Mutational analysis of the preferential binding of human topoisomerase I to supercoiled DNA

Mutational analysis of the preferential binding of humantopoisomerase I to supercoiled DNAZheng Yang, James F. Carey and James J. Champoux

Department of Microbiology, School of Medicine, University of Washington, Seattle, WA, USA

Introduction

Type I DNA topoisomerases relax supercoils by intro-

ducing a transient single-strand break in the DNA.

These enzymes are classified into type IA and type IB

subfamilies based on the polarity of attachment to the

cleaved DNA [1–3]. The members of the two subfami-

lies share no sequence homology and are further dis-

tinguished by their substrate requirements and

mechanisms of relaxation. Type IA subfamily members

require a single-stranded region to bind DNA, become

attached to the 5¢ end upon cleavage, and only relax

negatively supercoiled DNA in the presence of divalent

cations such as Mg2+. Escherichia coli DNA topo-

isomerase I is the prototype of the type IA subfamily.

Type IB subfamily members bind double-stranded

DNA, become attached to the 3¢ end of the cleaved

strand, and relax both positive and negative supercoils.

ATP or divalent cations are not required for the

type IB enzymes, although Mg2+ and Ca2+ enhance

the rate of relaxation [4].

The cleavage–religation reaction catalyzed by human

DNA topoisomerase I, the prototypical type IB

enzyme, is essential for many biological processes,

Keywords

competition binding assay; DNA topology;

node binding; supercoiled DNA;

topoisomerase I

Correspondence

J. J. Champoux, Department of

Microbiology, University of Washington, Box

357242, Seattle, WA 98195-7242, USA

Fax: +1 206 543 8297

Tel: +1 206 543 8574

E-mail: [email protected]

(Received 7 July 2009, revised 9 August

2009, accepted 11 August 2009)

doi:10.1111/j.1742-4658.2009.07270.x

Human topoisomerase I binds DNA in a topology-dependent fashion with

a strong preference for supercoiled DNAs of either sign over relaxed circu-

lar DNA. One hypothesis to account for this preference is that a second

DNA-binding site exists on the enzyme that mediates an association with

the nodes present in supercoiled DNA. The failure of the enzyme to dimer-

ize, even in the presence of DNA, appears to rule out the hypothesis that

two binding sites are generated by dimerization of the protein. A series of

mutant protein constructs was generated to test the hypotheses that the

homeodomain-like core subdomain II (residues 233–319) provides a second

DNA-binding site, or that the linker or basic residues in core subdo-

main III are involved in the preferential binding to supercoiled DNAs.

When putative DNA contact points within core subdomain II were altered

or the domain was removed altogether, there was no effect on the ability

of the enzyme to recognize supercoiled DNA, as measured by both a gel

shift assay and a competition binding assay. However, the preference for

supercoils was noticeably reduced for a form of the enzyme lacking the

coiled-coil linker region or when pairs of lysines were changed to glutamic

acids in core subdomain III. The results obtained implicate the linker and

solvent-exposed basic residues in core subdomain III in the preferential

binding of human topoisomerase I to supercoiled DNA.

Abbreviations

Dcap, NH2-terminal truncation of human topoisomerase beginning at residue 433; GST, glutathione S-transferase; topo31, a fragment of

human topoisomerase I extending from residues 175–433; topo56, COOH-terminal truncation of topo70 missing the last 126 amino acids;

topo58, COOH-terminal truncation of topo70 missing the last 106 amino acids; topo70, NH2-terminal truncation of human topoisomerase I

missing the first 174 amino acids; topo70DL, a form of topo70 missing linker residues 660–688.

5906 FEBS Journal 276 (2009) 5906–5919 ª 2009 The Authors Journal compilation ª 2009 FEBS

including DNA replication, transcription and recombi-

nation [2,3]. Strand cleavage is initiated by nucleophilic

attack of the O4 atom of the active site tyrosine on the

scissile phosphate in the DNA, resulting in the cova-

lent attachment of the enzyme to the 3¢ end of the

broken strand [2]. Rotation of the duplex region

downstream of the break site relieves any supercoiling

strain in the DNA prior to religation and release of

the topoisomerase [5,6].

Human DNA topoisomerase I is composed of 765

amino acids and has a molecular mass of 91 kDa. On

the basis of sequence comparisons, limited proteolysis

studies and the crystal structure of the enzyme [7,8],

four domains have been identified in the protein: an

NH2-terminal domain (Met1-Gly214), a core domain

(Ile215-Ala635), a linker domain (Pro636-Lys712) and

a COOH-terminal domain (Gln713-Phe765) (Fig. 1).

The NH2-terminal domain is unstructured, poorly con-

served, highly charged and dispensable for the DNA

relaxation activity in vitro. It contains nuclear localiza-

tion signals and was shown to interact with nucleolin,

the SV40 large T antigen, p53, and possibly certain

transcription factors [9–13]. Topo70 is a truncated

form of human topoisomerase I that lacks residues

1–174 of the NH2-terminal domain, yet retains full

enzymatic activity [7] and a preference for binding su-

percoiled DNA. The core domain is highly conserved

and more protease-resistant than the other domains.

The poorly-conserved linker domain is highly charged

and forms an anti-parallel coiled-coil structure that

connects the core domain to the COOH-terminal

domain. The linker protrudes from the body of the

protein and, instead of tracking with the axis of a

bound DNA helix, angles away from DNA. The

COOH-terminal domain is highly conserved and con-

tains the active site tyrosine, Tyr723. When separately

expressed, the COOH-terminal and core domains can

associate in vitro to reconstitute wild-type levels of

enzymatic activity, demonstrating that the linker

domain is not required for activity [4,7,14].

The co-crystal structure of human topoisomerase I

with bound DNA indicates that the core domain can

be further divided into three subdomains [8] (Fig. 1).

Core subdomain I (residues 215–232, 320–433) and

core subdomain II (residues 233–319) form the Cap

structure of the enzyme and cover one side of the

DNA. Core subdomain III (residues 434–635) contains

all the residues implicated in catalysis except Tyr723

and cradles the DNA on the side opposite of the Cap

[5,8]. Although there is little sequence similarity, the

fold of the subdomain II is very similar to that of a

homeodomain found in a family of DNA-binding

proteins. For example, residues 244–314 of the core

subdomain II superimpose on the POU homeodomain

of the Oct-1 transcription factor with an rmsd of only

3.0 A [8,15]. This observation suggests that core sub-

domain II, which forms part of the exposed Cap,

could represent a second DNA-binding site distinct

from the substrate binding channel observed in the

co-crystal structure. However, the conserved residues

that are involved in base-specific contacts in the POU

homeodomain are absent in core subdomain II of

human topoisomerase I, suggesting that, if sub-

domain II interacts with DNA, it does so with low

affinity and likely without sequence specificity [15,16].

It has been proposed that topoisomerases relax the

negative and positive supercoils generated by the trans-

location of an RNA polymerase along the DNA

during transcription [17]. In support of this model,

eukaryotic type IB topoisomerases have been found to

associate with transcriptionally active genes and have

been reported to interact directly with the transcription

machinery [18–26]. Eukaryotic type IB topoisomerases

have also been shown to provide the swivels for the

Fig. 1. Crystal structure of human topoisomerase I. Core subdo-

mains I, II and III are colored yellow, blue and red, respectively,

with the linker and C-terminal domains colored orange and green,

respectively. The Cap and Linkers regions are labeled along with

the amino acid residues that were changed in the present study.

Amino acids in core subdomain II (His266, Lys299 and Ser306) that

were changed to glutamic acid in the combinations indicated in the

text are shown in ball and stick and colored magenta. The three

amino acids in the linker (Lys650 ⁄ Lys654 ⁄ Gln657) that were

simultaneously changed to alanine are similarly depicted and

colored brown. The four amino acids in the linker (Lys679 ⁄Lys682 ⁄ Lys687 ⁄ Lys689) that were simultaneously changed to

serine are colored gray. Surface-exposed lysine residues in core

subdomain III (Lys466 ⁄ Lys468 and Lys545 ⁄ Lys549) that were

pairwise mutated to glutamic acid are colored black.

Z. Yang et al. Supercoil binding by topoisomerase I

FEBS Journal 276 (2009) 5906–5919 ª 2009 The Authors Journal compilation ª 2009 FEBS 5907

relaxation of positive supercoils during DNA replica-

tion [20,27–31]. The mechanism for recruiting DNA

topoisomerase I to transcriptionally active and repli-

cating DNA remains unclear, although several studies

have shown that the enzyme prefers to bind super-

coiled over relaxed DNA [32–37]. Because the enzyme

binds supercoiled DNA irrespective of the sign of the

supercoils, Zechiedrich and Osheroff [36] hypothesized

that topoisomerase I specifically binds at a node where

two duplex regions of the supercoiled DNA cross and

also provided electron microscopic evidence in support

of this hypothesis [36].

The structural basis for the preferential binding of

human topoisomerase I to supercoiled DNA is

unknown but, if node recognition is important, then it

is likely that the binding involves an interaction with

two regions of DNA at the point of crossing. One

hypothesis to explain how the enzyme provides two

DNA-binding sites to stabilize an interaction with a

DNA node is to assume that it binds as a dimer

(Fig. 2A). An alternative hypothesis is that, in addition

to the substrate binding channel identified in the crys-

tal structure of the protein (Fig. 1) [8], there is a

second DNA-binding site present on the protein that

stabilizes an interaction at a DNA node (Fig. 2B). In

the present study, we performed experiments designed

to distinguish between these possible explanations for

the preference of topoisomerase I for supercoils.

Results

Human topoisomerase I does not dimerize in the

absence or presence of DNA

We previously used a gel filtration assay to demon-

strate that, although topo70DL, a mutant form of

topo70 missing a portion of the linker (i.e. linker

residues 660–688), formed dimers through a domain

swapping mechanism, no dimerization of WT topo70

was detectable under the same conditions [4,38].

Because these earlier experiments were carried out in

the absence of DNA, we wanted to test whether

dimers could form in the presence of DNA. In the

present study, we used a glutathione S-transferase

(GST) pull-down assay to determine whether topo70

that was already covalently bound to a DNA oligonu-

cleotide could dimerize. GST-topo70 was incubated

with free topo70 in the absence or presence of an oli-

gonucleotide suicide substrate, and any protein bound

to GST-topo70 was collected by adsorption to gluta-

thione S-Sepharose beads and analyzed by SDS–

PAGE. Control experiments showed that free topo70

did not bind to either GST alone or to the beads

(Fig. 3, lanes 6 and 7). Under the same conditions, no

topo70 was found associated with the bead-bound

GST-topo70 either in the absence or presence of DNA

(Fig. 3, lanes 2 and 3, respectively). The slower migrat-

ing species of the doublet observed in lane 3 in Fig. 3

is the result of suicide cleavage and shows that approx-

imately half of the GST-topo70 contained covalently

bound oligonucleotide DNA. Thus, these results con-

firm our earlier finding that topo70 does not dimerize

when free in solution and also extend the results to

show that, even when bound to DNA after suicide

cleavage, dimerization does not occur.

Fig. 2. Alternative modes for topoisomerase I binding to a DNA

node. (A) Node binding occurs through dimerization of topoisomer-

ase I. (B) Node binding is mediated by two DNA-binding sites on a

single molecule of topoisomerase I.

Fig. 3. GST pull-down experiment to test for dimerization. The indi-

cated combinations of GST-topo70, topo70 and GST were incu-

bated with and without a suicide DNA oligonucleotide and mixed

with glutathione Sepharose 4B beads (GSH beads). The beads

were collected by centrifugation, washed and the samples were

analyzed by SDS-PAGE. Lane 1, protein markers with sizes (kDa)

indicated along the left side of the gel. Lanes 4 and 5 contain GST-

topo70 and topo70 size markers, respectively. The GST protein in

lane 6 was run off the gel in this analysis. Although all of the

samples were analyzed on the same gel, lanes with unrelated data

were removed digitally at the places indicated by the thin vertical

lines.

Supercoil binding by topoisomerase I Z. Yang et al.


DNA-binding properties of mutant proteins as

measured by a gel shift assay

A structural alignment of core subdomain II of human

topoisomerase I with the POU homeodomain of Oct-1

indicated that the residues making base specific

contacts with the DNA in the homeodomain are not

conserved in the core subdomain II. However, basic

residues K25, R46 and R53 of the POU homeodomain

that make hydrogen bonds with phosphates in the

bound DNA correspond to residues His266, Lys299

and Ser306 in core subdomain II of human topo-

isomerase I (Fig. 1). All three of these residues are

conserved among known eukaryotic topoisomerase I

sequences. To test whether these amino acids mediate

an interaction with DNA that accounts for node bind-

ing by the enzyme, site-directed mutagenesis was used

to replace these residues with glutamic acid in topo70.

These changes would be predicted to disrupt an inter-

action with the DNA phosphate backbone, but have a

minimal effect on the overall enzyme structure because

all three are in a solvent-exposed region. Because the

assays to detect the preferential binding to supercoiled

DNA require a catalytically inactive form of the pro-

tein [37], a mutation in the active site tyrosine (Y723F)

was also introduced into the proteins. Topo70 capKS-

E ⁄Y723F and topo70 capHKS-E ⁄Y723F were

expressed and purified from recombinant baculovirus-

infected insect SF-9 cells. The following proteins were

similarly purified for use in these assays: a reconsti-

tuted form of the protein lacking the linker, compris-

ing a COOH-terminal truncation of topo70 missing

the last 126 amino acids (topo56) plus the Y723F

mutant form of the COOH-terminal domain (topo6.3),

the catalytically inactive NH2-terminal truncation of

human topoisomerase beginning at residue 433 (Dcap)[39] and a fragment of human topoisomerase I extend-

ing from residues 175–433 (topo31) (Fig. 4).

The various forms of the topoisomerase protein

described above were mixed with an equimolar mixture

of supercoiled, nicked circular and linear pBluescript

KSII(+) DNAs, and a gel shift assay [40–44] was

used to analyze the preference of the proteins for the

different topological forms of DNA. For the positive

Fig. 4. Human topoisomerase I fragments

used in the DNA-binding studies. (A) The

four domains of full-length human topoisom-

erase I (topo I) are shown above the various

constructs used in the binding studies:

topo70, a 70 kDa NH2-terminally truncated

protein that starts with an engineered Met

upstream of Lys175; topo58, a COOH-termi-

nal deletion of topo70, ending at Ala659;

topo31, a COOH-terminal deletion of topo70

ending at Ser433; Dcap, an NH2-terminal

truncation starting at Ser433; topo56 ⁄ 6.3, a

reconstituted protein comprising the core

domain from Lys175 to Thr639 and the

COOH-terminal domain from Lys713 to the

COOH terminus (Phe765). (B) SDS-PAGE

analysis of 2 lg of the indicated purified pro-

teins. Lane 1, protein markers with sizes

(kDa) indicated along left side of the panel;

lane 2, topo70 Y723F; lane 3, topo70 capKS-

E ⁄ Y723F; lane 4, topo70 capHKS-E ⁄ Y723F;

lane 5, topo56 ⁄ 6.3 Y723F (6.3 kDa fragment

of topo6.3 Y723F was run off the bottom of

the gel); lane 6, Dcap; lane 7, topo31; lane

8, protein markers; lane 9, topo70 K466-

468E ⁄ Y723F; lane 10, topo70 K545-

549E ⁄ Y723F.



control protein, topo70 Y723F, the mobility of the

supercoiled DNA was reduced, with essentially no effect

on the mobility of either the nicked or linear DNAs at

the two lowest protein concentrations (Fig. 5A, com-

pare lanes 2 and 3 with lane 1). As the amount of topo70

Y723F protein was increased, the supercoiled DNA was

shifted further and, to a lesser extent, both the linear

and nicked DNA bands became shifted as well (Fig. 5A,

lanes 4 and 5). These results confirmed the earlier find-

ing that topo70 Y723F has a preference for supercoiled

over linear and nicked DNA [37]. Topo31, which corre-

sponds to the Cap region of human topoisomerase I,

provides a convenient nonspecific negative control for

this analysis. As shown in Fig. 5A, lanes 22–26, all three

forms of the plasmid DNA responded equally to

increasing concentrations of the topo31 fragment, con-

sistent with a lack of preference for one form over

another. A higher concentration of topo31 was required

to effect a gel shift, reflecting the lower affinity of the

protein for DNA compared to topo70.

Both topo70 capKS-E ⁄Y723F and topo70 capHKS-

E ⁄Y723F retained the preference for binding super-

coiled DNA (Fig. 5A, lanes 7–10 and 12–15), ruling

out Cap residues His266, Lys299 and Ser306 as con-

tributors to the preferential binding to supercoils. To

further test the possible involvement of the core subdo-

main II in the preferential binding to supercoiled

DNA, the Dcap mutant lacking core subdomains I and

II was also tested in the gel shift analysis (Fig. 5A,

lanes 17–20). Dcap contains core subdomain III, the

linker domain, and the COOH-terminal domain (resi-

dues 433–765) (Fig. 4A), and is catalytically inactive,

despite containing all of the residues known to be

directly involved in catalysis [39]. At the lower concen-

trations of the Dcap protein, the supercoiled DNA was

selectively shifted upon binding, although the magni-

tude of the shift was less compared to that observed

with the topo70 protein (Fig. 5A, compare lanes 17–20

with lanes 1–5). This reduction in the shift most likely

resulted from the two-fold lower affinity of the Dcapfor DNA [39] and the lower molecular weight of Dcap(41 kDa) compared to topo70 (71 kDa). Thus, deletion

of the Cap region that includes subdomain II did not

eliminate the preference for supercoiled DNA, indicat-

ing that core subdomain II is dispensable for the

preferential binding of topoisomerase I to supercoils.

Although the band corresponding to the supercoiled

DNA was selectively shifted in the presence of topo70

Y723F and all of the mutant proteins except topo31,

we wanted to formally rule out the possibility that the

Fig. 5. DNA-binding measured by an agarose gel shift assay. (A) Two-fold serial dilutions of the indicated proteins were incubated with equal

amounts of supercoiled, linear and nicked pBluescript KSII(+) plasmid DNA and analyzed by electrophoresis in an agarose gel as described in

the Experimental procedures. The mobilities of unshifted supercoiled, linear and nicked DNAs are indicated along the right side. Lanes 1, 6,

11, 16, 21 and 27 contain DNA alone; lanes 2–5 contain 0.88, 1.75, 3.5 and 7 pmol of topo70 Y723F, respectively; lanes 7–10 contain 0.88,

1.75, 3.5 and 7 pmol of topo70 capKS-E ⁄ Y723F, respectively; lanes 12–15 contain 0.88, 1.75, 3.5 and 7 pmol of topo70 capHKS-E ⁄ Y723F,

respectively; lanes 17–20 contain 0.88, 1.75, 3.5 and 7 pmol of Dcap, respectively; and lanes 22-26 contain 0.88, 1.75, 3.5, 7 and 14 pmol of

topo31, respectively. The white spaces demarcate separate gel analyses. (B) Same experimental design as in (A) for the indicated proteins.

Lanes 1, 6 and 11 are DNA alone; lanes 2–5 contain 0.88, 1.75, 3.5 and 7 pmol of topo70 Y723F, respectively; and lanes 7–10 contain 0.88,

1.75, 3.5 and 7 pmol of topo56 ⁄ 6.3 Y723F, respectively.



proteins bound to the supercoiled, linear and nicked

DNAs equally well, but only the supercoiled DNA

shift was detected visually because of its greater initial

mobility. Therefore, the gel shift assay was repeated

using topo70 Y723F or topo70 capHKS-E ⁄Y723F that

had been previously labeled with 32P using protein

kinase C. The autoradiograph of the agarose gel

showed that the majority of the labeled proteins were

associated with the shifted supercoiled DNA and that

the amount of bound label correlated with the extent

of the shift (Fig. 6, lanes 2, 3, 5 and 6). Furthermore,

label was only associated with the nicked and linear

DNAs at the protein concentration where a mobility

shift of these species was also detected (Fig. 6, lanes 3

and 6). These results validated the gel shift assay and

confirmed that the selective shift of the supercoiled

DNA band results from preferential binding.

To further define the region that is involved in the

preferential binding to supercoiled DNA, we repeated

the assays using a form of human topoisomerase I

reconstituted from a mixture of topo56 and topo6.3

Y723F (Fig. 4A). This reconstituted protein contains

only the core and COOH-terminal domains and com-

pletely lacks the linker region (Fig. 1). When tested in

the gel shift assay, topo56 ⁄ 6.3 Y723F retained a pref-

erence for supercoiled DNA, although the preference

was reduced compared to that of the topo70 Y723F

(Fig. 5B). For example, although only the supercoiled

DNA was shifted by both topo70 Y723F and

topo56 ⁄ 6.3 Y723F at the lowest protein concentration

tested (Fig. 5B, lanes 2 and 7), at the higher protein

concentrations where mainly the supercoiled DNA was

shifted by topo70 Y723F, the reconstituted enzyme

shifted the linear and nicked DNAs as well (Fig. 5B,

in particular, compare lane 4 with lane 9). These

results suggest that an intact linker region is necessary

for the full manifestation of the preference for super-

coiled DNA but, in its absence, the enzyme can still

distinguish to a limited extent a supercoiled from a

nonsupercoiled DNA.

Competition binding assays

To verify these results by an independent method and

to provide a more quantitative measure for the binding

of the various proteins to supercoiled DNA, we

employed a filter binding assay similar to the one we

used previously [37]. Unlabeled nicked and supercoiled

SV40 DNAs were used separately as competitors for

the binding of 3H-labeled nicked SV40 DNA to cata-

lytically inactive (Y723F) mutant forms of topo70. The

competition assays were carried out for topo70 cap-

HKS-E ⁄Y723F and 4cap and the results were com-

pared with those obtained for topo70 ⁄Y723F. For all

three proteins, the competition profile for the like com-

petitor (nicked DNA) exhibited a half-maximum at the

expected 1 : 1 ratio of competitor to labeled DNA

(Fig. 7A, closed symbols), whereas only approximately

one-tenth as much supercoiled competitor was required

to reduce the binding of the labeled nicked DNA to

the 50% level (Fig. 7A, open symbols). The competi-

tion profile of topo56 ⁄ 6.3 Y723F for the supercoiled

DNA showed that the amount of supercoiled DNA

needed to compete to the 50% level was approximately

one-third as much as for the nicked DNA (Fig. 7B).

These results are consistent with the gel shift assays

and confirm that topo70 Y723F, topo70 capHKS-

E ⁄Y723F and Dcap have a strong preference for

supercoiled DNA over nicked DNA, whereas the

reconstituted topo56 ⁄ 6.3 Y723F lacking the linker has

a reduced ability to discriminate supercoiled from

nicked DNA.

Because the above results implicate the linker in the

preference for binding supercoiled DNA, we wanted to

investigate whether the clusters of positively-charged

amino acids in the linker region are required for this

effect. To test this possibility, we generated two

mutant forms of topo70 Y723F, each of which elimi-

nates the positive charges associated with clusters of

basic amino acids within one of the a-helices of the lin-

ker region (a18). The changes in one of the mutant

proteins were K650A ⁄K654A ⁄Q657A and in the

Fig. 6. Gel shift assay with 32P labeled proteins. (A) Agarose gel

shift assay as described for Fig. 5 using 32P labeled topo70 Y723F

and topo70 capHKS-E ⁄ Y723F. Lanes 1 and 4, DNA alone; lanes 2

and 3 contain 1.75 and 3.5 pmol of topo70 Y723F, respectively;

lanes 5 and 6 contain 1.75 and 3.5 pmol of topo70 capHKS-

E ⁄ Y723F, respectively. (B) Autoradiogram of the gel shown in (A).

The mobilities of unshifted supercoiled, linear and nicked DNAs are

indicated along the right side.



second were K679S ⁄K682S ⁄K687S ⁄K689S (Fig. 1).

These proteins are referred to as topo70 linkerKKQ-

A ⁄Y723F and topo70 linker4K-S ⁄Y723F, respectively.

When these proteins were used in the competition

binding assay, the ratio of unlabeled supercoiled com-

petitor to labeled nicked DNA that was required for

half-maximal binding was offset from the ratio for the

nicked or like competitor by the same amount for the

mutants as for the topo70 Y723F protein (Fig. 8A).

The magnitude of this offset was slightly less for the

competition profiles in Fig. 8A compared to that

observed in Fig. 7A because the preparation of unla-

beled supercoiled competitor used in this experiment

contained a slightly higher percentage of nicked mole-

cules (� 20% compared with the previous � 5%, data

not shown). On the basis of these results, we conclude

that the absence of either of these two clusters of basic

amino acid within the linker does not affect the ability

of the protein to preferentially bind supercoiled DNA.

The solvent-exposed region of the core subdo-

main III distal from the Cap represents yet another

Fig. 8. (A) Filter binding assays comparing unlabeled supercoiled

and nicked SV40 DNAs as competitors for 3H-labeled nicked SV40

DNA-binding to topoisomerase variants containing multiple amino

acid changes in the linker domain: topo70 Y723F (nicked competi-

tor, solid squares; supercoiled competitor, open squares); topo70

linker4K-S ⁄ Y723F (nicked competitor, solid triangles; supercoiled

competitor, open triangles); and topo70 linkerKKQ-A ⁄ Y723F (nicked

competitor, solid diamonds; supercoiled competitor, open diamonds,

dashed line). (B) Filter binding assays for topoisomerase variants

containing mutations at exposed lysine residues in the core domain

of the enzyme: topo70 Y723F (nicked competitor, solid diamonds,

supercoiled competitor, open diamonds); topo70 K466-468E Y723F

(nicked competitor, solid squares, supercoiled competitor, open

squares); and topo70 K545-549E Y723F (nicked competitor, solid tri-

angles, supercoiled competitor, open triangles). For topo70 Y723F,

the values plotted are the mean of seven independent determina-

tions and, for the two mutant proteins, the values are the mean of six

independent determinations.

Fig. 7. Filter binding assays comparing unlabeled supercoiled and

nicked SV40 DNAs as competitors for 3H-labeled nicked SV40

DNA-binding to topoisomerase I constructs. (A) The results of the

competition assay for topo70 Y723F (nicked competitor, solid

squares; supercoiled competitor, open squares), topo70 capHKS-

E ⁄ Y723F (nicked competitor, solid triangles; supercoiled competi-

tor, open triangles) and Dcap (nicked competitor, solid diamonds;

supercoiled competitor, open diamonds). (B) Results for the compe-

tition assay for topo56 ⁄ 6.3 Y723F (nicked competitor, solid circles;

supercoiled competitor, open circles).



region of the protein that might provide a binding

interface for a second DNA-binding site. To examine

this possibility, we generated mutant proteins in which

pairs of positively-charged lysine residues within core

subdomain III were changed to glutamates (Fig. 1)

and tested these proteins in the competition binding

assay. As shown in Fig. 8B, the competition profiles of

the nicked competitor DNA for the topo70 K466-

468E ⁄Y723F and topo70 K545-549E ⁄Y723F proteins

are identical to the profile for the control topo70

Y723F protein (Fig. 8B, closed symbols) but, impor-

tantly, the supercoiled DNA did not compete as well

for the binding to the two mutant proteins as it did

for the binding to the control topo70 Y723F protein

(Fig. 8B, compare the open squares and triangles with

the open diamonds). To be certain that these differ-

ences were significant, multiple experiments were per-

formed to determine the mean value for the ratio of

unlabeled nicked to supercoiled competitor required to

reduce binding to the 50% level. For the positive con-

trol topo70 Y723F, this ratio (±SD) was found to be

8.6 ± 3.9 (seven repeats), which is consistent with the

earlier determinations, whereas the corresponding

ratios for topo70 K466-468E ⁄Y723F and topo70

K545-549E ⁄Y723F were 4.1 ± 1.1 and 4.6 ± 1.7,

respectively (six repeats). Using the t-test, these differ-

ences of the ratios for the two mutant proteins from

the control are significant at P < 0.05, and thus the

mutant proteins have a reduced ability to discriminate

supercoiled from nonsupercoiled DNA.

Discussion

Although protein–protein interactions have been impli-

cated in targeting topoisomerase I to supercoiled sub-

strates in vivo [21,24–26], when given a choice of

supercoiled and relaxed substrates in the absence of

other proteins in vitro, the enzyme exhibits a prefer-

ence for binding to the supercoiled DNA [32–37].

Because this intrinsic preference for supercoils is inde-

pendent of the sign of the supercoiling [37,45], it is

likely the DNA feature being recognized by the

enzyme is a DNA node [36], a structural element that

is shared by DNAs with positive and negative super-

coils. In the absence of DNA, the topoisomerase I

protein is a bi-lobed structure that exists in an open

clamp conformation [5]. Upon binding DNA, the

clamp closes around the duplex to form a clearly-

defined channel that interacts with the DNA backbone

over a length of approximately 6 bp (Fig. 1) [8]. The

simplest model to explain node recognition by the

enzyme assumes that, in addition to this well-charac-

terized DNA-binding channel, the protein has a second

DNA-binding region that stabilizes the interaction

with a DNA crossing. Here, we consider four struc-

ture-related hypotheses that could explain node bind-

ing. First, the bent structure of a supercoiled duplex

could be a feature that is recognized by a single topo-

isomerase I protein without the need for a second

DNA-binding site. Second, a topoisomerase I homodi-

mer could provide two DNA-binding sites on the same

protein molecule (Fig. 2A). Third, core subdomain II,

which structurally resembles a homeodomain and is an

exposed feature of the Cap (Fig. 1), could constitute

a second DNA-binding site on the protein. Fourth,

clusters of basic residues in core subdomain III, and

the linker on the side of the protein distal from the

Cap, could mediate DNA-binding at a node.

For some proteins, the preference for binding to

supercoiled DNA is related to the tendency of the

proteins to cause DNA bending. For example, high-

mobility group proteins [44,46–50] and the p53 protein

[40–43,51] preferentially bind supercoiled DNA and, in

both cases, it was shown that the proteins bend DNA.

Moreover, in the case of the high-mobility group pro-

teins, the DNA bending capacity correlates with the

supercoiled DNA-binding [50]. In the crystal structure

of the human topoisomerase I-DNA complex, the

22 bp DNA substrate does not show any bending

deformation and is an almost perfect B-shaped helix

[8]. This observation suggests that the preference of

human topoisomerase I for supercoiled DNA is not

the result of an attraction of the enzyme for bent

DNA.

In a previous study [38], we showed that the

topo70DL form of human topoisomerase I missing

part of the coiled-coil linker domain could form dimers

through a domain swapping mechanism involving the

core and COOH-terminal domains of the two subunits.

We hypothesized that the shortened linker in the

mutant enzyme destabilized the interaction between

the COOH-terminal and core domains, enabling the

COOH-terminal domain of one protein to occupy its

binding site in the core domain of the other protein

and vice versa. Consistent with this suggestion, we

were unable to detect dimerization of free wild-type

enzyme containing the normal length linker [4,38].

However, these results did not rule out the possibility

that dimerization of the enzyme only occurs after the

first molecule of enzyme is already bound to DNA. In

this regard, it was shown that a molecule of topoisom-

erase I that is covalently trapped on DNA after suicide

cleavage recruits another molecule of enzyme to cleave

approximately 13 bp upstream of the trapped enzyme

[52]. Although the basis for dimerization in this case

is unknown, this interaction between two enzyme



molecules is unlikely to mediate node binding because

the second molecule of enzyme is bound to the DNA

immediately adjacent to the one already trapped on

the DNA. For our GST pull-down assay, we deliber-

ately chose an oligonucleotide that was too short to

permit this type of side-by-side contact (total duplex

length 14 bp) to assay for DNA-mediated dimeriza-

tion. Importantly, under these conditions, we show

that a topoisomerase I molecule covalently bound to

DNA after suicide cleavage does not bind another

molecule of the enzyme. These results rule against the

hypothesis that dimerization of topoisomerase I

accounts for the preference of the enzyme for super-

coiled DNA.

In previous studies [36,37] demonstrating a prefer-

ence of topoisomerase I for supercoils, the full length

enzyme was used. In the present study, we demonstrate

that topo70, a form of the enzyme missing residues

1–174 that constitute most of the N-terminal domain,

also preferentially binds supercoiled over relaxed

DNA. This observation rules out this portion of the

N-terminus as a region of the enzyme that provides a

second DNA-binding site involved in node recogni-

tion.

In the present study, we tested whether the homeo-

domain-like region within the Cap of the enzyme (core

subdomain II) constitutes a second DNA-binding site

on the enzyme that mediates the preference for super-

coils (Fig. 2B). Alignment of the sequences of human

topoisomerase I and the Oct-1 homeodomain revealed

three amino acids within core subdomain II of the Cap

that might be expected to interact with the negatively-

charged DNA backbone and form the basis for a sec-

ond DNA-binding site on the enzyme (His266, Lys299

and Ser306) (Fig. 1). Replacing all three of these resi-

dues with a glutamic acid residue or complete deletion

of the Cap region (Dcap) had no effect on the ability

of the resulting proteins to preferentially bind super-

coiled DNA when assayed by either a gel shift assay

or a competition binding assay. These results rule out

the hypothesis that an interaction with a node is medi-

ated by a second DNA-binding site localized to core

subdomain II of the enzyme.

The results obtained in the present study with

respect to topo56 ⁄ 6.3 Y723F, a reconstituted enzyme

completely missing the linker region, reveal that this

form of the enzyme has a reduced preference for

supercoiled DNA compared to the wild-type enzyme.

In a study carried out prior to the availability of the

co-crystal structure of topoisomerase I [8], we exam-

ined the substrate binding preference of topo58, a form

of the protein now known to contain the core domain

and one third of the linker region (residues 175–659)

(Fig. 4). At the time, we concluded that the binding

properties of a COOH-terminal truncation of topo70

missing the last 106 amino acids (topo58) was similar

to those of topo70 Y723F, but a re-examination of

these older data [37] reveals that, similar to the recon-

stituted topo56 ⁄ 6.3 Y723F investigated in the present

study, topo58 alone exhibits a reduced preference for

supercoiled DNA. Taken together, these observations

suggest that an intact linker region of the enzyme is

necessary for the full manifestation of the preference

for supercoils. It is noteworthy that the elimination of

either of the clusters of basic amino acids within the

linker region (Fig. 1) does not affect the preference of

the enzyme for supercoiled DNA. Our interpretation

of this finding is that the contribution of the linker to

node binding relates to how the linker influences local

protein structure rather than via the formation of a

second DNA-binding site that makes direct amino acid

side chain contacts with the DNA backbone. In this

regard, it is noteworthy that the linker region is not

only remarkably flexible [53], but also mutations that

affect its flexibility can influence the structure of the

protein at distant sites [54].

Unlike the linker where the evidence rules out a

direct interaction between basic amino acids and the

DNA in node binding, mutational studies within core

subdomain III indicate that reversing the charge on

pairs of basic, surface-exposed amino acids (K466 ⁄K468 and K545 ⁄K549) (Fig. 1) has a significant

impact on the preferential binding of the topoisomer-

ase to supercoiled DNA. Notably, these lysine residues

are conserved in the topoisomerase I protein in most

higher eukaryotes. (Fig. 9). These results suggest that

basic amino acids within core subdomain III contrib-

ute to node binding through direct contacts with the

DNA. The observation that the pairwise mutation of

these lysines to glutamic acid only partially eliminates

the preference for supercoiled DNA suggests that other

residues within this domain also contribute to the for-

mation of a second DNA-binding region in the pro-

tein. Taken together, the results obtained in the

present study strongly support the node binding

hypothesis to explain the preference of human topo-

isomerase I for supercoiled DNA [36].

The related type IB topoisomerase from vaccinia

virus also preferentially binds to node structures in

duplex DNA [36,55]. In a recent study, it was found

that the vaccinia topoisomerase binds cooperatively to

DNA to form long filaments in a reaction that is

nucleated by the formation of an intramolecular node

on DNA [56]. Although it is not known whether the

initial node binding event involves a monomer or

dimer of the enzyme, if a monomer is sufficient for



node binding, then a second DNA-binding region must

exist within the viral enzyme, as we have suggested

above for the human enzyme. If this were to be the

case, it is noteworthy that the structural similarity

between the human and vaccinia enzymes is confined

to the region referred to as subdomain III in the

human enzyme [57,58] and that two of the residues in

the human enzyme that we have implicated in node

binding (Lys466 and Lys549) are conserved in the viral

enzyme (Fig. 9). Thus, it is conceivable that the struc-

tural basis for node binding by the two enzymes is

similar.

Experimental procedures

Generation of baculovirus constructs expressing

mutant proteins

pFASTBAC1-topo70 K299E ⁄ S306E, pFASTBAC1-topo70

K299E ⁄S306E ⁄Y723F, pFASTBAC1-topo70 H266E ⁄K299E ⁄S306E and pFASTBAC1-topo70 H266E ⁄K299E ⁄ -S306E ⁄Y723F were generated as follows. The plasmid

pGEX-topo70 [14] was the template for making site-directed

mutations using the QuickChange� mutagenesis kit from

Stratagene (La Jolla, CA, USA). A pair of oligonucleotides

containing the nucleotide changes for replacing Lys299 and

Ser306 with glutamic acid was used to generate pGEX-topo70

K299E ⁄S306E. The resulting plasmid and another set of

oligonucleotides that changed His266 to glutamic acid were

similarly used to generate pGEX-topo70 H266E ⁄K299E ⁄S306E. Both pGEX-topo70 K299E ⁄ S306E and pGEX-

topo70 H266E ⁄K299E ⁄ S306E were digested with NdeI and

NheI and the fragments that contain the point mutations were

purified and used to replace the corresponding fragments in

NdeI andNheI digested pFASTBAC1-topo70 [59]. The result-

ing constructs, pFASTBAC1-topo70 K299E ⁄ S306E and

pFASTBAC1-topo70 H266E ⁄K299E ⁄ S306E, were used to

generate baculoviruses with the Bac-to-Bac system (Invitro-

gen, Carlsbad, CA, USA) in accordance with the manufac-

turer’s instructions. Recombinant baculovirus infection of Sf9

cells was used to produce proteins referred to as topo70 cap-

KS-E and topo70 capHKS-E, respectively. These same two

pFASTBAC1 constructs were also digested with NdeI and

PpuMI and the fragments containing the mutations were puri-

fied by gel electrophoresis. The isolated fragments were used

to replace the corresponding fragment of pFASTBAC1-

topo70 Y723F [59] that had been digested with the same two

restriction enzymes to generate pFASTBAC1-topo70 K299E ⁄S306E ⁄Y723F and pFASTBAC1-topo70 H266E ⁄K299E ⁄S306E ⁄Y723F. The catalytically inactive proteins expressed

in baculoviruses from these two constructs are referred to as

topo70 capKS-E ⁄Y723F and topo70 capHKS-E ⁄Y723F,

respectively.

Starting from pFASTBAC1-topo70, two sets of oligonu-

cleotide pairs were used to introduce clustered mutations in

the linker-coding region to produce pFASTBAC1-topo70

K650A ⁄K654A ⁄Q657A and pFASTBAC1-topo70 K679S ⁄K682S ⁄K687S ⁄K689S using the QuickChange method

Fig. 9. Sequence alignment within core subdomain III of representative eukaryotic members of the type IB subfamily of topoisomerases.

Human, Drosophila, Saccharomyces cerevisiae and vaccinia virus topoisomerase I sequences were aligned using CLUSTALW2 software avail-

able online from the European Bioinformatics Institute (http://www.ebi.ac.uk/Tools/clustalw2/). The homology of the bacterial type IB

enzymes to these eukaryotic members of the family was too weak for them to be included in the alignment. The key conserved active site

residues Arg488 and Lys532 (human numbering) are marked with closed circles. The open circles identify the residues in the human enzyme

(Lys466, Lys468, Lys545 and Lys549) that are implicated in the preferential binding to supercoils.



described above. Starting with these two pFASTBAC1 con-

structs, an oligonucleotide containing the codon change that

generates the Y723F mutation was subsequently used to pro-

duce pFASTBAC1-topo70 K650A ⁄K654A ⁄Q657A ⁄Y723F

and pFASTBAC1-topo70 K679S ⁄K682S ⁄K687S ⁄K689S ⁄Y723F. The catalytically inactive versions of the two proteins

produced in baculoviruses by these constructs are referred to

as topo70 linkerKKQ-A ⁄Y723F and topo70 linker4K-S ⁄Y723F, respectively. Similarly, starting with pFASTBAC1-

topo70 ⁄Y723F, oligonucleotide based mutagenesis was used

to introduce two pairs of clustered mutations into the coding

region for the core domain of topoisomerase I to produce

pFASTBAC1-topo70 K466E ⁄K468E ⁄Y723F and pFAST-

BAC1-topo70 K545E ⁄K549E ⁄Y723F. The mutant proteins

produced in baculoviruses by these constructs are referred to

as topo70 K466-468E ⁄Y723F and topo70 K545-549E ⁄Y723F, respectively. A construct of the topo70 K466E ⁄K468E mutant containing the wild-type Tyr723 codon

(pFASTBAC1-topo70 K466E ⁄K468E) was produced by

replacing a NheI-EcoRI fragment with the corresponding

fragment from a wild-type clone. The Tyr723-containing ver-

sion of pFASTBAC1-topo70 K545E ⁄K549E was toxic to

E. coli and therefore baculoviruses expressing this particular

mutant protein could not be obtained.

Site-directed mutagenesis, as described above, was used

to introduce a stop codon after residue 639 in pGEX-

topo70. The resulting plasmid was digested with Eco0109I

and NheI. The fragment containing the stop codon was

purified and used to replace the corresponding fragment of

pFASTBAC1-topo70 that had been digested with the same

two restriction enzymes to generate pFASTBAC1-topo56.

All the mutants were confirmed by dideoxy sequencing.

The generation of Dcap and topo31 has been described pre-

viously [39]. The structures of the constructs described

above are illustrated in Fig. 4A.

Expression and purification of proteins

Expression and purification of GST-topo70 has been

described previously [14]. Topo70, topo70 capKS-E, topo70

capHKS-E, topo70 capKS-E ⁄Y723F, topo70 capHKS-

E ⁄Y723F, topo70 linkerKKQ-A, topo70 linker4K-S,

topo70 linkerKKQ-A ⁄Y723F, topo70 linker4K-S ⁄Y723F,

topo70 K466-468E, topo70 K466-468E ⁄Y723F, topo70

K545-549E ⁄Y723F and Dcap were expressed and purified

as described previously for topo70 [7]. Topo56 ⁄ 6.3 Y723F

was purified by the procedure described previously for

topo58 ⁄ 6.3 [14]. The purification of top31 has been

described previously [39]. SDS-PAGE analysis of the puri-

fied proteins is shown in Fig. 4B. The DNA-binding assays

were carried out with the various mutant proteins contain-

ing the Y723F inactivating mutation but, to ensure that the

mutations did not affect the overall fold of the protein, the

mutant proteins containing the active site Tyr723 were also

purified and assayed for plasmid relaxation activity. In all

cases tested, the proteins containing Tyr723 were found to

retain almost full wild-type activity. As explained above, we

were unable to obtain the topo70 K545E ⁄K549E mutant

protein to test whether it was enzymatically active.

GST pull-down assay for dimerization

Fifteen picomols of purified GST-topo70 were incubated in

the absence or presence of a two-fold molar excess of an oligo-

nucleotide suicide substrate CP14 ⁄CL25 (5¢-GAAAAAAGA

CTTAG ⁄ 5¢TAAAAATTTTTCTAAGTCTTTTTTC-3¢) [60]

in 10 mm Tris-HCl, pH 7.5, 100 mm KCl, 1 mm EDTA,

0.1 mgÆmL)1 BSA for 60 min at 23 �C. An equimolar amount

of purified topo70 was added to the samples and the mixtures

were incubated at 23 �C for 30 min. Each reaction was added

to a 15 lL packed volume of glutathione Sepharose 4B beads

(Amersham Biosciences Corp., Piscataway, NJ, USA) and

mixed by rotation for 2 h at 23 �C. As a negative control,

topo70 alone was incubated with either purified GST or the

Sepharose beads. Reactions were centrifuged for 2 min at

8000 g, and the supernatant containing unbound protein was

discarded. The pelleted beads were washed one time with

1 mL of the same buffer, pelleted and resuspended in SDS gel

loading buffer, and boiled. The samples were fractionated

with size standards by 8% SDS–PAGE as described previ-

ously [39]. Proteins were visualized by Coomassie blue stain-

ing and photographed using the AlphImager IS-2200 (Alpha

Innotech, San Leandro, CA, USA) digital imager.

Generation of nicked and linear DNA

Unlabeled and 3H-labeled supercoiled SV40 DNA, pre-

pared as described previously [37], and pBluescript

KSII(+) DNA were relaxed by topo70 in relaxation buffer

(150 mm KCl, 1 mm EDTA, 1 mm dithiothreitol, 10 mm

Tris-HCl, pH 7.5, 50 lgÆmL)1 BSA), and phenol ⁄ chloro-form extracted before ethanol precipitation. The relaxed

DNAs (100 lgÆmL)1) were treated with BamH1 (1000

unitsÆmL)1) in a buffer containing 150 mm NaCl, 10 mm

Tris–HCl, pH 7.9, 10 mm MgCl2, 1 mm dithiothreitol,

100 lgÆmL)1 BSA, and 100 lgÆmL)1 ethidium bromide.

After incubation at 37 �C for 1 h, the majority of the DNA

was nicked by the restriction enzyme under these conditions

[61]. The reaction was phenol ⁄ chloroform extracted, etha-

nol precipitated, and stored in TE buffer (10 mm Tris-HCl,

pH 8.0, 1 mm EDTA) at 4 �C. Linear DNAs were gener-

ated by digestion with BamH1 under the same conditions

as above, without the addition of ethidium bromide.

Labeling proteins with protein kinase C

Fifty picomols of the indicated mutant topoisomerase I

proteins were incubated with 20 ng of protein kinase C

(Upstate Biotechnology, Inc., Lake Placid, NY, USA) and



20 lCi of [c-32P]ATP (3000 CiÆmmol)1) in 20 lL of labeling

buffer (20 mm Hepes, pH 7.4, 10 mm MgCl2, 0.5 mm

CaCl2, 50 ng of phosphatidylserine, 2 lL of diacylglycerol).

The reactions were incubated at 30 �C for 30 min in amber

Eppendorf tubes because of the light sensitivity of both

phosphatidylserine and diacylglycerol. Labeled proteins

were stored at 4 �C.

Agarose gel shift assay

Equal molar amounts of supercoiled, linear, or nicked

pBluescript KSII(+) DNA (0.08 pmol of each) were incu-

bated with the indicated amounts of the various protein

constructs in 10 lL of reaction buffer (10 mm Tris–HCl,

pH 7.5, 50 mm KCl, 1 mm EDTA, 1 mm dithiothreitol) at

23 �C for 20 min, and 2.5 lL of 50% glycerol was added to

the reaction before loading on a 1% agarose gel. The gel

was run with 0.5 · TBE buffer (45 mm Tris base, 45 mm

boric acid, 1 mm EDTA) at 4 �C. Bands were visualized

with a UV illuminator after staining with ethidium bro-

mide. For the gel shift assay with labeled proteins, the gel

was stained with ethidium bromide, visualized under a UV

illuminator, and then dried before exposure to film.

Filter binding assay

Filter binding assays were carried out by incubating

0.08 pmol of nicked 3H-labeled SV40 DNA with 0.32 pmol

of topo70 Y723F, topo70 capKS-E ⁄Y723F, topo70

capHKS-E ⁄Y723F, topo70 linkerKKQ-A ⁄Y723F, topo70

linker4K-S ⁄Y723F or 0.64 pmol of Dcap in 10 lL of reac-

tion buffer at 23 �C for 20 min. Ten microliters of reaction

buffer containing the indicated amounts of either unlabeled

supercoiled or unlabeled nicked SV40 DNA was added to

the reaction as competitor DNAs. The reactions were fur-

ther incubated at 23 �C for 30 min. The 20 lL reactions

were then applied to 0.45 lm, 13 mm nitrocellulose filter

papers (D25AGR; Whatman, Maidstone, UK), which had

been soaked in H2O, positioned on a suction apparatus and

filtered at the rate of 3–4 mLÆmin)1. The filters were imme-

diately washed with 0.8 mL of buffer containing 10 mm

Tris–HCl, pH 7.5, 50 mm KCl, dried and immersed in

5 mL of toluene ⁄Omnifluor (Perkin Elmer, Boston, MA,

USA) (4 gÆL)1) scintillation fluid before counting in a

Beckman LS 3801 scintillation counter (Beckman Coulter,

Fullerton, CA, USA).

Acknowledgements

This work was supported by Grants GM60330 and

GM49156 from the National Institutes of Health. We

thank Matthew Redinbo and Wim Hol for their assis-

tance with the structural comparison of core subdo-

main II of human topoisomerase I with homeodomains.

We gratefully acknowledge Sharon Schultz and Heidrun

Interthal for critically reading the manuscript.

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Mutational analysis of the preferential binding of human topoisomerase I to supercoiled DNA

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