Antonio Rodrıguez-Campos- DNA Knotting Abolishes in Vitro Chromatin Assembly

8/3/2019 Antonio Rodrguez-Campos- DNA Knotting Abolishes in Vitro Chromatin Assembly

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D N A Kn o t t i n g A b o l is h e s i n V i t r o C h r o m a t i n A s s e m b l y *

(Received for publication, October 30, 1995, and in revised form, March 27, 1996)

Anton io Rodr gue z-Campo s

From the Departamento de Biologa Molecular y Celular, Centro de I nvestigacion y Desarrollo, CS IC, J ordi Girona, 18E-08034, Barcelona, Spain

T o p o l o g i c a l k n o t s c a n b e f o r m e d i n v i t r o b y i n c u b a t -

i n g c o v a l e n t l y c l o s e d d o u b l e s t r a n d e d D N A a n d p u r i f i e d

t o p o i s om e r a s e I I f r o m t h e y e a s t S a c c h a r o m y c es c e r e vi -

s i a e i n a n A T P -d e p e n d e n t r e a c t i o n . Kn o t t i n g p r o d u c t i o n

requires a starting enzyme/DNA mass ratio of 1. Analysis

o f k n o t t e d D N A w a s c a r r ie d o u t b y u s i n g b o t h o n e - a n d

t w o - d i m e n s i o n a l a g a r o s e g e l e l e c t r o p h o r e s i s . T h e k n o t s

g e n e r a t e d a r e e f fi c i e n t ly u n t i e d , a n d g i v e r e l ax e d D N A

r i n g s , b y c a t a l y t i c a m o u n t s o f t o p o i s o m e r a s e I I, b u t n o t

b y t o p oi s om e r as e I . T im e c o u rs e a n a ly s is s h o w s t h e

k n o t t i n g f o r m a t i o n o v e r r e l a x e d a n d s u p e r c o i l e d D N A .

W h e n s u p e r c o i l e d D N A w a s u s e d a s a s u s b t r a t e , k n o t s

a p p e a r i m m e d i a t e l y w h e r e a s n o t r a n s i e n t r e l a x e d r i n g s

w e r e o b s e r ve d . T h e c e l l-f re e e x t r a c t f r o m X e n o p u s o o -c y t e s S - 15 0 c a n n o t a s s e m b l e n u c l e o s o m e s o n k n o t t e d

D N A t e m p l a t e s a s r e v e a l e d b y t o p o l o g i c a l a n d m i c r o c o c -

c a l n u c l e as e a n a ly s is . N e v e r t he l e ss , t h e p r e se n c e o f

k n o t t e d D N A t e m p l a t e s d o e s n o t i n h i b i t t h e a s s e m b l y

o v e r t h e r e la x e d p l as m id . F i n al ly , a p r e tr e a tm e n t o f

k n o t t e d D N A w i t h t r a c e a m o u n t s o f t o p o i s o m e r a s e I I

b e fo r e t h e a d d it i on o f t h e S -1 50 y i e ld s a c a n o ni c al

m i n i c h r o m os o m e a s s e m b l e d i n v i tr o. T a k in g i n t o a c -

c o u n t t h e s e r e s u l t s , I s u g g e s t a m e c h a n i s m o f c h r o m a t i n

a s s e m b l y r e g u l a t i o n d i r e c t e d b y t o p o i s o m e r a s e I I .

DNA topoisomerases are a kind of enzymes which, by chang-

ing the topological st ructure of the DNA by means of tra nsient

single (in class I) or double (in class II) st randed breaks, pro-duce different topological forms called topoisomers (1, 2). These

enzymes can easily knot/unknot, catenate/decatenate, and su-

percoil/relax DNA molecules in vitro (36). Their critical r ole in

processes a s replication, t ran scription, recombination, r epair,

and chromosome condensation have been established in vivo (2,

710). Protein was the first DNA topoisomerase discovered

(11); t his ba cte r ia l e nz ym e f a lls into th e cla ss I . The cla ss

II-enzyme bacterial gyrase is, u ntil now, the only topoisomer-

ase capable t o introduce negat ive supercoiling over a covalently

closed relaxed DNA in an ATP-driven process (6, 12, 13).

A broad ra nge of organisms and sources, including viruses,

show activities that change the topological state of the DNA.

Concomitantly with the fact of breaking and rejoining one or

two strands in the DNA molecule, topoisomerases II change thelinking number in steps of two (3, 14), whereas class I alters

this topological parameter in steps of one (15).

Knotting of either single or double stranded DNA rings can

be carried out by both classes of topoisomerases (3, 14, 16) by

breaking and rejoining one or two strands. The ATP depend-

ence on the knotting activity is controversial: topoisomerase II

knotting of circular duplex DNA, using purified enzyme from

Drosophila embryos, is greatly enhanced by ATP (14). How-

ever, t opoisomerase II from T4 bacteriophage can knot either

covalently closed or nicked DNA rings without ATP (3, 17).

Nevertheless, either the viral or the eukaryotic enzyme require

in the presence of ATP or not, to be present in larger amounts

(3, 14, 17, 18) than necessary to achieve DNA relaxation (4, 5).

Recently, an ATP-dependent enzymatic mechanism for yeast

topoisomerase II has been reported (19, 20; reviewed in Ref.

21). Briefly, th e enzyme, a homodimer of a 170-kDa p olypeptide

(4), forms an open clamp which traps a DNA duplex; then, asecond DNA duplex enters the open gate, which is closed when

ATP is bound to t he pr ote in. S im ulta ne ously to th e double

str a nd br e a ka ge of the f ir st DNA duple x, the se c ond duple x

cr osse s the br e a k a n d e xits th r ough a se cond pr ote in ga te

opposite to the e ntr a nc e ga te . The pr oce ss f inishe s a fte r the

resealing of the broken duplex, which exits through the first

gate depen ding on ATP hydr olysis. As class II DNA topoisomer-

ases are evolutionary related, the mechanism reported can be

extensive t o all su ch enzymes.

In th is study I sh ow that t he eukar yotic DNA topoisomerase

II purified from Saccharomyces cerevisiae can efficiently knot

any topologically closed DNA template, either relaxed or su-

percoiled. This ATP-driven reaction depends on the addition of

stoichiometric amounts of enzyme. When topoisomerase II is

e m ploye d a t a c a ta lytic le ve l, both unknotting a nd r e la xinga ctivitie s over knotte d or supe r coile d DNA te m pla te s a r e

achieved.

Fina lly, the a ddition of the cell-free extra ct S-150, capa ble of

assembly of chromatin over any topologically linked DNA tem-

plate (22, 23), fails to form periodically arranged nucleosomes

whe n t he DNA te m pla te is knotte d. None the less, a pr e tr e a t-

ment with trace amounts of topoisomerase II before the onset of

the chromatin assembly process permits the nucleosome for-

m a tion ove r knotte d DNA. The se r e sults sugge st tha t DNA

topoisomerase II may modulate, via knotting/unknotting, the

assembly of chromatin.

MATERIALS AND METHODS

En z yme s a n d DNA Plasmid DNA (pUC19) was purified following

standard procedures including CsCl buoyant density gradients.SV40 DNA wa s kindly provided by Dr. F . Azor n (CID-CSIC). Yeast

DNA topoisomerase II was a generous gift from Dr. J. Roca (Harvard

University); the enzyme had a purity degree 99% and a ctivity 70%.

A working stock solution was made diluting 1 l (containing 7 g) of

enzyme stock (kept at 70 C) in 70 l of 50 m M Tris-HCl, pH 8.0, 500

m M KCl, 5 m M dithiothreitol, 100 g/ml bovine seru m a lbumin, and 50%

glycerol (18). Calf thymus DNA topoisomerase I was purchased from

GIBCO-BRL.

Knotting Reactions K n ottin g r e a ction s w e re c a rried o u t in a m ix -

ture containing 20 m M H E P E S p H 7 . 5 , 2 mM magnesium chloride, 0.1

m M EGTA, 0.2 m M ATP, 20 m M KCl, and a topoisomerase II/DNA mass

ratio ranging from 1 to 3. The final concentration of closed circular DNA

(either relaxed or supercoiled) was currently 10 ng/l a n d th e in c u b a -

tion temperat ure was always 30 C. The time of reaction varies between

* The costs of publication of this a rticle were defrayed in part by the

payment of page char ges. This ar ticle must therefore be hereby markedadvertisement in a ccordance with 18 U.S.C. Section 1734 solely to

indicate this fact. T o w h o m c orre sp on d e n ce sh ou ld b e a d d re ssed . P re se n t a d d re ss:

D e pt. d e P a to lo g a Molecular y Terapeutica, I.I.B.B. CSIC, J ordiGirona, 18-26; E -08034 Barcelona, Spain. Tel.: 34-3-400-61-38; F ax:

34-3-204-59-04.

THE J OURNAL OF BIOLOGICAL CH EMISTRY Vol. 271, No. 24, Issue of June 14, pp. 1415014155, 1996 1996 by The American Society for Biochemistry a nd Molecular Biology, Inc. Printed in U.S.A.

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1 h and overnight; reactions were stopped by addition of one-fourth of

the mixture volume of 2.5% Sarkosyl, 100 m M EDTA; samples were

deproteinized using SDS, proteinase K, and phenol extraction.

Unknotting/ relaxing Reactions Unknottin g/relaxing reactions with

topoisomerase II were performed at the same ionic condition than the

k n o ttin g re a c tion b u t u sin g a to po isom e ra se II/D N A ra tio from on e -

tenth to 1/50 and a KCl concentration of 150 m M.

Electrophoresis1% agarose-TBE gels were performed in h orizontal

gel slabs immersed in the electrophoresis buffer either in one or two

dimensions.

Electrophoresis in the first dimension was usually performed for 16 hat 6065 V. For the second dimension the gel was soaked in the TBE

buffer plus an aliquot of a fresh solution of chloroquine (10 g/l),

giving a final concentration of 1.4 M, a n d e q u ilib ra te d for 6 8 h . T h e

equilibrated gel was shifted 90 from its original position in the first

d im en sion a n d re -ele ctro p h ore se d d u rin g 1 6 h a t 6 5 V . In o rd er to

remove chloroquine, the gel was extensively washed with deionized

w a te r. F in a lly i t w a s b lotte d a n d h y brid ize d w ith th e a p p rop ria te ra -

diolabeled probe.

Chromatin Assembly Reactions C h rom a tin a sse m b ly re a ction s

were carried out with t he cell-free extract S-150 (21, 22). In the exper-

iment described in Fig. 5, 3 g of either relaxed or relaxed plus kn otted

pUC19 DNA were added t o a mixtur e conta ining 20 m M HEPES pH 7.5,

5 m M magnesium chloride, 1 m M EGTA, 3 m M ATP, 10% glycerol, 40 m M

disodium creatine phosphate, 10 m M glycerophosphate, a nd 600 l of

S-150 in a final volume reaction of 1 ml. Samples were incubated at

3 7 C; a t 1 5 , 3 0 , 6 0 , 1 2 0 , a n d 2 4 0 m in o f re a ction , on e -fifth o f th e

star ting reaction was r emoved and stopped by addition of 50 l of 2.5%Sarkosyl, 100 m M EDTA, followed by deproteinization with SDS (final

concentration: 0.2%) and proteinase K (1 g/l, final concentrat ion).

After a 1-h incubation at 65 C, a phenol extraction was performed, and

DNA was pu rified and electrophoresed.

T h e e x p erim e n t sh o wn in F ig . 6 w a s p e rform e d w ith 8 0 0 n g o f

knotted pUC19 DNA; 400 ng of knotted DNA were untied in a m ixture

containing 20 m M H E P E S p H 7 . 5 , 2 m M magnesium chloride, 0.1 m M

EGTA, 0.2 m M ATP, 150 m M KCl, and a bout 20 ng of topoisomerase II

in a 3 0 -l final volume reaction. In the mock-incubated reaction with

400 ng of knotted DNA, topoisomerase II was t he only reagent omitt ed.

After 1 h at 30 C, concentrated solutions of HEPES pH 7.5, magnesium

chloride, EDTA, EGTA, ATP, creatine phosphat e, creatine kina se, and

glycerol were added to raise the ionic conditions described above, to-

gether with 150 l of S-150 in 250 l of final reaction volume. Those

mixtures were incubated 6 h at 37 C. For the topological assay, two

a liqu o ts o f e a ch re a ction w e re r e m ov ed a n d p ro ce ssed a s d e scrib ed

above. The remaining aliquots were digested with 50 units of micrococ-cal nuclease (Boehringer Mannheim) in 3 m M calcium chloride for the

indicated t imes at room t emperat ure. Aliquots were removed and proc-

essed as described.

RESULTS

Whe n a r e la xe d pUC 19 pla sm id DNA wa s inc uba te d with

increasing amounts of yeast topoisomerase II and the exten-

sively deproteinized samp les were electrophoretically resolved,

an emergent distribution of bands with progressively higher

mobilities appears (Fig. 1). At topoisomerase II/DNA ma ss

r a t i o of 1 2, t h e h e a d o f t h is l a dd er r u n s fa s t er t h a n t h e

covalently closed, supercoiled DNA (Fig. 1, lanes 4, 5, a n d 7),

showing a higher compactness. According to previous results

(3, 17), each rung of the ladder corresponds to a knot with a

different number of crosses (nodes). It is noteworthy that poorsta ining wa s shown by knots with a high num be r of c r osse s

when ethidium bromide was employed; on the opposite, this

e ffe ct doe s not a ppe a r whe n a r a diola beled pr obe wa s use d

instead of the colorant. Likely, th e high degree of compaction

can act as a barrier for the dye intercalation (compare Figs. 1

and 4).

Under these experimental conditions, the knotting reaction

is str ic tly de pe nde nt on ATP sinc e in i ts a bse nc e , I did not

obtain a detectable pr esence of th ose DNA forms (not shown),

in good a greement with previous data (14).

The t opologica l sta te of th e knots wa s fur the r a sse sse d by

two-dimensional agarose gel electrophoresis (24). Electro-

phor e sis in the se cond dim e nsion wa s c a r r ie d out a fte r the

equilibration of the gel in the electrophoresis buffer containing

chloroquine. F ig. 2 illustr ates the two-dimensional electro-

phoresis for knotted pUC19 DNA. Several set s of spots can be

distinguished. The topoisomers are resolved as discrete spotsalong a regular curve (depicted with asterisks in th e scheme of

Fig. 2B ) while knots display t wo different distributions, a per-

fect dia gonal cont ainin g the n icked popula tion of knots (18) and

a cur ve d distr ibution which cor r e sponds to the cova le ntly

closed knots (diagrammed as dark a n d whit e circles, respec-

tively, in Fig. 2B ). Both distributions converge at the right end

of the dia gona l be c a use the c a pa bili ty of the c hlor oquine to

discriminate between both nicked and covalently closed knot

distr ibutions de cr e a se s pr ogr essive ly a s the ir node num be r

increases.

In order to gain further insight into the characterization of

knots, I performed the experiment shown in Fig. 3, where equal

amounts of knotted pUC19 DNA (Fig. 3, lane 3) were tr eated in

two different reactions with topoisomerase I and II under the

experimental conditions described (see Materials and Meth-

ods) t ogether with supercoiled SV40 DNA (Fig. 3, l a n e 1)

added at the onset of the reaction as relaxation internal control.

Both topoisomerase I and II were working properly, since their

were able t o relax th e negat ively supercoiled SV40 DNA (Fig. 3,

lanes 4 a n d 5, respectively). However, while topoisomerase II

c a n r e la x the supe r c oile d S V40 DNA a nd untie the knotte d

pUC19, topoisomerase I is able to relax the internal control but

fails to unknot the knotted pUC19. These results exclude the

possibility that any topological form other than knots might

ha ve be e n f or m e d whe n topoisom e r a se I I a nd c ir c ula r DNA

were incubated at a mass ratio close to 1.

Previous data have suggested that knotting formation using

T4 bacteriophage topoisomerase II requires a supercoiled DNA

a s substr a te r a the r tha n the nic ke d or r e la xe d f or m s ( 3, 17) .However, with large amounts of topoisomerase II from Dro-

sophila embryos, knotted DNA can be generated over either

covalently closed circular DNA or it s n icked, r elaxed form (14).

Fig. 4 displays th e t opological forms observed during the time

course of two different knotting reactions with relaxed (Fig. 4,

lanes 15 a n d lane 12, r e la xed substr a te be for e the knotting

reaction) or supercoiled (Fig. 4, lanes 711 a n d la n e 6 , supe r -

coiled subst rate before th e k notting reaction) pUC19 DNA.

Knot format ion is very fast; in th e experiment shown in Fig. 4,

the earliest sampling, 15 min after the onset of the reaction,

a lr e a dy shows a knot la dder a lm ost undistinguisha ble fr om

samples removed after longer incubation times, especially over

the r e la xe d te m pla te r a the r tha n those obta ine d ove r the su-

percoiled form. A small, but significant, amount of supercoiled

F IG . 1. R e l a x e d D N A i s k n o t t e d b y a d d i t i o n o f s t o i c h i o m e t r i ca mo u n ts o f to p o is o me r a s e I I . Pur ified topoisomerase II was added tothe knotting r eaction containing 200 ng of relaxed pUC19, incubated at30 C for 1 h, deproteinized, and the DNA was purified and loaded in 1%

agarose-TBE slab gel. Lanes 25, incubation made with 0, 0.07, 0.14,0.21, and 0.42 g of topoisomerase II, respectively; lane 1, 200 ng of

relaxed pUC19; l a n e 6 , 50 ng of linearized pUC19; l a n e 7 , 2 0 0 n g o f supercoiled pUC19; I, II , a n d II I denotes the electrophoretic mobilities

of supercoiled, relaxed, and linearized forms of pUC19, respectively;white bar depicts the knot distribution.

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D N A a p pe a r s t o b e r e s is t a n t t o k n o t t in g e v en a ft e r 4 h of

incubation (Fig. 4, lane 11) . I t should be note d tha t the knot

formation over supercoiled DNA is as fast as over the relaxed

form. Moreover, part ially super coiled DNA topoisomers a re n ot

seen (see Fig. 4, lanes 611), suggestin g th at super coils become

k n ot s in on e s t ep w it h ou t r ela xe d i nt e rm ed ia t es (s ee

Discussion).

Extracts from Xenopus eggs and oocytes can form regularly

spaced nu cleosomes on circular , topologically linked DNA in a n

ATP-driven process (22, 23, 25, 26): when a relaxed DNA is

added t o those extra cts it becomes su percoiled concomitan tly to

the assembly of nucleosomes (25, 27). Thus, the measuremen t

of the superhelix density reflects th e actua l nu mber of nucleo-

somes loaded on the DNA ring (26).

To test if the knotted DNA could assemble periodically ar-

ranged n ucleosomes, equal am ounts of either r elaxed or k not-

ted (but conta ining a fraction of relaxed DNA) pUC19 DNA

were incubated in two separat ed chromatin assembly reactions

with the cell-free extract from Xenopus oocytes S-150. Fig. 5

depicts the topological changes during the time. While both

relaxed (Fig. 5, lanes 16) and unknotted (relaxed) fraction

c onta ine d in the knotte d m ixtur e ( F ig. 5, la n e s 9 1 4 , w h ite

circles) become progressively supercoiled by nucleosome load-

i n g, t h e k n ot t e d D N A (w h ite d a s h es), which r uns e le ctr o-

phoretically in between t he topoisomers and remains u naltered

after th e assembly process. The experiment addresses how th e

knotted fraction is resistant to assembly but does not inhibit

this process over th e u nkn otted (relaxed) fraction.

It is remarkable the very low levels of endogenous topoi-

somerase II activity detected in crude oocyte extracts which is

pelleted during the preparation of the S-150 (23, 28, 29) and, in

contr a st, the str ong topoisom e r a se I a ctivity of t his e xtr a c t

which relaxes completely in a few minu tes after a supercoiledDNA exogenously was added at the beginning of the chromatin

assembly; over t his en dogenously relaxed template occurs the

nucleosome deposition, giving a negatively supercoiled DNA

after the deproteinization of the sample (not shown) (Ref. 25

and references th erein).

An a dditiona l que stion is whe the r the ne ga tive e f fe ct of

knotte d DNA substr a te on c hr om a tin a sse m bly c ould be r e -

ve r se d or not by untying the te m pla te with tr a c e a m ounts of

topoisomerase II immediately before the addition of the S-150.

Fig. 6 (to p) depicts the design of the experiment. Supercoiled

pUC19 DNA was knotted by means of stecheometric amounts

of topoisomerase II under standard conditions; the deprotein-

ized sam ple was then divided into two halves and incubated a t

30 C for 1 h in two diffe r e nt r e a ctions, e ithe r with a tr a c e

F IG . 2. Ele c tr o p h o r e tic c h a r a c te r iz a tio n o f k n o tte d DNA u s in gt w o - d i m e n s i o n a l g e l s . A, 1 g of covalently closed relaxed pU C19 wask n o tte d a s d e scrib ed u n d e r Ma te ria ls a n d Me th od s. A fte r c a refu lpurification, th e DNA s ample wa s electrophoresed in 1% agarose-TBE

g e l a t 6 5 V d u rin g 1 6 h , so a k e d in th e ru n n in g b u ffe r w ith 1 . 4 Mchloroquine for 68 h, shifted 90 from its original position, and re-

electrophoresed in the second dimension for 16 h at 65 V, blotted to afilter, an d hybridized with a pUC19 probe. B , scheme from A . Indicated

are nicked circle (star), n ick e d k n o ts (black dots), covalent ly closedk n o ts (white dots), and supercoils (asterisks); for illustra tive purposes,

drawings of three node knots (trefoil) are shown.

F IG . 3. E n z y m a t i c c h a r a c t e r i za t i o n o f k n o t t e d D N A b y t o p o i -s o me r a s e I a n d I I . 300 ng of relaxed pUC19 were knotted and, afterDNA purification, the sample was divided into t hree ident ical a liquots.Knotted DNA (100 ng) was incubated, in two separate reactions, with

either commercial topoisomerase I from calf thymus (10 units/l) oryeast t opoisomerase II (10 units/l) together with 100 ng of supercoiled

SV40 DNA as interna l contr ol of relaxation. After incubation, the s am-ples were deproteinized and the DNA was pur ified and electrophoresed

in 1% agarose-TBE gel. After running, the gel was blotted and hybrid-

ized with a SV40-pUC19 radiolabeled probe. La n e 1, supercoiled SV40DNA; lane 2, relaxed pUC19 DNA; lane 3, knotted pUC19 DNA; lane 4,supercoiled SV40 DNA knotted pUC19 DNA after topoisomerase I

tre a tm e n t; lane 5, th e sa m e m ix tu re a s in lane 4 but after relaxationwith topoisomerase II; lane 6, supercoiled pUC19 DNA. I, II, a n d II I

denotes th e super coiled, nicked, and linear forms of each DNA, respec-tively. Bracket depicts the position of knots. Observe that only topoi-

s om e r a s e I I i s a b le t o u n k n ot d ou b le s t r a n d ed D N A w h e r ea s b ot htopoisomerases I and II relax the DNA with equal efficiency.

F IG . 4. T i m e c o u r s e a n a l y s i s o f k n o t t i n g w i t h r e l a x e d a n d s u -p e r c o ile d DNA. 200 ng of both relaxed (lane 12) or sup ercoiled (lane 6)pUC19 were knotted with topoisomerase II as described. At the indi-

cated times, one-sixth of the mixture, containing about 30 ng of DNA,was removed and the reaction stopped by addition of 2.5% Sarkosyl, 100

m M E D T A. S a m p les w e re d e p ro tein ize d a n d th e p u rifie d D N A w a sresolved in 1% agarose-TBE gel, blotted, a nd hybridized with a pUC19

probe. Lanes 15, knotting over relaxed DNA template (lane 12) after15, 30, 60, 120, and 240 min of incubation at 30 C, respectively. Lanes

711, a s lanes 15 b u t u sin g su p e rc o ile d D N A a s su b stra te . L a n e 6 ,supercoiled DNA template t ogether with 10 n g of added B am HI-linear-

ized pUC19. Indicated are knots (asterisks) and topoisomers (circles).Arrowheads denote the position of the sim plest kn ot (trefoil). I, II, an d

III, su percoiled, relaxed, and linearized forms of pUC19, respectively.

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amount of topo II or, in a mock incubated reaction, with buffera lone be f or e the a ddition of the S - 150. S ix hour s la te r , the

chromatin assembly products were processed for both topolog-

ical and micrococcal nuclease digestion analysis. Pretreatment

with catalytic amounts of topoisomerase II prior to the assem-

bly process can r everse t he ina bility of the kn otted DNA tem-

plate (Fig. 6, lane 3) to assemble nucleosomes as revealed both

by its topological state (Fig. 6, la n e 8) a nd the pr e se nc e of a

series of DNA fragments which are multiples of approximately

160170 base pairs (Fig. 6, lanes 9 11) obtained upon partial

micrococcal nuclease digestion. In contrast, the topoisomerase

II mock-incubated half (Fig. 6, lane 4) shows, after th e micro-

coccal n uclease digestion, a mixt ur e of DNA fragment s with out

a clear nu cleosomal periodicity (Fig. 6, lanes 57). This patt ern

of ba nds m a y be e xpla ine d by the pa r tia l a sse m bly ove r the

unknotted species contained in the knotted mixture (see Fig. 5).I t should a lso be note d the higher se nsitivity a ga inst t he m i-

crococcal nu clease digestion shown by t he topoisomerase II

mock-incubated reaction (compare Fig. 6, la n es 6 a n d 10 )

whic h, a ga in, r e ve a ls the a sse m bly only ove r the unknotte d

template. Therefore, the previous unt ying of knotted DNA tem-

plate seems to be critical for the correct nucleosome loading.

DISCUSSION

In this paper I show how the topoisomerase II from S. cer-

evisiae ca n i n t r od u ce k n ot s i n a p la s m id ic D N A w h en t h e

reaction is performed at an enzyme/DNA mass ratio equal or

highe r tha n 1, a lwa ys in the pr e senc e of ATP . This pr oce ss

needs a topologically linked DNA substrate regardless its form.

DNA knotting, topoisomerase class II-mediated, ha s beendescribed by means of purified enzymes from T4 bacteriophage

(3, 17), Drosophila (14), and yeast (18). In those cases knotting

is a c hie ve d a t a n e nz ym e /DNA m a ss r a tio quite highe r tha n

necessary to produce relaxation/unknotting/decatena tion r eac-

tions (5, 14, 18). Under the experimental conditions described

in this paper, I estimate that about 15 molecules of topoisomer-

ase II per pUC19 DNA molecule are needed to produce a highly

com pa cte d knotting, while in the r e la xa tion r e a ction using

topoisomerase II from Drosophila one molecule of enzyme,

working in a processive fashion, can relax a bout 15 m olecules of

super coiled DNA (5). Two different condit ions h ave been foun d

to promote t he kn otting production by both the prokaryotic and

eukaryotic enzymes. The knotting process requires ATP hy-

drolysis or the addition of a nonhydrolyzable analog of ATP

such as AMPPNP 1 when a eukaryotic enzyme is employed (14,

18 and t his report) but not wh en th e T4 enzyme is used (3, 17).

Furt hermore, whereas th e eukar yotic enzyme can operat e over

a DNA ring regardless of its topological form, the viral enzyme

knots DNA with a linking number deficit much more efficiently

(17).

I t ha s be e n sugge ste d tha t the juxta position of the DNA

helices in the plectonemic supercoiling is the general feature

for the spatial recognition of the DNA topology by topoisomer-

ases (3, 14, 30). In addition, yeast topoisomerase II promotes

the sta biliza tion of c r ossings of DNA duplexes, a c ting a s a

ba r r ier for the r e la xa tion with topoisom e r a se I a nd, on the

othe r ha nd, pr om oting the knotting f or m a tion str ongly over

several topological forms (18). Th erefore, the finding tha t the

e nz ym e c a n f or m knots ove r DNA r ings without a ny supe r -

structure (see Fig. 4) may account for the ability of topoisomer-

1 T h e a b b re v ia tio n u se d is: A MP P N P , 5-adenylyl-,-imidodiphos-

p h a te .

F IG . 5. Th e c e ll-fr e e e x tr a c t S -1 5 0 c a n p r o d u c e s u p e r c o ils o v e rr e l a x e d D N A t e m p l a t e s i n t h e p r e s e n c e o f k n o t s . 3 g of eitherre la xe d a n d k n o tte d p lu s re la xe d p U C 1 9 w e re in cu b a te d w ith S -1 5 0under standard conditions for chromatin assembly (see Materials and

Methods) in two identical reactions. At the indicated times, one-fifth ofthe assembly mixture was removed and stopped, samples were depro-

teinized and, after their purification, DNA were electrophoresed in a 1%agarose-TBE gel without et hidium bromide. La n es 2 6 , supercoiling

over relaxed DNA after 15, 30, 60, 120, and 240 min of incubation at37 C, respectively. Lanes 10 14, a s lanes 26 b u t u sin g a m ix tu re o f

re la xe d a n d k n o tte d p la sm id a s te m p la te . Lanes 1, 8, a nd 16, relaxedDNA. Lanes 9 an d 15 , relaxed plus knotted DNA. La n e 7 , supercoiled

DNA. Dots a n d dashes denote relaxed and knotted species, respectively.

F IG . 6. T h e u n k n o t t i n g b y c a t a l y t i c a m o u n t s o f t o p o i s o m e r a s eI I p e r mits th e i n v i t r o c h r o m a t i n a s s e m b l y . A, schematic diagramof the experimental protocol used. Supercoiled (sc) p U C 1 9 D N A w a sk n o tte d (kn ) w ith to p oisom e ra se II a s d e scrib e d d u rin g 1 h a t 3 0 C.

After extensive deproteinization, the purified knotted DNA (lane 3) wasincubated either with 15 units of topoisomerase II or, in a mock incu-

bated reaction, without enzyme at 30 C for 1 h and t hen both mixtur eswere processed in two independent but identical chromatin assembly

re a ction s w ith S -1 5 0 for 6 h a t 3 7 C. A p o rtion (on e -te n th o f e a chmixture) was removed and deproteinized for the analysis of DNA topol-

ogy (lanes 4 a n d 8, without and with topoisomerase II relaxation priorth e S -1 5 0 a d d ition , re sp e ctiv ely). T h e re m a in in g w a s d ige ste d w ith

micrococcal nuclease for 2, 8, and 20 min (lanes 57, without and lanes911 with t opoisomerase II r elaxation pr ior t he S-150 addition, respec-tively). All the samples were deproteinized, DNA purified, electrophore-

sed in 1% agarose-TBE gel, blotted, and hybridized with a pUC19 probe. Lanes 1 a n d 2, relaxed and supercoiled pUC19, respectively. Super-

coiled (I), relaxed (II), a n d lin e a riz e d (III) fo rm s o f th e p la sm id a reindicated. 1 n , 2 n , 3 n , a n d 4n , mono-, di-, tri-, and tetranucleosome,

respectively. Dashes denote a standa rd ladder of fragment s mult iples of142 base pair s. Micrococcal n uclease ban ds of about 160 base pair s a re

diagnostic of nucleosome presence. Only topoisomerase II-preincubatedsubstrate is packaged into chromatin upon the S-150 incubation.



5/6

ase II to promote those crosses if the DNA molecule is wrapped

around the enzyme as in the DNA-bacterial gyrase complexes

observed in vitro (3133).

Knots have several well defined characteristics: 1) they show

the highest compactation degree as revealed by their electro-

phoretic mobilities: the knots conta ining more nodes run elec-

trophoretically ahead of the supercoiled form (Figs. 1, 3, 4, and

6). 2) In part because of its compactness, the knots are less well

stained than the other topological forms when ethidium bro-mide is employed; nonetheless, when a radiolabeled probe was

used instead of ethidium bromide such differences were not

observed (compa re F igs. 1 and 4). 3) Two-dimensional gel an al-

ysis displays th e peculiar electr ophoretic mobility shown by th e

nicked kn ots, which run in diagonal. When the second dimen-

sion is pe r for m e d in the pr e senc e of c hlor oquine , a cur ve d

distribution of covalently closed knots appears converging to

the tip of the diagonal, as th e nu mber of crosses increases (18)

(Fig. 2). 4) The knots can only be unt ied, without endonucleo-

lytic linearization, by topoisomerase II at a enzyme/DNA ratio

much lower than the necessary for the knotting formation and

in the presence of 150 m M KCl (data not sh own).

Under the experimental conditions reported here, the level of

KCl is critical: the knotting process requires low levels of KCl

(20 m M) probably because the high ionic strength (150 m M) can

affect, by shielding of charged groups, the binding between the

DNA and the large number of topoisomerase II molecules re-

quir ed to pr oduce knots (18). I n the r e la xa tion/unknotting/

decatena tion processes, where the enzyme must be present at a

ca ta lytic le ve l, t his e ffe ct is pr e se nt a t a lesse r e xte nt. The

increase of the ionic strength alters t he na tur e of the relaxation

reaction from processive t o distributive by changing the bind-

ing rat e between DNA and t he topoisomerase II from Drosoph-

il a (5). This could explain the different requirements of KCl

observed for both relaxation and knotting process.

Time cour se an alyses over eith er rela xed or su percoiled DNA

show that after 15 min of incubation the knotting distribution

is already the same as observed 4 h later, especially when the

subst rat e is a covalently closed rela xed DNA (Fig. 4, lanes 15).The knotting over negatively su percoiled DNA occurs without

detectable DNA relaxation (Fig. 4, lanes 711), and is easily

distinguishable in well resolved gels, because the knots (Fig. 4,

asterisks) migrate between the topoisomers (Fig. 4, white cir-

cles), according to other reports (3, 14).

Whe n t he knotting is c a r r ied out on supe r c oile d DNA, a

small fraction keeps this topological form throughout the time.

It could be explained by considering a potentially very short

ste p of r e la xe d sta te , whic h I did not se e , be twe e n both the

supe r c oile d a nd the knotte d f or m . None the le ss, the le ve l of

KCl, optimal for knotting but not for relaxation, would explain

why suc h a supe r c oile d f r a c tion doe s not r e a c h the knotte d

stat e (Fig. 4, lanes 711).

By using the cell-free extract S-150 from Xenopus oocytes as

a n in v itr o chromatin assembly system (22, 23) is shown by

means of both topological and micrococcal nuclease digestion

analysis th at the knotted DNA is the only topologically linked,

single ring un able to a ssemble nucleosomes. Interestingly, th e

treatment of 800 ng of knotted substrate with 20 ng of topoi-

somerase II before the onset of the chromatin assembly leads to

a c om pe te nt substr a te tha t a llows the or de r e d de position of

histones in a reaction m ixture conta ining about 400 g of total

protein, and yields a minichromosome undistinguisha ble from

those obtained on competent substrates (Figs. 5 and 6, lanes

811) . This e f f e c t a ppe a r s to be m or e dr a m a tic tha n the ob-

served using the same S-150 system in the remodeling of the

minichromosome by the exogenous addition of histone H1 (34).

The knotted species present in the chromatin assembly reac-

tion does not inhibit this process over other unknotted forms;

as F ig. 5 shows, the u nknotted template r eaches progressively

a negative supercoiled stat e in a very similar fashion tha n th at

obtained on th e relaxed plasmids. In contrast , no nu cleosomes

are formed over the knotted population.

Topoisomerase II is, among t he nonhistone proteins, one of

the most abundant components of the mitotic chromosome. It

se em s to pla y a n im por ta nt r ole in the or ga niza tion of the

chromosome stru cture (Refs. 3538 and references th erein).

This protein bin ds very preferentia lly to the so called matr ix- or

scaffold-associated regions anchoring chromatin loop domains

in v iv o (Ref. 36 and references therein). Furthermore, it has

been observed in living embryonic cells from Drosophila t h a t

the topoisomerase II a ppears to be localized a t precise sites in

the nucleus, in a temporally regulated fashion (39). This in vivo

redistribution has also been reported during the induction of

heat-shock genes (10).

This r e por t docum e nts the a bili ty of th e e uka r yotic topoi-

somerase II to induce very different topological structures over

DNA r ings de pending on the e nz ym e conce ntr a tion. Thus,

when the enzyme/DNA mass rate is high, knots are produced;

on the opposite , the de c r e a se of this r a tio le a ds to a typic a l

relaxation/unknotting/decatena tion activity.

The spacially and temporally regulated distribution of topoi-somerase II in nuclei, observed in vivo, could reflect the local

variation of the enzyme concentration during the cell cycle. It

would not be u nreasonable to suppose tha t t his local variation

in t he t opoisomerase II concentration would promote the knot-

ting of specific regions or domain s of DNA, which could impede

the regular nucleosome assembly, favoring the genetic activity

of those domains. When the transcription process is finished,

the enzyme, via u nkn otting, may open up these r egions toward

the regular chromatin recondensation.

These results suggest tha t t opoisomerase II ma y be essential

in the topological transitions undergone by the eukaryotic chro-

mosome during the cell cycle.

Acknowledgments I am especially grateful to Drs. L. Badimon and

L. Cornudella for their generous support and stimulating discussions.Dr. J. Roca for the provision of pure topoisomerase II from yeast. Drs.

F. Azor n, B. Pina , J . Bernu es, J . Portu gal, D. W. Sumn ers, an d J . C.Wang for their invaluable critical comment s; to my colleagues E . Pra ts,

C. Bonet, M. Ort iz, J. Arsua ga, and I. Dazey for their help. Very specialthan ks to A. Sanchez and M. Daban.

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Antonio Rodrıguez-Campos- DNA Knotting Abolishes in Vitro Chromatin Assembly