Enzyme-Mediated DNA Looping

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10.1146/annurev.biophys.33.110502.132711
Annu. Rev. Biophys. Biomol. Struct. 2004. 33:124

doi: 10.1146/annurev.biophys.33.110502.132711
c 2004 by Annual Reviews. All rights reserved
Copyright
First published online as a Review in Advance on December 12, 2003
ENZYME-MEDIATED DNA LOOPING
Annu. Rev. Biophys. Biomol. Struct. 2004.33:1-24. Downloaded from www.annualreviews.org

Access provided by University of Maryland - College Park on 09/30/15. For personal use only.
Stephen E. Halford, Abigail J. Welsh,

and Mark D. Szczelkun
Department of Biochemistry, School of Medical Sciences, University of Bristol,
University walk, Bristol BS8 1TD, United Kingdom; email: s.halford@bristol.ac.uk;
abi.welsh@bristol.ac.uk; mark.szczelkun@bristol.ac.uk
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 complexes that interact with two or more sites in the DNA and thus loop out the DNA
between the sites. The enzymes that catalyze these reactions usually have no activity
until they interact with both sites. This review examines the mechanisms for the assembly of protein complexes spanning two DNA sites and the resultant triggering of
enzyme activity. There are two main routes for bringing together distant DNA sites in
an enzyme complex: either the proteins bind concurrently to both sites and capture the
intervening DNA in a loop, or they translocate the DNA between one site and another
into an expanding loop, by an energy-dependent translocation mechanism. Both capture
and translocation mechanisms are discussed here, with reference to the various types of
restriction endonuclease that interact with two recognition sites before cleaving DNA.
CONTENTS
INTRODUCTION: ENZYME REACTIONS AT TWO DNA SITES . . . . . . . . . . . . .
LOOPING BY RESTRICTION ENZYMES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
LOOP CAPTURE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Preassembled Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Assembly on DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Assembly on Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
LOOP TRANSLOCATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The Tracking Model for Loop Expansion and Contraction . . . . . . . . . . . . . . . . . . .
Topological Constraints of the Tracking Model . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Alternative Ways to Distribute Topology into Loops . . . . . . . . . . . . . . . . . . . . . . . .
FUNCTIONS OF ENZYME LOOPS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1
2
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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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enzymes are dimers of identical protein subunits that interact symmetrically with
palindromic nucleotide sequences. They position the active site from one subunit
against one strand of the DNA and likewise that from the other subunit on the
complementary strand (68). Consequently, they cut a DNA with two copies of the
sequence first at one site and then, in an entirely separate reaction, at the other
site (10, 30). They can, however, act processively, cutting two sites in succession
without leaving the domain of the DNA molecule (83, 91).
Nevertheless, the vast majority of reactions on DNA are mediated not by individual enzymes acting at solitary sites but rather by multimeric proteins interacting
with 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 transposition (17). In many of these situations the sites are specific DNA sequences, often
two copies of the same (or a similar) palindromic sequence. These are commonly
bridged by a tetrameric protein composed of identical subunits: Two of the subunits
interact with one copy of the palindrome (in the same way as a dimeric restriction
enzyme at its recognition site), and the other two subunits interact with the second
copy (15, 19, 94). Alternatively, the two targets may be unrelated sequences that are
recognized by distinct subunits in a heterologous protein assembly, for example,
transcription factors activating RNA polymerase (64). In other situations, one or
more of the subunits may be a molecular motor, capable of coupling the hydrolysis of nucleoside triphosphates to the processive translocation of DNA. In these
cases, motion along DNA results in the bridging interaction spanning specific and
nonspecific sites, or even two nonspecific sites (36).
Two main kinds of pathway can lead to communications between distant DNA
sites (1). In one kind, the sites become juxtaposed in three-dimensional space
as a result of the dynamic flexibility of the DNA chain, and the protein(s) binds
concurrently to both sites. This captures the intervening DNA in a loop. The loop
has a fixed size, which corresponds to the length of DNA between the sites. In
the other kind, energy-dependent translocation of one or more subunits along the
one-dimensional DNA contour relative to the other subunits results in DNA being
pumped into or out of a sequestered loop. In this case, loop size is not fixed but
either increases or decreases with time, depending on the polarity of the motor. In
many instances these two pathways can be distinguished if the bridging interaction
can occur on a catenane containing two interlinked rings of DNA, with one target
site in each ring (1). Such a catenane allows for action by systems that bridge
the sites through three-dimensional space but not by systems that follow the onedimensional 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 will
initially cut a DNA with two copies of its site at just one site. This reaction proceeds
at 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
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Figure 1 Enzyme reactions on DNA with one and two target sites. The reactions
contained a Type IIS restriction enzyme, (a) BsaI or (b) FokI, and a plasmid with either
one or two copies of the relevant recognition sequence. For each reaction the amount
of the plasmid substrate was measured as a fraction of the total DNA at the time points
indicated: black circles, one-site plasmid; white squares, two-site plasmid.
although flanking sequences may modulate the rates. However, many restriction
enzymes have either no activity or only weak activity against DNA with a single
target sequence. The DNA with one site is then cleaved either slowly, often with
the liberation of nicked intermediates, or not at all, whereas DNA with two or more
sites is cleaved rapidly and efficiently (Figure 1b). The simplest explanation for
this behavior is that these enzymes interact with two recognition sites. Looping
interactions through three-dimensional space normally occur more readily with
sites in cis (in the same molecule of DNA) than with sites in trans (in separate
DNA molecules), because the effective concentration of one DNA site in the
vicinity of another is usually higher for sites in cis than in trans (70, 71). However,
looping reactions that follow the one-dimensional DNA contour can only operate
with 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 route
the DNA cut at one site (63, 104). For concerted action, the enzyme must interact
simultaneously with both sites. However, some enzymes rapidly cleave just one of
the two sites: in several cases, the residual site is then cleaved slowly, at the same
rate as a DNA with one site (25); in other cases, the residual DNA is resistant to
further cleavage despite the presence of an intact site (67). Hence, even though
certain 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 catalytic
process (35).
The restriction enzymes that interact with two sites in the DNA include all of the
endonucleases from the Type I and the Type III restriction-modification systems
(56). These cleave DNA some distance away from their recognition sites because
of an energy-dependent translocation of the adjacent nonspecific DNA, driven by
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nucleoside triphosphate hydrolysis (13, 21). Both types recognize asymmetric sites
that, unlike palindromic sites, possess an orientation within the DNA. While the
Type 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 sites
can then be in either inverted or repeated orientations. Three-dimensional looping
between pairs of sites has also been observed with EcoKI (7). In addition, the GTPdependent systems that restrict methylated DNA operate via two sites (66, 84).
The Type II restriction enzymes cleave DNA at fixed locations relative to their
recognition 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 examples
from several subsets of the Type II systems (73). Among these are the Type IIE
and the Type IIF enzymes, which mostly recognize palindromic sites. The Type
IIE enzymes, such as EcoRII (50), NaeI (39), and Sau3AI (29), are usually (but not
always) dimers. The dimer has two separate DNA-binding clefts, at the top and at
the bottom of the subunit interface. Both clefts can hold the relevant recognition
sequence, but only one possesses the catalytic functions for phosphodiester hydrolysis (39). The catalytic cleft is, however, inactive unless the other (allosteric) cleft
is also filled with cognate DNA. On the other hand, the Type IIF enzymes, such as
Bse634I (34), Cfr10I (80), NgoMIV (19), SfiI (104), SgrAI (10), and many others
(D. Gowers & A. Welsh, unpublished data), are generally tetramers. Two subunits
interact symmetrically with one copy of the palindromic recognition sequence, in
the style of a dimeric restriction enzyme at an individual site, and likewise the
other two subunits with the second copy. Yet these tetramers are fully active only
when both clefts are filled. They then cleave both sites in both strands within the
lifetime 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). Because
Type IIS enzymes recognize asymmetric sequences, they have the potential to be
affected by the relative orientation of their sites. Indeed, given two sites 700 bp
apart in supercoiled DNA, BspMI has a 20-fold-higher affinity for the repeated
orientation over the inverted orientation, but strikingly, this preference is abolished
by linearizing the DNA (46). Supercoiling may allow only repeated sites to become
aligned appropriately for the catalytic reaction, whereas no such constraint applies
to linear DNA (1, 46).
In addition, almost all of the Type IIB enzymes, which cleave DNA both upstream and downstream of their recognition sites, need two copies of their target
site. 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 enzymes
thus cleave eight phosphodiester bonds per turnoverboth strands on both sides
of both sites.
The frequencies with which these various types of restriction-modification systems 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 systems
probably constitute by themselves 50% of the restriction-modification systems
in nature, and Type IIS and Type IIB systems may comprise another 10%15%. In
addition, whereas genome sequence analysis cannot reveal whether a conventional
Type II enzyme that recognizes a palindromic sequence needs to interact with two
sites, biochemical assays have already shown that a significant fraction of such
enzymes definitely require two sites (35). Historically, many restriction enzymes
have been assayed only on DNA substrates with multiple recognition sites, and
it had not been noted before just how ineffectual they often are on DNA with a
single site: FokI (Figure 1b) and Sau3AI are just two examples (5, 29).
Even though the restriction enzymes that are often considered as the archetypes
of this group, such as EcoRI, EcoRV, and BamHI, act at individual sites, these now
seem to be in the minority. The reason why some enzymes that act at solitary sites
are better known than those needing two sites is most likely because they are much
more convenient as tools for DNA manipulations in vitro. EcoRI and its ilk have, of
course, vast applications as molecular biology reagents. In contrast, the nucleases
acting at two sites are difficult, or impossible, to use as tools in recombinant DNA
technology, because their activities can depend on the number and the positions of
recognition sites in the DNA and many of them cleave DNA considerable distances
away from their recognition sites. However, SfiI, a restriction enzyme that needs
two sites, yielded a novel application as a sensor to measure the compaction of
DNA (79). Moreover, the many restriction enzymes that interact with two sites can
be used to illustrate the range of mechanisms of enzyme reactions across distant
DNA 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 different
schemes. In the first, the protein exists in an aggregation state that enables it to
bind directly to both DNA sequences (78), i.e., the native protein possesses two
separate surfaces for binding DNA (Figure 2a). In the second, separate molecules
of the protein bind to the individual sites and then associate to a higher-order
assembly via protein-protein interactions (Figure 2b). In some such cases, the
dimeric form of the protein binds initially to each site and the DNA-bound dimers
then associate to a tetramer (38). In the third, the protein binds first to the DNA
and then to another molecule of either the same or a different protein. The second
protein binds to another site in the DNA and so captures the loop. All three routes
can be illustrated by Type II restriction enzymes.
Preassembled Protein
An enzyme with two separate DNA-binding surfaces can capture a loop on a
DNA with two target sequences by binding first to one of the targets and then
to the other target in cis (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 palindromic
recognition sequence (head-to-head arrows) via two protein subunits (the flat edges
mark the DNA-binding surface), and then to the second copy via its other subunits: This
causes 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 second
tetramer at the vacant site. (b) A dimeric protein (two gray subunits) binds to one copy
of a palindromic recognition sequence; another dimer binds to the other copy. The two
DNA-bound dimers then associate to form a tetramer, with a new conformation in all
four subunits. KD and KL are defined in the text. The unequal arrows for the looping
equilibrium 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 its
reaction when bound to a single site? Second, how quickly and by what route does
the enzyme bound to one site contact the second site? Third, how stable is the
complex of a single molecule of the enzyme spanning two sites in cis relative to
the alternatives, separate molecules of the enzyme bound to each site (Figure 2a)
or a single molecule spanning sites in trans (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 the
other segment. For example, in the synaptic complex of the MuA transposase with
both 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 the
tetrameric enzymes such as SfiI and NgoMIV (19, 102), the two subunits that
interact with one copy of the recognition sequence contain the catalytic functions
for 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 60 A.
Nevertheless, SfiI binds two cognate duplexes that contain its recognition site
with a high degree of cooperativity (26). But SfiI cannot bind concurrently two
noncognate duplexes that differ from the cognate site by 1 bp; instead, it binds only
one such duplex. In addition, the complex with one noncognate sequence cannot
bind the cognate sequence at its vacant cleft (26). Moreover, in a complex with
two specific duplexes, a phosphorothioate in place of the target phosphodiester in
one duplex prevents SfiI from cleaving not only that duplex but also the bona fide
duplex (106). Hence, the nature of the DNA at one cleft is transmitted to the other
cleft on the opposite side of the protein. By initiating its catalytic reaction only
when both clefts contain scissile DNA, SfiI is largely precluded from cleaving
DNA at any sequence other than its recognition site.
If a preassembled protein with two DNA-binding surfaces is to bind concurrently to two sites in cis, the intrinsic flexibility of the DNA chain needs to bring
the protein bound to one site into close proximity with the second site. Brownian
dynamic simulations of supercoiled DNA indicate that the first juxtaposition of
the 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 is
more likely to be the initial binding to the first site than the capture of the second
site, at least in supercoiled DNA.
The protein at the initial site is, however, unlikely to encounter straightaway
the free target site but will instead collide first with some random nonspecific
site. DNA-binding proteins have long been thought to locate their target sites by
first binding to the chain at random and then sliding along the DNA by onedimensional diffusion (97). However, sliding is unlikely to play any significant
role in the location of the second DNA sequence. If one DNA-binding surface
of 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, the
intervening DNA will then soon become twisted severely out of its minimal freeenergy state (see below). Hence, it is much more likely that the protein bound to
one specific site finds the second site through a series of dissociation/reassociations
with the nonspecific DNA before encountering the specific sequence. This now
seems the predominant pathway for target-site location by DNA-binding proteins
(32, 83). Indeed, many restriction enzymes that act at two sites operate efficiently
on DNA catenanes with one site in each ring (25, 89), where the protein at one site
cannot reach the site in the other ring by sliding alone.
A preassembled protein bound to one site in a DNA with two target sites will
almost 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 vicinity
of DNA-bound protein is usually higher for a site in cis than for one in trans
(71). Moreover, the preference is even larger in supercoiled DNA, as supercoiling
enhances cis interactions while impeding trans interactions. The probability of
juxtaposition of two DNA sites in cis decreases as their separation increases above
0.5 kb, but at all separations the probability on supercoiled DNA is much higher
than that on relaxed DNA (96). On the other hand, an interaction between sites
in separate DNA molecules requires one molecule to penetrate the domain of the
other molecule, a process that occurs more readily if at least one of the molecules
is linear (104). The linear DNA can presumably worm its way end-first through the
volume occupied by the other DNA. SfiI follows the above principles: On a 7-kb
DNA with two sites 1 kb apart, it forms its most stable bridging interactions on the
supercoiled circle; its next most stable on a linear form with sites 1 kb apart; then
the alternative linear form with sites 6 kb apart; after that, two linear fragments
with isolated sites; finally, two supercoiled DNA with solitary sites (62).
On DNA molecules with two closely spaced SfiI sites 200 bp apart, the stability of the loop trapped by SfiI varies cyclically with the length of the DNA
between the sites, with a periodicity corresponding to the helical repeat of DNA
(103), as observed previously with many other looping systems (78). The looping
interaction requires both sites to present the requisite face of the DNA, usually
the major groove, to the corresponding DNA-binding surfaces in the protein, but
altered separations of the sites cause the position of one face relative to the other to
rotate around the DNA once every helical repeat. Hence, inappropriate separations
require the intervening DNA to be twisted out of its minimal free-energy state to
align the two faces on the protein. However, for SfiI, the maxima in the periodic
response of loop stability against intersite spacing occurs not when the sites are
separated by an integral number (n) of helical turns, but rather at spacings of n
1
/2 turns (102). The additional half-turn arises from the geometry of the synapse
by SfiI (Figure 3). Because one DNA segment crosses over the other, the curvature
of 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 DNA
on the opposite sides of the SfiI tetramer traps either (a) a negative node (4), as in
negatively 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, SfiI
loops are most stable when the intervening DNA is either over- or undertwisted by
this amount: Unwinding results in the structure shown in Figure 3a, overwinding
that in Figure 3b.

Assembly on DNA
Instead of a protein with two DNA-binding surfaces associating first with one
site and then with the second (Figure 2a), many looping reactions proceed by the
proteins binding individually to the separate sites and then associating with each
other via protein-protein interactions (Figure 2b). Two restriction enzymes that
follow this route, albeit in different ways, are Sau3AI (29) and SgrAI (18). Sau3AI
cleaves both strands at a palindromic recognition sequence but is a monomer in
solution. However, the monomer may possess two DNA-binding domains but only
one active site, and the minimal structure for making one double-strand break seems
to be two monomers spanning two copies of the recognition site. This enzyme thus
cleaves DNA with two Sau3AI sites faster than DNA with one site, but the twosite DNA is cut at just one site, in the manner of a Type IIE enzyme (29). SgrAI
also cleaves DNA with two copies of its recognition site faster than DNA with
one copy, but it cleaves the DNA with two sites concertedly at all four scissile
phosphodiester 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 its
recognition site as a dimer to give a complex with low activity, which cleaves DNA
much more slowly than enzymes such as EcoRV. But on a DNA with two SgrAI
sites, the dimers at the separate sites associate to a tetramer that rapidly cleaves
the DNA at both sites (Figure 2b).
The equilibrium constant for the association of two protomers bound to separate
sites in the same chain of DNA is usually much larger than that for the association
of the same two protomers in free solution (38), due to the local concentration
effect noted above (71). The maximal distance between DNA-bound protomers
is the length of the intervening DNA, whereas no such limit applies to the free
protomers in solution.
The aspects of DNA structure (supercoiling, site separation, and so forth) that
influence loop stability by a preassembled protein (Figure 2a) pertain equally to
looping by protein assembly on the DNA (Figure 2b). However, altered protein
concentrations affect the two sorts of loop differently (Figure 4). Looping by a
preassembled protein with two DNA-binding surfaces is inevitably blocked at
high concentrations of the protein (89) because the DNA must then bind a separate
molecule of the protein at each target site (Figure 2a). Both the maximum in the
yield of looped DNA and the decline in the yield at excess protein concentrations
are mutual functions of KD, the equilibrium dissociation constant for the binding
of the protein to an individual target, and KL, the equilibrium constant between
the 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 ratios
of 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). The
calculation was made by numerical integration (as in Reference 57) using the following
parameters: 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 assembly
on the DNA is not blocked by excess protein concentrations (Figure 4). Indeed,
this pathway operates best when the protein is at higher concentration than the
DNA-binding sites. At lower protein concentrations, only a fraction of the DNA
molecules 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 of
little consequence for an enzyme reaction. The dynamic equilibria ensure that all
of the individual DNA molecules in the reaction will repeatedly visit the looped
state and, provided that the half-time for the disassembly of the loop is not very
much shorter than that of the catalytic reaction, all of the DNA molecules will
sooner or later be converted to product. The disruption by excess protein may,
however, have severe consequences for a regulatory system, for example, where
the level of gene expression is determined by the equilibrium distribution between
looped and unlooped states (78).
Assembly on Protein
A further possibility for DNA looping systems is that a protein first binds to one
site in the DNA and then recruits a second protein from free solution, which in turn
binds to a second site in the DNA (Figure 5). This route is common among transcription 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. FokI
is a Type IIS restriction enzyme that recognizes an asymmetric sequence and
cleaves both strands of the DNA at specified positions on one side of the sequence
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11
Figure 5 Loop capture by assembly on protein. The scheme shown here is for the FokI
endonuclease. The protein is shown as a large DNA recognition domain connected by
a flexible linker to a small catalytic domain. To cut both strands, the monomer bound
to its asymmetric recognition sequence (arrow) associates with a second monomer
through its catalytic domain, but the resultant dimer normally disassembles back to
monomers before it can cleave the DNA. On a DNA with a second site, the dimer can
bind to that site through its second recognition domain.
(73). It exists in solution as a monomer with two domains, one of which contacts
the entire recognition sequence while the other possesses the catalytic functions
for cleaving a single phosphodiester bond (99). Some Type IIS enzymes use a
single active site to act successively on the two strands of the DNA (77), but FokI
uses a dimer created by the association of the catalytic domain in one monomer
with a second catalytic domain (12). The interface of the two catalytic domains has
a 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 dimer
cleaves DNA with a single FokI site at a slow rate (Figure 1b) is that it usually
dissociates back to monomers before carrying out the catalytic reaction. On the
other hand, if the DNA possesses a second FokI site, the DNA recognition domain
of the second monomer can bind to the vacant site (Figure 5). The tethering of
both monomers to the DNA, via their DNA recognition domains, stabilizes the
association of the catalytic domains. The active form of FokI clearly involves the
dimer bound to two copies of the recognition sequence (93). Hence, the reason why
this 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 to
monomers (5).
LOOP TRANSLOCATION
Although less well documented, loop formation by one-dimensional translocation
is likely to be as common as loop capture through three-dimensional space. Enzymes 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 on

bidirectional translocation of a Type I restriction enzyme. An asymmetric recognition
sequence (arrow) is bound by a single HsdS subunit (rectangle) that aligns two HsdM
subunits (ovals) over the site and two HsdR subunits (gray) to the nonspecific DNA on
either side. ATP hydrolysis by the HsdR subunits pumps the DNA ahead of the motors
toward the HsdS subunit, which remains attached to its site throughout. Consequently,
the DNA on either side of the enzyme is translocated independently into two expanding
loops of variable size.
recombinase (72), and the chromatin remodeling enzymes (37, 105). The Type
I and Type III restriction endonucleases are large multimeric proteins that have
four enzyme functions within the same complex (13, 21): a methyltransferase, an
endonuclease, an ATPase, and a translocase. Both the HsdR subunits of the Type
I enzymes and the Res subunits of the Type III enzymes carry helicase motifs that
are capable of coupling the last two functions. The Type I endonucleases were
among the first enzymes demonstrated to form loops (27, 75, 107). A model based
on the biochemical evidence (13) is shown in Figure 6. In short, an enzyme remains
bound to its recognition site while translocating adjacent nonspecific DNA past
the 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 side
of the asymmetric site so that a single loop is extruded (with EcoBI). An equivalent unidirectional mechanism has been suggested for the Type III enzymes (56).
For a bidirectional scheme, the translocating motors appear to act independently
so that the expanding loops can differ in size (87). DNA cleavage is stimulated
when an HsdR or Res subunit is stalled by collision with a related motor subunit
translocating from the opposite direction.
The Tracking Model for Loop Expansion and Contraction

From DNA structures observed by electron microscopy during translocation by
EcoBI and EcoKI, it was realized that loop formation might be accompanied by
changes in DNA twisting or wrapping (27, 107). If a motor enzyme moves along
DNA one base pair at a time, the protein must track the helical path of the DNA
strands. 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 an
increase 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 experiments
carried out with a chimera of Gal4 repressor and T7 RNA polymerase (65). On a
relaxed, circular DNA carrying Gal4 binding sites and a T7 promoter, binding of
the chimera trapped a figure-of-eight structure. Subsequent transcription caused
Figure 7 Alternative mechanisms for partitioning DNA twist between loops. The motor
domain 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 be
translocated from right to left, from the contracting domain into the expanding loop. The
resulting topological penalties are indicated to the right of each scheme. (a) The tracking
model. The motor follows a helical pathway, causing the DNA to rotate around its helix, as
indicated. (b) The relaxation model. The motor translocates the DNA as in (a), except that it
transiently 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, the
DNA 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 the
expanding 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 were
topologically constrained, the resultant DNA twisting was manifested as negative
supercoiling in the expanding loop and positive supercoiling in the contracting
loop (Figure 7a). Similarly, a restriction-deficient mutant of EcoAI was shown
to partition a circular DNA into positive and negative domains; the supercoiling
increased with time and the effect was specific to ATP-driven translocation (42).
Intriguingly, when the same approach was applied to a Type III endonuclease, no
changes in supercoiling were detected (43); one-dimensional DNA translocation
has 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 sum
of two geometric terms for the changes in DNA twist (1Tw) and writhe (1Wr)
relative to the unconstrained configuration (4). One way to define the additional
change in twist (11Tw) induced by translocation is by a twisting step size
(StepTw); this is the number of base pairs translocated before a unitary change
in 1Tw is constrained in the loop(s) (i.e., 11Tw = 1). Equally, StepTw is the
distance translocated before a 360 rotation of the DNA is trapped. Thus, the change
in DNA twist accompanying translocation is described by 11Tw = d/StepTw (or
d/StepTw), where d is the distance translocated. The sign of this equation depends
on the domain and on the model (see below). For the tracking model (Figure 7a),
StepTw corresponds to h, the number of base pairs per helical turn, and 11Tw is
negative in the expanding loop and positive in the contracting domain.
A fundamental concern with the tracking model is that relatively large changes
in 11Tw per base pair translocated must be accommodated in the loops. For loops
many kilobases in length, this is less of a problem. Correspondingly, for translocation on linear DNA the contracting domain is freely rotating. However, for Type
I enzymes the problem of accommodating twist is particularly acute with the expanding loop. The size of this domain prior to the start of translocation is suggested
to be 1020 bp (69). Assuming a tracking model with StepTw = 10 bp (Figure
7a), then the specific linking difference ( ) of the expanding loop drops rapidly to
hypersupercoiled levels, even after relatively short distances (Figure 8a). Because
the free energy of supercoiling varies with the first power of DNA length and
with the second power of (4), translocation over longer distances would require
an energetic input far in excess of that available from coupled ATP hydrolysis.
Moreover, the torque required far outstrips the stall forces typical of DNA-based
motors (28). If the loop could expand sufficiently, 11Tw would distribute more
freely and would plateau, but this steady-state level is energetically unobtainable
with these twisting parameters. Even if the restriction enzymes were to start with
a larger expanding loop (e.g., by initially wrapping DNA around the complex), an
impassable topological barrier would still rapidly accumulate (Figure 8a).
A further concern is how 11Tw distributes through small DNA loops. For
smaller domains, torsional DNA rigidity disfavors bending and 1Wr 0 such
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Figure 8 The effect of translocation on expanding loop topology. was calculated

following translocation of d bp of DNA with h = 10.5 bp/turn and StepTw, as indicated,
into an expanding loop of initial size L. = 0.06 at the start of the reactions.
(a) Varying expanding loop size; StepTw = 10 bp and L = 10 bp (black circles),
20 bp (black squares), 50 bp (black triangles), or 250 bp (inverted black triangles).
(b) Varying StepTw; L = 10 bp and StepTw = 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).
that 1Lk is mostly 1Tw (4). For small expanding or contracting loops, rapidly
accumulating 11Tw could not partition into writhe. Regular B-form duplex DNA
would not be able to accommodate these changes entirely as 1Tw and the DNA
would deform (76).
It is commonly argued that these may be moot problems, as the cellular activity
of topoisomerases would counteract any changes induced by translocation. Although 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 prevent
protein access; where enzyme activity is prevented by 1Lk being partitioned almost exclusively into 1Tw, with little 1Wr (14, 85); or where translocation causes
a gradient of 11Tw to accumulate (61, 101).
Alternative Ways to Distribute Topology into Loops

Although it has been suggested that stalling of an endonuclease by topology could
be 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 with
one site appears to correlate with the collision of the translocating HsdR subunits
from a single enzyme complex when they have each traveled, on average, half the
length of the circle (S. McClelland, unpublished data). Because this is a distance
generally measured in kilobases, it becomes difficult to reconcile a tracking model
in which 360 of twist is introduced for every 10.5 bp translocated. In fact, for
the 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 HsdR
subunits over half the length of the 2.8 kb plasmid (42). If StepTw were 10 bp,
the supercoiling should have peaked much earlier in the reaction. Furthermore, the
majority of the DNA loops observed by either electron microscopy (27, 75, 107)
or atomic force microscopy (24) were relaxed or only moderately twisted; highly
twisted expanding loops were only rarely observed. Clearly, the Type I enzymes
can avoid hypersupercoiling their expanding loops early in the reaction.
One way to avoid overtwisting is to release the strain before 11Tw can accumulate. Speculations for how this may occur have included topoisomerase-like
nick-closure activity (107) or an initial nicking of the expanding loops to allow
free rotation of the DNA (88). However, translocation by nuclease-deficient mutants indicates that neither approach is likely to be used by Type I enzymes (42;
S. McClelland, unpublished data). Alternatively, any mechanism that allows for
StepTw to increase will also allow 11Tw to be introduced more slowly into the expanding loop (Figure 8b). Consequently, supercoiling can reach a plateau at a more
realistic level and translocation can proceed over longer distances, particularly if
StepTw > 100 bp (Figure 8b). Two models that could achieve this are illustrated in
Figure 7b,c.
In the first scheme (Figure 7b), the motor still tracks the DNA helix but dissociates transiently to allow 11Tw to repartition between the topological domains by
DNA swiveling (42). This may occur in a regular fashion (e.g., after every 10 bp
translocated) or may fluctuate as a function of strain (e.g., more relaxation steps
would be required when the loops were small). In either case, StepTw would be
greater than h and would represent the 11Tw trapped on average after multiple
relaxation steps. The distribution of DNA topoisomers formed by EcoAI appears
broader than expected from a simple Poisson relationship. This supports a swiveling model in which the DNA rotations are unconstrained and variable 11Tw is
trapped. Further support comes from the apparent Km for ATP determined from the
supercoiling assays (42). This constant was higher than that estimated by ATPase
assays, indicating that more 11Tw is constrained as the ATP concentration is
increased. Because the processivity of translocation (the probability of the protein
remaining bound to the DNA) is likely to be a function of ATP concentration,
11Tw can only remain trapped if the nucleotide concentration is sufficiently high
to prevent motor domain dissociation and DNA swiveling.
In the alternative scheme (Figure 7c), the motor does not track the helix but
instead walks along the DNA surface, similar to kinesin along a microtubule
(51). Although some enzymes, such as polymerases, must take single base pair
steps and thus conform to a helical pathway (53, 92), there is no formal reason
why every DNA-based motor should act in the same way. Indeed, translocation on
intact duplex DNA and with translocation step sizes 1 bp have been suggested
for some DNA helicases (8, 81). For a walking model, StepTw is defined by the
architecture of the translocation complex. For example, a motor domain may move
a significant distance (tens of base pairs) forward along one face of the DNA. For
steps 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 adjustments
are equivalent to the n 1/2 turns seen in some three-dimensional looping reactions
(102) (Figure 3). When the motor domain returns to its starting position, drawing
the 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 accumulate
until 11Tw = 1. As a result, StepTw could be tens, or even hundreds, of base
pairs. It is possible that this model also introduces variable 11Tw at each step.
The other key feature of a walking model is that the DNA rotations may, like
those in Figure 3, be either left- or right-handed (Figure 7c); accordingly, 11Tw
in any given loop may be negative, positive, or, by rotations alternating between
either handedness, canceled out to zero. The supercoiling assays used with both
Type I and Type III enzymes (42, 43) assume the formation of an undertwisted
expanding loop and an overtwisted contracting loop (65, 92). However, we cannot
formally rule out a stepping model in which it is actually the expanding loop
that becomes positively supercoiled. To determine unambiguously, loop topology
requires alternative assays that utilize enzyme reactions that are activated only by
particular DNA topologies (37, 101).
FUNCTIONS OF ENZYME LOOPS

Some of the enzymes that act on DNA have clear-cut reasons for interacting with
two sites before initiating their reactions. For instance, many recombinases and
transposases mediate cut-and-paste reactions, in which the DNA is first cut at
two separate sites and the termini from each site are then pasted onto those from
the other site (17). In these cases, the enzyme needs to be prevented from carrying
out the cut stage unless it can complete the paste stage (2). If the enzyme is
active when bound to a solitary site, instead of becoming active only after bridging
two sites, it may cleave the DNA at the individual site without being able to join
the 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 to
interact with two sites, particularly when enzymes such as EcoRI and EcoRV show
that endonucleases acting at individual sites can accomplish the biological process
of restriction in vivo. Indeed, SfiI is ineffective in vivo at restricting DNA with
a single SfiI site, though it restricts DNA with two and three SfiI with the same
efficiencies 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 sites
may be related through evolution to the recombinases that catalyze cut-and-paste
reactions at two specific sites (9, 60, 104). There is, however, no current justification for this view. In the structures of the Cre and the Flp recombinases bound to
their recombination sites, the two DNA duplexes lie next to each other so that the
strand 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 NaeI
restriction 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 excluding
any possibility of transferring termini between the sites (19, 39). Moreover, the
reactions of the cut-and-paste enzymes usually conserve the energy of the phosphodiester bond by employing a covalent enzyme-DNA intermediate where the
terminal phosphate is attached transiently to a tyrosine residue. Such reactions retain the stereochemical configuration at the phosphate (58). Conversely, the direct
hydrolysis of phosphodiester bonds by the EcoRI and EcoRV restriction enzymes
inverts the stereochemistry (16, 33). DNA cleavage by SfiI proceeds with stereochemical inversion, thus excluding a covalent intermediate (59). The Type II
restriction enzymes that interact with two DNA sites are thus unrelated to the
site-specific recombination enzymes in terms of both protein structure and chemical mechanism.
At present, perhaps the most plausible rationale for why many Type II restriction enzymes need two sites is that these enzymes effectively double-check
the DNA sequence to which they are bound before cleaving the DNA (26). The
pivotal requirement for a restriction enzyme is not to cleave DNA at its recognition sequence but rather to avoid cleaving DNA at any sequence other than the
unmodified recognition site. In vivo, the modification methyltransferase protects
every copy of the recognition sequence in the host chromosome, but not every
possible sequence that differs from the recognition site by one or two base pairs
(90). Hence, if the restriction endonuclease cleaves these alternative sequences, the
chromosome will soon be degraded. Even though EcoRI and EcoRV achieve sufficient levels of fidelity while recognizing individual sites (35, 68), an alternative
route to this end is that the enzyme bridges two sites and that it initiates its DNA
cleavage reaction only when both sites possess the cognate sequence. In this way,
a relatively low level of discrimination against alternative sequences at one site is
multiplied by the same (relatively low) level at the second site to obtain an overall 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 restriction
endonuclease from cleaving its host chromosome when the latter is incompletely
methylated.
It is likely that the Type I and Type III enzymes have evolved to benefit from
similar enhanced specificity, except that a one-dimensional linear search is used to
communicate between sites instead of a three-dimensional search. However, why
employ such baroque protein architectures to spool loops (Figure 6)? For some
looping enzymes, such as those involved in mismatch repair (3), a linear search
is the only way to maintain strand polarity over long distances. For the Type III
enzymes at least, one-dimensional tracking prevents the formation of site pairings
with 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 translocate
DNA may lie in the modular adaptability of the complexes. By translocating and
cleaving nonspecific DNA through discrete HsdR subunits, the HsdS subunits can
readily 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 would

require both an endonuclease and a methyltransferase to converge separately on
the same, new sequence (48).
Notwithstanding the energetic exertion required to trap 11Tw in loops, the
resulting changes in DNA topology may be key to driving otherwise unfavorable processes. For example, it has been suggested that formation of a negatively
supercoiled expanding loop by RAD54 facilitates strand invasion by a RAD51
nucleoprotein complex (95). Alternatively, the generation of torque in small contracting or expanding loops can be used to destabilize nucleoprotein complexes
by altering DNA twist to a level unacceptable for protein association. Examples
include chromatin remodeling (37, 105) and displacement of stalled polymerases
by transcription coupled repair factors (86). Because high levels of torque need to
be generated in these cases, StepTw may be 10 bp and translocation over tens of
base pairs is sufficient (105). Although Type I enzymes undoubtedly form topologically constrained DNA loops (42), translocation over thousands of base pairs
requires StepTw to be 10 bp. Nonetheless, the more moderate torque introduced
may still affect the action of other proteins on a target bacteriophage genome. Alternatively, since collision with Type I enzymes can displace Lac repressor from
its operator (20), translocation may be required simply to displace proteins from
the 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 space
limitations. We thank other members of The Bristol DNA-Protein Interactions
Group, past and present, for input and discussion. Our research is supported by the
Wellcome Trust and by the BBSRC. M.D.S is a Wellcome Trust Senior Fellow in
Basic Biomedical Sciences.
The Annual Review of Biophysics and Biomolecular Structure is online at
http://biophys.annualreviews.org
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13:39
Annual Reviews
AR214-FM
Annual Review of Biophysics and Biomolecular Structure

Volume 33, 2004

CONTENTS
ENZYME-MEDIATED DNA LOOPING, Stephen E. Halford, Abigail J. Welsh,
and Mark D. Szczelkun
DISEASE-RELATED MISASSEMBLY OF MEMBRANE PROTEINS,

Charles R. Sanders and Jeffrey K. Myers
25
CONFORMATIONAL SPREAD: THE PROPAGATION OF ALLOSTERIC

STATES IN LARGE MULTIPROTEIN COMPLEXES, Dennis Bray
and Thomas Duke
A FUNCTION-BASED FRAMEWORK FOR UNDERSTANDING

BIOLOGICAL SYSTEMS, Jeffrey D. Thomas, Taesik Lee, and Nam P. Suh
STRUCTURE, MOLECULAR MECHANISMS, AND EVOLUTIONARY
RELATIONSHIPS IN DNA TOPOISOMERASES, Kevin D. Corbett
and James M. Berger
53
75
95
STRUCTURE, DYNAMICS, AND CATALYTIC FUNCTION OF

DIHYDROFOLATE REDUCTASE, Jason R. Schnell, H. Jane Dyson, and
Peter E. Wright
THREE-DIMENSIONAL ELECTRON MICROSCOPY AT MOLECULAR

RESOLUTION, Sriram Subramaniam and Jacqueline L.S. Milne
TAKING X-RAY DIFFRACTION TO THE LIMIT: MACROMOLECULAR
STRUCTURES FROM FEMTOSECOND X-RAY PULSES AND
DIFFRACTION MICROSCOPY OF CELLS WITH SYNCHROTRON
RADIATION, Jianwei Miao, Henry N. Chapman, Janos Kirz,
David Sayre, and Keith O. Hodgson
119
141
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
THE USE OF IN VITRO PEPTIDE-LIBRARY SCREENS IN THE

ANALYSIS OF PHOSPHOSERINE/THREONINE-BINDING DOMAIN
STRUCTURE AND FUNCTION, Michael B. Yaffe and Stephen J. Smerdon
ROTATION OF F1 -ATPASE: HOW AN ATP-DRIVEN MOLECULAR
MACHINE MAY WORK, Kazuhiko Kinosita, Jr., Kengo Adachi, and
Hiroyasu Itoh
199
225
245
ix
P1: FDS
April 12, 2004
13:39
Annual Reviews
AR214-FM
CONTENTS
MODEL SYSTEMS, LIPID RAFTS, AND CELL MEMBRANES,

Kai Simons and Winchil L.C. Vaz
MASS SPECTRAL ANALYSIS IN PROTEOMICS, John R. Yates, III

INFORMATION CONTENT AND COMPLEXITY IN THE HIGH-ORDER
ORGANIZATION OF DNA, Abraham Minsky
THE ROLE OF WATER IN PROTEIN-DNA RECOGNITION, B. Jayaram
and Tarun Jain
269
297
317
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
SPIN DISTRIBUTION AND THE LOCATION OF PROTONS IN

PARAMAGNETIC PROTEINS, D. Goldfarb and D. Arieli
415
441
INDEXES
Subject Index
Cumulative Index of Contributing Authors, Volumes 2933
Cumulative Index of Chapter Titles, Volumes 2933
ERRATA
An online log of corrections to Annual Review of Biophysics
and Biomolecular Structure chapters may be found at
http://biophys.annualreviews.org/errata.shtml
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30 Apr 2004

Annu. Rev. Biophys. Biomol. Struct. 2004. 33:124

Annu. Rev. Biophys. Biomol. Struct. 2004.33:1-24. Downloaded from www.annualreviews.org

Stephen E. Halford, Abigail J. Welsh,

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

INTRODUCTION: ENZYME REACTIONS AT TWO DNA SITES

Annu. Rev. Biophys. Biomol. Struct. 2004.33:1-24. Downloaded from www.annualreviews.org

LOOPING BY RESTRICTION ENZYMES

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RESTRICTION ENZYMES LOOPING DNA

Annu. Rev. Biophys. Biomol. Struct. 2004.33:1-24. Downloaded from www.annualreviews.org

Annu. Rev. Biophys. Biomol. Struct. 2004.33:1-24. Downloaded from www.annualreviews.org

RESTRICTION ENZYMES LOOPING DNA

Annu. Rev. Biophys. Biomol. Struct. 2004.33:1-24. Downloaded from www.annualreviews.org

Annu. Rev. Biophys. Biomol. Struct. 2004.33:1-24. Downloaded from www.annualreviews.org

RESTRICTION ENZYMES LOOPING DNA

Annu. Rev. Biophys. Biomol. Struct. 2004.33:1-24. Downloaded from www.annualreviews.org

RESTRICTION ENZYMES LOOPING DNA

Annu. Rev. Biophys. Biomol. Struct. 2004.33:1-24. Downloaded from www.annualreviews.org

Annu. Rev. Biophys. Biomol. Struct. 2004.33:1-24. Downloaded from www.annualreviews.org

measuring the modulation of looping with protein concentration (57). In contrast,

Annu. Rev. Biophys. Biomol. Struct. 2004.33:1-24. Downloaded from www.annualreviews.org

RESTRICTION ENZYMES LOOPING DNA

Annu. Rev. Biophys. Biomol. Struct. 2004.33:1-24. Downloaded from www.annualreviews.org

Figure 6 Generation of loops by a motor enzyme. The model shown is based on

The Tracking Model for Loop Expansion and Contraction

RESTRICTION ENZYMES LOOPING DNA

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Annu. Rev. Biophys. Biomol. Struct. 2004.33:1-24. Downloaded from www.annualreviews.org

Topological Constraints of the Tracking Model

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RESTRICTION ENZYMES LOOPING DNA

Figure 8 The effect of translocation on expanding loop topology. was calculated

Alternative Ways to Distribute Topology into Loops

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Annu. Rev. Biophys. Biomol. Struct. 2004.33:1-24. Downloaded from www.annualreviews.org

RESTRICTION ENZYMES LOOPING DNA

FUNCTIONS OF ENZYME LOOPS

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RESTRICTION ENZYMES LOOPING DNA

In comparison, to change the specificity of the majority of Type II enzymes would

heteroduplex loop formation by a translocation mechanism. EMBO J. 16:446776

Annu. Rev. Biophys. Biomol. Struct. 2004.33:1-24. Downloaded from www.annualreviews.org

2001. Replisome-mediated DNA replication. Annu. Rev. Biochem. 70:181208

DNA cleavage by restriction endonuclease SgrAI. J. Mol. Biol. 327:57991

RESTRICTION ENZYMES LOOPING DNA

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continuous elongation by Escherichia coli

38. Cold Spring Harbor, NY: Cold Spring

Annu. Rev. Biophys. Biomol. Struct. 2004.33:1-24. Downloaded from www.annualreviews.org

49. Kong H, Smith CL. 1998. Does BcgI,

for determining the stereochemistry of

RESTRICTION ENZYMES LOOPING DNA

Annu. Rev. Biophys. Biomol. Struct. 2004.33:1-24. Downloaded from www.annualreviews.org

nucleic acid polymer. Trends Biochem.

81. Singleton MR, Scaife S, Wigley DB.

Annu. Rev. Biophys. Biomol. Struct. 2004.33:1-24. Downloaded from www.annualreviews.org

EcoRI endonuclease. Nonspecific interactions in EcoRI catalysis. J. Biol. Chem.

of the multimodular endonuclease FokI

April 12, 2004

Annual Review of Biophysics and Biomolecular Structure