Voet D., Voet Ju.G. Biochemistry

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corresponding relaxed circles. This phenomenon has been

established by observing the effect of ethidium ion bind-

ing on the sedimentation rate of circular DNA (Fig. 29-

21). Intercalating agents such as ethidium (a planar aro-

matic cation; Section 6-6Ca) alter a circular DNA’s degree

of superhelicity because they cause the DNA double helix

to unwind (untwist) by ⬃26° at the site of the intercalated

molecule (Fig. 29-22). W  0 in an unconstrained under-

wound circle because of the tendency of a duplex DNA to

maintain its normal twist of 1 turn per 10.5 bp. The titra-

Section 29-3. Supercoiled DNA 1161

W>0W<0

DNA

sedimentation

velocity

Ethidium bromide concentration

Figure 29-21 Sedimentation rate of underwound closed

circular duplex DNA as a function of ethidium bromide

concentration. The intercalation of ethidium between the base

pairs locally untwists the double helix (Fig. 29-22), which, since

the linking number of the circle is constant, is accompanied by an

equivalent increase in the writhing number.As the negatively

coiled superhelix untwists, it becomes less compact and hence

sediments more slowly. At the low point on the curve, the DNA

Figure 29-22 X-ray structure of a

complex of ethidium with 5-iodo-UpA.

Ethidium (red) intercalates between the

base pairs (green with I purple) of the

double helically paired dinucleoside

phosphate and thereby provides a model

for the binding of ethidium to duplex DNA.

[After Tsai, C.-C., Jain, S.C., and Sobell,

H.M., Proc. Natl.Acad. Sci. 72, 629 (1975).]

circles have bound sufficient ethidium to become fully relaxed.

As the ethidium concentration is further increased, the DNA

supercoils in the opposite direction, yielding a positively coiled

superhelix.The supertwisted appearances of the depicted DNAs

have been verified by electron microscopy. [After Bauer, W.R.,

Crick, F.H.C., and White, J.H., Sci. Am. 243(1), 129 (1980).

JWCL281_c29_1143-1172.qxd 7/8/10 8:39 PM Page 1161

tion of a DNA circle by ethidium unwinds the duplex (de-

creases T), which must be accompanied by a compensat-

ing increase in W. This, at first, lessens the superhelicity of

an underwound circle. However, as the circle binds more

and more ethidium, its value of W passes through zero

(relaxed circles) and then becomes positive, so that the

circle again becomes superhelical.Thus the sedimentation

rate of underwound DNAs, which is a measure of their

compactness and therefore their superhelicity, passes

through a minimum as the ethidium concentration in-

creases. This is what is observed with native DNAs (Fig.

29-21). In contrast, the sedimentation rate of an over-

wound circle would only increase with increasing ethid-

ium concentration.

b. DNAs Are Separated According to Their Linking

Number by Gel Electrophoresis

Gel electrophoresis (Sections 6-4 and 6-6C) also sepa-

rates similar molecules on the basis of their compactness,

so that the rate of migration of a circular duplex DNA in-

creases with its degree of superhelicity. The agarose gel

electrophoresis pattern of a population of chemically iden-

tical DNA molecules with different linking numbers there-

fore consists of a series of discrete bands (Fig. 29-23). The

molecules in a given band all have the same linking num-

ber and differ from those in adjacent bands by L  1.

Comparison of the electrophoretic band patterns of

simian virus 40 (SV40) DNA that had been enzymatically

relaxed to varying degrees and then resealed (Fig. 29-23)

reveals that 26 bands separate native from fully relaxed

SV40 DNAs. Native SV40 DNA therefore has W  26

(although it is somewhat heterogeneous in this quantity).

Since SV40 DNA consists of 5243 bp, it has 1 negative su-

perhelical turn per ⬃19 duplex turns. Such a superhelix

density (W/T) is typical of circular DNAs from various bi-

ological sources.

c. DNA in Physiological Solution Has 10.5 Base

Pairs per Turn

The insertion, using genetic engineering techniques

(Section 5-5C), of an additional x base pairs into a super-

helical DNA with a given linking number will increase

the DNA’s twist and hence decrease its writhing number

by x/h°, where h° is the number of base pairs per duplex

turn. Such an insertion shifts the position of each band in

the DNA’s gel electrophoretic pattern by x/h° of the

spacing between bands. By measuring the effects of sev-

eral such insertions, James Wang established that h° 

10.5  0.1 bp for B-DNA in solution under physiological

conditions.

C. Topoisomerases

The normal biological functioning of DNA occurs only if it

is in the proper topological state. In such basic biological

processes as RNA transcription and DNA replication, the

recognition of a base sequence requires the local separa-

tion of complementary polynucleotide strands. The nega-

tive supercoiling of naturally occurring DNAs results in a

torsional strain that promotes such separations since it

tends to unwind the duplex helix (an increase in T must be

accompanied by a decrease in W). If DNA lacks the proper

superhelical tension, the above vital processes (which them-

selves supercoil DNA; Sections 30-2C and 31-2Ca) occur

quite slowly, if at all.

The supercoiling of DNA is controlled by a remarkable

group of enzymes known as DNA topoisomerases (or sim-

ply topoisomerases).They are so named because they alter

the topological state (linking number) of circular DNA but

not its covalent structure. There are two classes of topoiso-

merases:

1. Type I topoisomerases, the first of which was discov-

ered by James Wang in 1971, act by creating transient

single-strand breaks in DNA.Type I enzymes are subclassi-

fied into types IA, IB, and IC topoisomerases on the basis

of their amino acid sequences and reaction mechanisms

(see below). Type I topoisomerases are denoted by odd

Roman numerals (e.g., topoisomerase I, III, etc.).

1162 Chapter 29. Nucleic Acid Structures

Figure 29-23 Agarose gel electrophoresis pattern of SV40

DNA. Lane 1 contains the negatively supercoiled native DNA

(lower band; the DNA was applied to the top of the gel). In lanes

2 and 3, the DNA was exposed for 5 and 30 min, respectively, to

an enzyme, known as a type IA topoisomerase (Section 29-3Cb),

that relaxes negative supercoils one at a time by increasing the

DNA’s linking number (L).The DNAs in consecutively higher

bands of a given gel have successively increasing linking numbers

(L 1). [From Keller, W., Proc. Natl. Acad. Sci. 72, 2553

(1975).]

JWCL281_c29_1143-1172.qxd 7/8/10 8:39 PM Page 1162

2. Type II topoisomerases, the first of which was discov-

ered by Martin Gellert in 1976, act by making transient

double-strand breaks in DNA with the accompanying hy-

drolysis of ATP to ADP  P

.Type II enzymes are subclas-

sified into types IIA and IIB topoisomerases on the basis

of their amino acid sequences. Type II topoisomerases are

denoted by even Roman numerals (e.g., topoisomerase II,

IV, etc.).

a. Type I Topoisomerases Incrementally Relax

Supercoiled DNA

Type I topoisomerases catalyze the relaxation of super-

coils in DNA by changing their linking number in incre-

ments of one turn until the supercoil is entirely relaxed. In

most organisms, type IA enzymes, which are present in

all cells, relax only negatively supercoiled DNA, whereas

type IB enzymes, which are present in all eukaryotes and

many prokaryotes (but not E. coli), relax both negatively

and positively coiled DNA. However, many hyperther-

mophiles, both eubacteria and archaea, have a type IA

topoisomerase known as reverse gyrase that induces pos-

itive supercoiling in DNA through the ATP-driven action

of a helicase domain that is fused to the N-terminus of

the topoisomerase domain (helicases are discussed in

Section 30-2C). This suggests that positive supercoiling,

which tightens the DNA double helix, protects DNA

from thermal denaturation. Although types IA and IB

topoisomerases are both monomeric, ⬃100-kD enzymes,

they share no apparent sequence or structural similari-

ties and function, as we shall see, via different enzymatic

mechanisms.

A clue to the mechanism of type IA topoisomerase was

provided by the observation that it reversibly catenates

(interlinks) single-stranded circles (Fig. 29-24a). Appar-

ently the enzyme operates by cutting a single strand, pass-

ing a single-strand loop through the resulting gap, and

then resealing the break (Fig. 29-24b), thereby twisting

double helical DNA by one turn. In support of this strand

passage mechanism, the denaturation of type IA enzyme

that has been incubated with single-stranded circular

DNA yields a linear DNA that has its 5¿-terminal phos-

phoryl group linked to the enzyme via a phosphoTyr di-

ester linkage.

In contrast, denatured type IB enzyme is linked to the 3¿

end of DNA via a phosphoTyr linkage.By forming such co-

valent enzyme–DNA intermediates, the free energy of the

cleaved phosphodiester bond is preserved, so that no energy

input is required to reseal the nick.

b. Type IA Topoisomerase Functions via a Strand

Passage Mechanism

Cells of E. coli contain two type IA topoisomerases

named topoisomerase I (also called  protein) and topo-

isomerase III. Topoisomerase III’s Tyr 328 is the active site

residue that forms a 5¿-phosphoTyr linkage with the cleaved

DNA. The X-ray structure of the inactive Y328F mutant of

topoisomerase III in complex with the single-stranded oc-

tanucleotide d(CGCAACTT), determined by Alfonso

Tyr

Type IA topoisomerase

DNA

Base

HOH

Base

–

P OP

...

Section 29-3. Supercoiled DNA 1163

Figure 29-24 Type IA topoisomerase action. By cutting a

single-stranded DNA, passing a loop of a second strand through

the break, and then resealing the break, a type IA topoisomerase

can (a) catenate two single-stranded circles or (b) unwind duplex

DNA by one turn.

(a)

(b)

Duplex DNA

(n turns)

Duplex DNA

(n – 1 turns)

JWCL281_c29_1143-1172.qxd 7/8/10 8:39 PM Page 1163

1164 Chapter 29. Nucleic Acid Structures

Figure 29-26 Proposed mechanism for the strand passage

reaction catalyzed by type IA topoisomerases. The enzyme is

shown in blue with the yellow patch representing the binding

groove for single-stranded (ss) DNA. The two DNA strands,

which are drawn in red and green, could represent the two

strands of a covalently closed circular duplex or two ss circles.

(1) The protein recognizes a ss region of the DNA, here the red

strand, and binds it in its binding groove. This is followed by or

occurs simultaneously with the opening of a gap between

domains I and III. (2) The DNA is cleaved with the newly formed

5¿ end becoming covalently linked to the active site Tyr and the

segment with the newly formed 3¿ end remaining tightly but

noncovalently bound in the binding groove. (3) The unbroken

(green) strand is passed through the opening or gate formed by

Figure 29-25 X-ray structure of the Y328F mutant of E. coli

topoisomerase III, a type IA topoisomerase, in complex with the

single-stranded octanucleotide d(CGCAACTT). The two views

shown are related by a 90° rotation about a vertical axis.The

protein’s four domains are drawn in different colors. The DNA is

the cleaved (red) strand to enter the protein’s central hole.

(4) The unbroken strand is trapped by the partial closing of the

gap. (5) The two cleaved ends of the red strand are rejoined in

what is probably a reversal of the cleavage reaction. (6) The gap

between domains I and III reopens to permit the escape of the

red strand, yielding the reaction product in which the green

strand has been passed through a transient break in the red

strand. (7) The enzyme returns to its initial state. If the two

strands form a negatively supercoiled duplex DNA, its linking

number, L, has increased by 1; if they are separate ss circles, they

have been catenated or decatenated. For duplex DNA, this

process can be repeated until all of its supercoils have been

removed (W  0). [After a drawing by Alfonso Mondragón,

Northwestern University.]

drawn in space-filling form with C white, N blue, O red, and P

yellow. The enzyme’s active site is marked by the side chain of

Phe 328, which is shown in space-filling form in yellow-green.

[Based on an X-ray structure by Alfonso Mondragón,

Northwestern University. PDBid 1I7D.]

JWCL281_c29_1143-1172.qxd 8/2/10 8:06 PM Page 1164

Mondragón (Fig. 29-25), reveals that this 659-residue

monomer folds into four domains which enclose an ⬃20 by

28 Å hole that is large enough to contain a duplex DNA and

which is lined with numerous Arg and Lys side chains. The

octanucleotide binds in a groove that is also lined with Arg

and Lys side chains with its sugar–phosphate backbone in

contact with the protein and with most of its bases exposed

for possible base pairing. Curiously, this single-stranded

DNA assumes a B-DNA-like conformation even though its

complementary strand would be sterically excluded from

the groove. The DNA strand is oriented with its 3¿ end near

the active site, where, if the mutant Phe 328 were the wild-

type Tyr, its side chain would be properly positioned to nu-

cleophilically attack the phosphate group bridging the

DNA’s C6 and T7 to form a 5¿-phosphoTyr linkage with T7

and release C6 with a free 3¿-OH.This structure and that of

the homologous and structurally similar E. coli topoiso-

merase I suggest the mechanism for the type IA topoiso-

merase-catalyzed strand passage reaction that is dia-

grammed in Fig. 29-26.

c. Type IB Topoisomerase Functions via a Controlled

Rotation Mechanism

Human topoisomerase I is a 765-residue type IB topo-

isomerase (and hence is unrelated to E. coli topoisomerase

I). It mediates the transient cleavage of one strand of a du-

plex DNA through the nucleophilic attack of Tyr 723 on a

DNA P atom to yield a 3¿-linked phosphoTyr diester bond

and a free 5¿-OH group on the succeeding nucleotide. Lim-

ited proteolysis studies revealed that topoisomerase I con-

sists of four major regions: its N-terminal, core, linker, and

C-terminal domains. The ⬃210-residue, highly polar, N-

terminal domain, which is poorly conserved, contains

several nuclear targeting signals and is dispensable for en-

zymatic activity.

The X-ray structure of the catalytically inactive Y723F

mutant of topoisomerase I lacking its N-terminal 214

residues and in complex with a 22-bp palindromic duplex

DNA was determined by Wim Hol (Fig. 29-27). The core

domain of this bilobal protein is wrapped around the DNA

in a tight embrace. If the mutant Phe 723 were the wild-

type Tyr, its OH group would be colinear with the scissile

P¬O5¿ bond and hence ideally positioned to nucleophili-

cally attack this P atom so as to form a covalent linkage

with the 3¿ end of the cleaved strand.As expected, the pro-

tein interacts with the DNA in a largely sequence inde-

pendent manner: Of the 41 direct contacts that the protein

makes to the DNA, 37 are protein–phosphate interactions

and only one is base-specific. The protein interacts to a

much greater extent with the five base pairs of the DNA’s

upstream segment (which would contain the cleaved

strand’s newly formed 5¿ end; 29 of the 41 contacts) than it

does with the base pairs of the DNA’s downstream seg-

ment (to which Tyr 723 would be covalently linked; 12 of

the 41 contacts).

Topoisomerase I does not seem sterically capable of un-

winding supercoiled DNA via the strand passage mecha-

nism that type IA topoisomerases appear to follow (Fig.

29-26). Rather, as is diagrammed in Fig. 29-28, it is likely

that topoisomerase I relaxes DNA supercoils by permitting

the cleaved duplex DNA’s loosely held downstream seg-

ment to rotate relative to the tightly held upstream

segment. This rotation can only occur about the

sugar–phosphate bonds in the uncleaved strand (, , , ε,

and  in Fig. 29-5) that are opposite the cleavage site be-

cause the cleavage frees these bonds to rotate. In support

of this mechanism, the protein region surrounding the

downstream segment contains 16 conserved, positively

charged residues that form a ring about this duplex DNA,

which would presumably hold the DNA in the ring but not

in any specific orientation. Nevertheless, the downstream

segment is unlikely to rotate freely because the cavity con-

taining it is shaped so as to interact with the downstream

segment during some portions of its rotation. Hence,

Section 29-3. Supercoiled DNA 1165

Figure 29-27 X-ray structure of the N-terminally truncated,

Y723F mutant of human topoisomerase I in complex with a

22-bp duplex DNA. The protein’s various domains and

subdomains are drawn in different colors.The DNA’s uncleaved

strand is cyan, and the upstream and downstream portions of the

scissile strand are purple and pink, respectively. [Courtesy of

Wim Hol, University of Washington. PDBid 1A36.]

JWCL281_c29_1143-1172.qxd 8/2/10 8:06 PM Page 1165

type IB topoisomerases are said to mediate a controlled

rotation mechanism in relaxing supercoiled DNA. This

unwinding is driven by the superhelical tension in the

DNA and hence requires no other energy input. Eventu-

ally, the DNA is religated by a reversal of the cleavage re-

action and the now less supercoiled DNA is released.

Type IC topoisomerase, whose only known family mem-

ber, topoisomerase V, occurs exclusively in archaea, resem-

bles type IB topoisomerases (which do not occur in ar-

chaea) in that it forms 3’-phosphoTyr intermediates and

appears to function via a controlled rotation mechanism.

However, it has no sequence or structural resemblance to

type IB topoisomerases.

d. Type II Topoisomerases Function via a Strand

Passage Mechanism

Bacteria have two types of type IIA topoisomerases:

DNA gyrase (or just gyrase) and topoisomerase IV, both of

which are A

heterotetramers. Eukaryotic type IIA

topoisomerases, which are named topoisomerase II, are

homologous to bacterial type IIA topoisomerases but with

their A and B subunits fused so that they are homodimers.

The type IIB topoisomerase family has only one member,

topoisomerase VI, an A

heterotetramer that occurs

mainly in archaea (although some archaea express both

type IIA and IIB topoisomerases).The A subunits of types

IIA and IIB topoisomerases share a common ATPase

module, but their B subunits are unrelated.

Gyrase is unique among topoisomerases in that it gener-

ates negative supercoils in DNA. All other topoisomerases

but reverse gyrase only relax supercoils (DNA supercoiling

in eukaryotes is generated differently from that in prokary-

otes; Section 34-1Ba). It’s A and B subunits are named

GyrA and GyrB.

Type II topoisomerases can also catenate and decate-

nate double-stranded circles as well as tie and untie knots

in them.The importance of this function can be seen as fol-

lows. The ⬃6.1 billion base pairs of DNA in a diploid

human cell have an aggregate length of ⬃ 2 m and are con-

fined to a nucleus that is 5 to 10 m in diameter. Imagine

that the ⬃20-Å-wide DNA was expanded by a factor of

5 million to the width of a 1-cm-diameter rope. It would

1166 Chapter 29. Nucleic Acid Structures

(a)

(b)

(c)

(d)

(f)

(g)

ReleaseBinding

Cleavage Religation

Controlled rotation

Structure

Covalent

intermediate

Covalent

intermediate

Noncovalent

complex

Noncovalent

complex

(e)

Figure 29-28 Controlled rotation mechanism for

type IB topoisomerases. A highly negatively

supercoiled DNA (red, with a right-handed writhe)

is converted, via stages (a) through (g), to a less

supercoiled form (green).Topoisomerase I is

drawn as a bilobal space-filling structure, in which

the cyan lobe is formed by core subdomains I and

II (Fig. 29-27) and the purple lobe is formed by

core subdomain III, the linker domain, and

the C-terminal domain.The structure shown

in (d), which is expanded by a factor of 2, shows

the downstream portion of the rotating DNA

(that containing the cleaved strand’s new 5¿ end)

at 30° intervals, all differently colored. Since the

enzyme is not always in direct contact with the

rotating DNA, small rocking motions of the

protein (small curved arrows) may accompany

the controlled rotation. [Courtesy of Wim Hol,

University of Washington.]

JWCL281_c29_1143-1172.qxd 8/2/10 8:06 PM Page 1166

Cut duplex

loop and pass

strand through

gap

Reseal

gap

Separate

duplex

strands

lengthwise

Two

turns

then be ⬃10,000 km long (the distance from San Francisco

to Rome) and confined to an expanded nucleus that is 25 to

50 m in diameter. Then imagine trying to manipulate the

ropes in this system without generating tangles or knots.

Yet such difficulties normally occur in cells during DNA

replication, repair, and recombination (Chapter 30), as well

as the segregation of the daughter chromosomes in divid-

ing cells (Section 1-4B). It is the function of type II topoiso-

merases to untangle the DNA during these processes.

Bacterial but not eukaryotic type IIA topoisomerases

are inhibited by a variety of substances including novo-

biocin, a member of the Streptomyces-derived coumarin

family of antibiotics, and ciprofloxacin (trade name Cipro),

a member of the synthetically generated quinolone family

of antibiotics (their coumarin and quinolone groups are

drawn in red):

Novobiocin

Ciprofloxacin

COOH

These agents profoundly inhibit bacterial DNA replication

and RNA transcription, thereby demonstrating the impor-

tance of properly supercoiled DNA in these processes. Stud-

ies using E. coli gyrase mutants resistant to these sub-

stances have demonstrated that ciprofloxacin associates

with GyrA and novobiocin binds to GyrB.

The gel electrophoretic pattern of duplex circles that

have been exposed to gyrase shows a band pattern in

which the linking numbers differ by increments of 2 rather

than 1, as occurs with type I topoisomerases. Evidently, gy-

rase acts by cutting both strands of a duplex,passing the du-

plex through the break, and resealing it (Fig. 29-29). This

hypothesis is corroborated by the observation that when

gyrase is incubated with DNA and ciprofloxacin, and sub-

sequently denatured with guanidinium chloride, a GyrA

subunit remains covalently linked to the 5¿ end of each of

the two cut strands through a phosphoTyr linkage. These

cleavage sites are staggered by 4 bp, thereby yielding

sticky ends.

Saccharomyces cerevisiae (baker’s yeast) topoisomerase

II is a homodimer of 1428-residue subunits whose N- and

C-terminal segments are homologous to E. coli’s GyrB

(804 residues) and GyrA (878 residues) subunits, respec-

tively. The breakage/reunion domain, which encompasses

residues 410 to 1202, can, by itself, cleave duplex DNA but

cannot transport it through the break without the action of

the enzyme’s ATPase domain (residues 1–409). However,

the C-terminal segment (residues 1203–1428), which is

poorly conserved, appears to be dispensable.

Although the structure of a full length type IIA topoiso-

merase is as yet unknown, those of its ATPase and break-

age/reunion domains from both E. coli gyrase and yeast

topoisomerase II have been determined. The X-ray struc-

ture of the homodimeric topoisomerase II ATPase in com-

plex with the nonhydrolyzable ATP analog AMPPNP,

determined by James Berger, consists of two domains (Fig.

Section 29-3. Supercoiled DNA 1167

Figure 29-29 A demonstration, in which DNA is represented

by a ribbon, that cutting a duplex circle, passing the double helix

through the resulting gap, and then resealing the break changes

the linking number by 2. Separating the resulting single strands

(slitting the ribbon along its length; right) indicates that one

single strand makes two complete revolutions about the other.

JWCL281_c29_1143-1172.qxd 8/2/10 8:06 PM Page 1167

29-30a). The N-terminal domain binds AMPPNP and the

C-terminal domains form the walls of a large hole through

the dimer, which in the X-ray structure of the structurally

similar E. coli GyrB is 20 Å across, the same width as the B-

DNA double helix.

Berger also determined the X-ray structure of the yeast

topoisomerase breakage/reunion domain in complex a 15-

bp DNA that has a self-complementary 4-nucleotide over-

hang on the 5¿ end of one its strands. The DNA thereby

forms a 2-fold symmetric 34-bp duplex with nicks on oppo-

site strands separated by 4 bp (Fig. 29-30b,c).These are pre-

cisely the sites at which the enzyme would cleave an intact

DNA by linking its newly formed 5¿-ending strands to the

active site Tyr 782 residues.The protein binds the DNA in a

positively charged groove that spans the width of the dimer

and, in doing so, bends it through an arc of 150° (we shall

see in later chapters that DNA-binding proteins often de-

form their bound DNA, although such an extreme defor-

mation is unusual). Interestingly, the DNA between the two

cleavage sites is essentially in the A form. There are almost

no direct contacts between the protein and the DNA bases,

as is expected for a protein with little sequence specificity.

Note also that the C-terminal portions of the protein come

together to enclose a large centrally located empty space.

Consideration of the foregoing two structures and those

of the corresponding portions of E. coli gyrase suggests a

1168 Chapter 29. Nucleic Acid Structures

(a)

(b)

(c)

Figure 29-30 X-ray structures of S. cerevisiae topoisomerase

II. (a) Structure of a homodimer of the N-terminal ATPase do-

main (residues 7–406) in complex with AMPPNP as viewed with

its 2-fold axis vertical.The protein is displayed in ribbon form

with one subunit gray and the other colored in rainbow order

from its N-terminus (blue) to its C-terminus (red).The bound

AMPPNP molecules are shown in space-filling form with C

green, N blue, O red, and P orange. (b) Structure of a homodimer

of the DNA breakage/reunion domain (residues 419–1177) in

complex with a doubly nicked 34-bp DNA. The structure is viewed

with its 2-fold axis vertical.The DNA, which describes a 150° arc,

is drawn in stick form with C green, N blue, O red, and P orange

embedded in its semitransparent molecular surface (orange).The

protein is displayed in ribbon form with one subunit gray and the

other colored in rainbow order from its N-terminus (blue) to its

C-terminus (red).The side chains of the two active site Tyr

residues (Y782) are shown in space-filling form with C magenta

and O red.The polypeptides in Parts a and b, which in the intact

protein are joined by an 11-residue linker (represented by the

dotted lines), presumably share the same 2-fold axis as drawn,

although their relative orientation about this axis is unknown.

side chain O atoms are positioned to link to opposite DNA

strands at sites separated by 4 base pairs. [Based on X-ray struc-

tures by James Berger, University of California at Berkeley.

PDBids 1PVC and 2RGR.]

See Interactive Exercise 32

JWCL281_c29_1143-1172.qxd 10/19/10 10:23 AM Page 1168

type of strand passage model for the mechanism of type IIA

topoisomerases (Fig. 29-31) in which the DNA duplex to be

cleaved, the so-called G-segment (G for gate). binds in the

above-described groove across the top of the cleavage/re-

union domain. ATP binding to the ATP-binding domain

then induces a sequence of conformational changes in which

the G-segment is cleaved and the resulting two fragments

are spread apart by at least 20 Å through the action of the

protein. This permits the passage of the DNA’s so-called T-

segment (T for transported) through the break in the DNA

and breakage/reunion domain’s upper gate (which may also

contain portions of the ATPase domain) into its central hole,

thereby incrementing the DNA’s linking number by 2.Then,

in a process that is accompanied by ATP hydrolysis, the up-

per gate closes to reseal the cleaved DNA, and the T-seg-

ment passes through the breakage/reunion domain’s bottom

gate. Finally, the resulting ADP and P

are released and the

bottom gate closes to yield recycled enzyme. Many of these

enzymatic states have been observed in the several known

X-ray structures of type IIA topoisomerase components.

e. Topoisomerase Inhibitors Are Effective Antibiotics

and Cancer Chemotherapy Agents

Coumarin derivatives such as novobiocin, and

quinolone derivatives such as ciprofloxacin, specifically

inhibit gyrase and are therefore antibiotics. In fact,

ciprofloxacin is the most efficacious oral antibiotic against

gram-negative bacteria presently in clinical use (novo-

biocin’s adverse side effects and the rapid generation of

bacterial resistance to it have resulted in the discontinua-

tion of its use in the treatment of human infections). A

number of substances, including doxorubicin (also called

adriamycin; a product of Streptomyces peucetius) and

etoposide (a synthetic derivative),

Etoposide

OCH

Doxorubicin (Adriamycin)

CCH

Section 29-3. Supercoiled DNA 1169

Figure 29-31 Model for the enzymatic

mechanism of type II topoisomerases. The

protein’s ATPase domain and the upper and

lower portions of the breakage/reunion

domain are colored yellow, red, and purple,

respectively, and the DNA’s G- and T-

segments are colored gray and green,

respectively. In 1, the G-segment binds to the

enzyme, thereby inducing the conformational

change drawn in 2. The binding of ATP

(represented by asterisks) and a T-segment

(3) induces a series of conformational changes

in which the G-segment is cleaved as the

upper gate opens.The ATPase domains

dimerize, and the T-segment is transported

through the break into the central hole (4).

The DNA transport step is shown as

proceeding through the hypothetical

intermediate in square brackets.The G-

segments are then resealed and the T-segment

is released through the lower gate (5).This

gate then closes as the ATP is hydrolyzed and

the resulting ADP and P

released to yield the

enzyme in its starting state (2). [Courtesy of

James Wang, Harvard University.]

JWCL281_c29_1143-1172.qxd 8/2/10 8:06 PM Page 1169

inhibit eukaryotic type IIA topoisomerases and are there-

fore widely used in cancer chemotherapy. Indeed, ⬃50% of

cancer chemotherapy regimens contain at least one drug

targeted to type IIA topoisomerases.

Type IIA topoisomerase inhibitors act in either of two

ways. Many of them, including novobiocin, inhibit their tar-

get enzyme’s ATPase activity (novobiocin is a competitive

inhibitor of ATP because it tightly binds to GyrB in a way

that prevents the binding of ATP’s adenine ring). They

therefore kill cells by blocking topoisomerase activity,

which results in the arrest of DNA replication and RNA

transcription. However, other substances, including

ciprofloxacin, doxorubicin, and etoposide,enhance the rate

at which their target type IIA topoisomerases cleave

double-stranded DNA and/or reduce the rate at which

these enzymes reseal these breaks. Consequently, these

agents induce higher than normal levels of transient

protein-bridged breaks in the DNA of treated cells. These

protein bridges are easily ruptured by the passage of the

replication and transcription machinery, thereby rendering

the breaks permanent. Although all cells have elaborate

enzymatic systems to repair damaged DNA (Section 30-5),

a sufficiently high level of DNA damage overwhelms these

systems and hence results in cell death. Consequently, since

rapidly replicating cells such as cancer cells have elevated

levels of type IIA topoisomerases, they are far more likely

to incur lethal DNA damage through the poisoning of their

type IIA topoisomerases than are slow-growing or quies-

cent cells.

Type IB topoisomerases are specifically inhibited by the

quinoline-based alkaloid camptothecin

(a product of the Chinese yew tree, Camptotheca acumi-

nata) and its derivatives, which act by stabilizing the cova-

lent topoisomerase I–DNA complex. These compounds,

the only known naturally occurring topoisomerase IB in-

hibitors, are potent anticancer agents.

Camptothecin

1170 Chapter 29. Nucleic Acid Structures

1 Double Helical Structures B-DNA consists of a right-

handed double helix of antiparallel sugar–phosphate chains

with ⬃10 bp per turn of 34 Å and with its bases nearly perpen-

dicular to the helix axis. Bases on opposite strands hydrogen-

bond in a geometrically complementary manner to form A ⴢ T

and G ⴢ C Watson–Crick base pairs. At low humidity, B-DNA

undergoes a reversible transformation to a wider, flatter right-

handed double helix known as A-DNA. Z-DNA, which is

formed at high salt concentrations by polynucleotides of alter-

nating purine and pyrimidine base sequences, is a left-handed

double helix. Double helical RNA and RNA ⴢ DNA hybrids

have A-DNA-like structures. The conformation of DNA, par-

ticularly that of B-DNA, varies with its base sequence largely

because DNA’s flexibility varies with its base sequence.

2 Forces Stabilizing Nucleic Acid Structures The orien-

tations about the glycosidic bond and the various torsion an-

gles in the sugar–phosphate chain are sterically constrained in

nucleic acids. Likewise, only a few of the possible sugar pucker

conformations are commonly observed. Watson–Crick base

pairing is both geometrically and electronically complemen-

tary. Yet hydrogen bonding interactions do not greatly stabi-

lize nucleic acid structures. Rather, the structures are largely

stabilized by hydrophobic interactions. Nevertheless, the hy-

drophobic forces in nucleic acids are qualitatively different in

character from those that stabilize proteins. Electrostatic in-

teractions between charged phosphate groups are also impor-

tant structural determinants of nucleic acids.

CHAPTER SUMMARY

3 Supercoiled DNA The linking number (L) of a cova-

lently closed circular DNA is topologically invariant. Conse-

quently, any change in the twist (T) of a circular duplex must

be balanced by an equal and opposite change in its writhing

number (W), which indicates its degree of supercoiling.Super-

coiling can be induced by intercalation agents. The gel elec-

trophoretic mobility of DNA increases with its degree of su-

perhelicity. Naturally occurring DNAs are all negatively

supercoiled and must be so in order to participate in DNA

replication and RNA transcription.

Type IA topoisomerases relax negatively supercoiled

DNAs via a strand passage mechanism in which they cleave a

single strand of DNA to form a 5¿-phosphoTyr bond, pass a

single-strand DNA segment through the gap, and then reseal

the gap. Type IB topoisomerases relax both negatively and

positively supercoiled DNAs via a controlled rotation mecha-

nism involving a single-strand cleavage in which a transient

phosphoTyr bond is formed with the newly generated 3¿ end.

Type II topoisomerases relax duplex DNA in increments of

two supertwists at the expense of ATP hydrolysis by making a

double-strand scission in the DNA so as to form two transient

5¿-phosphoTyr linkages, passing the duplex through the break,

and resealing it. DNA gyrase also generates negative super-

twists in an ATP-dependent manner. Topoisomerases are the

targets of several antibiotics and chemotherapeutic agents.

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