreA large multiprotein complex that assembles onto the centromere of the chromosome and links the chromosome to the microtubules of the mitotic spindle.

The DNA translocase FtsK illustrates such stochastic movement and its advantages. As a result of an unequal number of crossing-over events during homologous recombination, ~15% of replication cycles in E. coli produce a circular dimeric chromosome. This would be lethal, except that a pair of site-specific recombinases, XerC and XerD, resolves the dimer at specific sites near the terminus of replication called dif sites40. The dif sites can be far apart, and one role of the FtsK translocase is to bring them together at the septum, where FtsK is attached to the cell membrane. Crucially, FtsK moves DNA both forward and backward on a local scale41,42, but with an overall directionality that brings the dif sites to the septum42 (FIG. 2). How does FtsK find the dif sites? It was recently shown that the search is governed by the 5′-GNGNAGGG-3′ sequence, which is known as FRS (FtsK recognition sequence) or KOPS (FtsK orienting polar sequence)43,44. When FtsK encounters a FRS/KOPS from the 5′→3′ direction, no change in the direction of DNA translocation occurs. When encountering this sequence from the opposite direction, FtsK reverses the direction of DNA translocation in ~40% of the cases43. These sequences are oppositely skewed in both halves of the E. coli genome (from the origin to the dif site) such that ~80% are oriented in a direction that will direct FtsK to the dif site. An advantage of direction-dependent recognition of FRS/KOPS is that chromosome move-ment will proceed correctly no matter where transloca-tion begins. The seemingly low fidelity of translocation reversal (~40%) upon encounter of a FRS/KOPS is no accident. In fact, if the turnaround probability were 100%, then FtsK would become trapped between inverted FRS/KOPS. The theoretical turnaround prob-ability that leads to the fastest movement to a dif site is ~30%, which is close to the observed probability43.

Efficient movement of the dif sites to the septum allows the resolution of the chromosome dimer by XerC and XerD recombination, the fail-safe, non-reversible con-clusion that allows the directed stochasticity of FtsK translocation to work so well.

Amusingly, the microscale stochasticity of movement by translocases is mirrored in their nanoscale catalytic mechanism. Translocases of the AAA+ family of ATPases are often hexamers, with the substrate positioned in the centre of the ring. ATP hydrolysis by each translocase subunit was until recently presumed to occur either sequentially around the ring or by all subunits together. Recent work with the protein translocase ClpX shows that, in fact, the monomers fire randomly and that trans-location can occur with only a single active protomer45. It is possible that this property can be extended to related DNA translocases.

Another instance in which stochastic action helps large proteins move DNA around is during eukaryotic cell division. The mitotic microtubule spindle both pulls chromosomes towards its poles and pushes them towards the cell centre46. Microtubule attachment to chromo-somes at the kinetochore is mostly a result of a process of steady microtubule polymerization followed by rapid depolymerization, which is termed dynamic instability47. Each time a microtubule repolymerizes, it extends in a slightly different direction, probing a different portion of the cell. Multiple iterations of this process consume prodigious quantities of energy, but ultimately result in the capture of every chromosome. This process is a three-dimensional analogue of the one-dimensional search mechanism of FtsK, and it highlights the importance of reversibility in such processes. Similar to FtsK reversal,

Box 2 | Equilibrium in DNA-molecule distribution

Circular DNA molecules can be found in three topological forms: supercoiled, knotted and catenated (see figure). Knotted and catenated DNA can also be supercoiled. Type II topoisomerases change the linking number (see BOX 1) by steps of two. Adapted with permission from REF. 56 © (1990) Cold Spring Harbor Laboratory Press.

Figure 2 | DNA translocation by FtsK is globally unidirectional but locally bidirectional. Tracking FtsK during biophysical experiments shows the ability of FRS (FtsK recognition sequence) or KOPS (FtsK orienting polar sequence) to direct translocation. When FtsK approaches specific sites near the terminus of replication — known as dif sites — from the right or the left (red and green arrows, respectively) its motion is oriented with the direction of the FRS/KOPS sequences. Conversely, when FtsK begins translocation near dif (blue), movement against FRS/KOPS causes the direction of translocation to reverse, keeping FtsK near dif. Reproduced with permission from REF. 42 © (2005) AAAS.

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Origin 1Time

Origin 2

Obstruction Resumption

Reversal Synthesis

b

amicrotubule depolymerization is crucial for finding the kinetochore, and it provides a fail-safe conclusion that is monitored by the spindle-attachment checkpoint.

DNA movement by the E. coli replisome also has stochastic aspects. Some early models for DNA replica-tion emphasized its strict bidirectionality22. Certainly in bacteria it was thought that replication began at the same time to the left and right of the origin of replication oriC. We know now that replication initiation begins in a ran-dom fashion left or right of oriC and that the probability of initiation decreases exponentially with distance from oriC48. As shown in FIG. 3a, replication in all organisms starts out unidirectionally until the opposite fork fires, and then becomes bidirectional until one of the forks stalls or collides with an oncoming fork. So, replication is often bidirectional, but is unidirectional at initiation and termination. Replication termination is stochastic as well, and occurs simply as a result of collision between forks. A rigidly defined bidirectionality is not just unnecessary but is counterproductive, as replication synchrony could not reasonably be maintained between forks due to fork pausing and DNA repair. So, there need not be strong selection pressure for strictly bidirectional initiation.

More generally, it was thought that there was a unique sequence, the origin, that was the starting point for every round of replication in all organisms. The sequence that specifies origins of replication differs among species and is in no case deterministic. Even in tiny phage T7, more than one sequence can be the origin, and, in bacteria, mutations that inactivate oriC are readily suppressed by various mechanisms49. In higher eukaryotes, each round of replication initiates at different positions with almost no regard for sequence specificity. The result of the relaxed requirements that allow for the variable place-ment of origins has the advantage of not interfering with the evolution of the rest of the chromosome.

Polymerase progress can be stochastic even within one replication fork. When the fork encounters DNA damage, it can be blocked from further synthesis. One mechanism of lesion bypass involves replication-fork reversal to form a ‘chicken-foot’ structure (FIG. 3b)50,51. Polymerization of the paired, newly synthesized strands allows for the extension of DNA synthesis past the lesion. Reversal of one replication fork therefore actually promotes DNA replication in general.

The discontinuous movements of the enzymes dis-cussed above do not share a mechanistic commonality. The ability to ‘capture’ molecules that have successfully arrived at a precise location following movement pre-sumably allowed this simple and robust strategy to arise several times throughout the course of evolution.

DNA movement and topologyA common but unexpected feature of all DNA trans-locases is that motion of the DNA leads to changes in its topology. Any enzyme that moves DNA along its axis exerts a force that not only stretches but also twists DNA (that is, torque or rotational force). These effects are comparable in terms of energetic costs. Movement of DNA along its axis has been far better appreciated than that of twist because the distance DNA moves lon-

gitudinally can involve a great length of DNA, whereas the rotational movement that accompanies twist occurs within the 20-Å diameter of DNA. A second reason why twist and torque changes have been underappreciated is that until recently they were not directly measurable. Single-molecule-manipulation techniques have allowed for the direct measurement of torque and twist52, and have already led to the elucidation of the mechano-chemical cycle of DNA gyrase53.

To consider the topological consequences of DNA movement quantitatively, we introduce the equation that was derived by White and Calugareanu (Lk = Tw + Wr), whereby the linking number (Lk) is the sum of two geometric properties, twist (Tw) and writhe (Wr), that describe independent, yet interconvertable, structural features of DNA54–56 (BOX 1). As a topological parameter, Lk can only be changed by breakage and reunion of one or both strands of DNA, and therefore any change in Tw by an enzyme that moves DNA must be balanced by an exact compensatory change in Wr.

Figure 3 | Stochastic initiation and movement of the replication fork. a | Initiation of DNA synthesis begins unidirectionally. In the first line, only the left-most fork has fired at origin 1, but both have at origin 2. Eventually, all replication forks start and DNA synthesis becomes bidirectional (middle line). After the converging replication forks collide (bottom line), DNA synthesis reverts to unidirectionality (with respect to the initiating origin). Unreplicated chromosome (grey line); replicated region (thick black arrow); origin of replication (dashed line). b | Bypass of a lesion in DNA by replication-fork reversal. Template DNA (blue line); replicated DNA (red line); DNA that is in the process of being synthesized (red dashed line); direction of DNA synthesis (red arrowhead). Obstruction of the fork by a lesion in the DNA (blue square) leads to fork reversal and the formation of a ‘chicken-foot’ structure by the pairing of the newly synthesized strands. DNA synthesis that is templated from the daughter strand allows bypass of the lesion once the normal fork geometry has been recreated.

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(–) supercoils

(+) supercoils

d

L0

DNA

Translocase

Topologicaldomain barrier

ProtomerAny of the subunits of which an oligomeric protein is built up.

Translocases move DNA using different mechanisms and step sizes (that is, the distance translocated in one cycle of activity), but they always produce a change in twist (BOX 3). The reason is simple. The DNA is moved by the successive binding of the enzyme to some part of the DNA. An enzyme that moves by exactly track-ing the DNA helix will have to rotate around the DNA once for every helical pitch, or as viewed by the enzyme, the DNA will have to be rotated as a bolt through a nut. Even when the translocase has a symmetric oligomeric structure that lets it swap DNA contacts between protom-ers, unavoidable mismatches between DNA pitch and enzyme geometry will introduce twist.

Although these changes in twist have been previously reported (for example, for RNA polymerase I57), they have only recently been measured directly58. Single-molecule-manipulation techniques can now be used to measure the degree of supercoiling that is introduced per translocated distance. Using these methods, it was determined that RNA polymerase tracks the DNA helix without slippage as it moves at ~15 bp/s58,59, and therefore introduces ~1.5

supercoils/s. By contrast, T7 DNA polymerase translo-cates at ~100 bp/s60. Assuming that the tracking of the DNA helix involves no slippage, T7 polymerase would induce a moderate amount of supercoiling (~10 super-coils/s). The DNA translocase domain of FtsK has been shown to introduce only one supercoil per ∼150 bp trans-located41. However, its fast translocation (~6,000 bp/s42) implies that it would induce a staggering ~40 supercoils/s in the absence of slipping. At a distance from the trans-locase ( >200 bp, for example), these changes in DNA twist and writhe can be swiftly removed by DNA topo-isomerases, which are able to relax DNA supercoiling at ~6 supercoils/s, thereby avoiding side effects such as stalling of the replication fork.

Close to the translocase, transient changes in twist and writhe might play a role in activating, inhibiting or dislocating other protein complexes that are bound to the DNA. For example, eukaryotic chromatin remodelling factors of the SWI2/SNF2 family of ATPases, such as the RSC complex61, Mot1 (REFS 62,63) or Rad54 (REF. 64), are proposed to function as ATP-driven motors that destabi-lize pre-existing protein–DNA complexes by transiently disrupting protein–DNA interactions.

Translocation rotates the DNA and can locally and/or globally change its writhe and twist. Such changes can be beneficial or deleterious to the cell. Local changes can help to clear road blocks or remodel pre-existing protein–DNA complexes. Global changes can have deadly conse-quences (such as replication-fork stalling) if topological stress is not reduced by topoisomerases.

Concluding remarksProteins that move DNA have many features in com-mon that explain a number of their properties. What have we learned in answering the five central questions of this review, and what do we still not know? First, our earlier perceptions about DNA movement might have been too simplistic or wrong. We find examples where DNA moves past enzymes that operate on it. Most peculiarly, these enzymes allow the DNA duplex to literally slide or slice through them during transpor-tation. Second, DNA movers are big, which is prob-ably due to a combination of three factors. We note that the protein–DNA interface benefits from being large; that these proteins often need to assemble on or encircle the DNA and thereby multimerize; and that most have composite enzymatic functions fused into a single large enzyme. Third, DNA-moving enzymes use ATP for many different, sometimes not obvious, reasons. This energy is used to transport tremendous lengths of DNA against burdens that include bound proteins or RNA makes perfect sense. However, for isoenergetic reactions that include most of the DNA-passage reactions that are carried out by type II topoi-somerases and for the exonuclease activity of RecBCD, the role of ATP can be subtle. Fourth, we see that the DNA movers realize a number of advantages by acting in a stochastic fashion. Notably, the FtsK translocase can move in either direction at the expense of ATP, but it uses a genetic barcode to move DNA in the correct direction. Fifth, we examined how moving DNA leads

Box 3 | Changes in DNA twist generated by DNA translocation

The degree of supercoiling that occurs as a consequence of translocation can be quantified by a single parameter (α), which is the number of supercoils that the translocase introduces per DNA helical pitch of relaxed DNA (36.8 Å). Assuming that the DNA translocase moves unidirectionally and with no slippage within a topological domain of length L0 (see figure), the supercoiling density ahead (σ+) and behind (σ−) the translocating protein can be written as a function of the distance that is translocated (d) (see figure), the initial supercoiling density (σ0) and α as σ±(d) = (σ0 ± αd/L0)/(1 – d/L0)

41. So, only a value of α equal to -σ0 would leave the DNA-supercoiling density ahead and behind unchanged. Values of α that are higher or lower than -σ0 will considerably decrease or increase the amount of translocation-induced supercoiling-density change, respectively.

The step size (s) of a DNA translocase can be defined as the distance (in base pairs (bp)) that is translocated per mechanochemical cycle. The calculation from first principles of α in terms of the enzyme step size requires specific assumptions about the oligomeric organization and the mechanism of action of the enzyme, and will therefore be different for each case considered.

DNA translocases that track the DNA helix induce one supercoil ahead per pitch of DNA travelled (that is, α = 1), independently of their step size. In fact, direct single-molecule experiments showed that this is the case for RNA polymerase58 (s = 1 bp) and the motor protein EcoR124I (REF. 73) (s = 2–3 bp). However, other DNA translocases, such as FtsK, introduce only one supercoil per ~150 bp41 (α = 0.07). In the case of FtsK, this experimentally measured value of α and a model for its architecture and mechanism were used to deduce a step size of 13 bp41. It was argued that this value of α has evolved to minimize the distortion to DNA-supercoiling density. However, because of temporal and spatial fluctuations in the supercoiling density, the translocation of an enzyme with a fixed α will always produce changes in the DNA-supercoiling density.

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to changes in DNA topology. Torque is inevitably intro-duced by translocases when their grasp does not match the helical pitch of DNA. The action of topoisomerases is required to restore the original topology.

We drew parallels between some fascinating proteins that move DNA, and in doing so suggested some impor-tant and unanswered questions. In attaining genome organization, is there coordination among the giants? We know, for example, that the timing of replication is tightly controlled65,66. In B. subtilis it has been shown that a biochemical network that is composed of at least Spo0J, Soj and Spo0A is responsible for the coordina-tion of DNA replication, chromosome partitioning and sporulation67–69. Does a similar network exist for the regulation of DNA topology, or is chromosomal super-coiling simply the net result of the ensemble action of topoisomerases? Does gyrase continually generate (–)

supercoils only to have them removed by topo I, or is there a more energetically parsimonious solution?

DNA-moving enzymes push, pull, weave and unweave a tremendously long molecule in a very crowded and comparatively small volume. The fate of the cell depends on a tight temporal and spatial coordination among the DNA movers. We really know very little about the details of temporal coordination, mostly because it is difficult to see movement in real time. Several emerging technolo-gies will probably lead us towards a better understanding of the details of putting DNA in its place. Techniques such as single-molecule imaging in live cells, combined with FRET-based interaction analyses, are now giving us an unprecedented look at processes inside the cell70–72. This kind of capability applied to DNA movement and the movement of proteins on DNA will answer basic, but paramount, questions.

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AcknowledgementsThe authors would like to dedicate this work to our friend and colleague N. Cozzarelli, who passed away during the comple-tion of this review. We acknowledge N. Crisona for critical reading of this manuscript. Work in our laboratories was sup-ported by the National Institutes of Health (N.R.C. and T.V.), Ruth Kirschtein awards (J.S. and G.C.) and the Human Frontiers Science Organization (M.N.).

Competing interests statementThe authors declare no competing financial interests.

DATABASESThe following terms in this article are linked online to:UniProtKB: http://ca.expasy.org/sprotBAF | Fis | FtsK | H-NS | Mot1 | MreB | Rad54 | Soj | Spo0J | Spo0A | SpoIIIEAccess to this links box is available online.

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giant proteins that move dna: bullies of the genomic playground

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