R.Daniel Camerini-Otero and Gary Felsenfeld- Supercoiling energy and nucleosome formation: the role of the arginine-rich histone kerne

8/3/2019 R.Daniel Camerini-Otero and Gary Felsenfeld- Supercoiling energy and nucleosome formation: the role of the argini

1/23

V o l u m e 4 N u m b e r 5 1 9 7 7 N u c l e i c A c i d s R e s e a r c hS u p e r c o i l i n g e n e r g y a n d n u c l e o s o m e f o r m a t i o n : t h e r o l e o f t h e a r g i n i n e - r i c h h i s t o n e k e r n e l t

R . D a n i e l C a m e r i n i - O t e r o a n d G a r y F e l s e n f e l dL a b o r a t o r y o f M o l e c u l a r B i o l o g y , N a t i o n a l I n s t i t u t e o f A r t h r i t i s , M e t a b o l i s m a n d D i g e s t i v eD i s e a s e s , N a t i o n a l I n s t i t u t e s o f H e a l t h , B e t h e s d a , MD 2 0 0 1 4 , USAR e c e i v e d 4 F e b r u a r y 1 9 7 7

ABSTRACTWe have f or me d c om pl ex es of r el ax ed c lo se d circular Col E l DNA withvarious c o m b i n a t i o n s o f h i s t o n e s , and examined t h e ef f ect s o f treating t h ecomplexes with nicking-closing e n z y m e . Germond e t a l ( 1 ) have shown t h a twhen a mixture o f t h e f o u r core histones o f t h e nu c l eos ome ( H 2 A , H 2 B , H 3a n d H 4 ) i s u s e d in s u c h an experiment, t h e subsequently isolated DN A i ss u p e r c o i l e d . We f i n d t h a t t h e arginine-rich histone p a i r , H3 a n d H 4 , i ss u f f i c i e n t t o induce t h e supercoiling observed i n t h i s experiment. Both H3a n d H 4 are r e q u i r e d , a n d i n t h e absence o f e i t h e r , n o o t h e r histones ar ee f f e c t i v e . H 3 a n d H 4 are a s efficient, p e r unit weight, a s a mixture o ft h e f o u r histones i n inducing s u p e r c o i l s .We a l s o show t h a t t h e r e i s a large difference between th e DN A bendingenergy needed t o form a nucleosome a n d t h a t needed t o form o n e turn o fn o r m a l s u p e r h e l i c a l D N A . These t w o processes are energetically quite di s -tinct a n d p r o b a b l y s e p a r a b l e . We estimate th e f ree ene rgy o f interactionbetween D N A - b o u n d histone p a i r s , an d find that one or t w o such interactionswould generate e n o u g h energy t o f o l d t h e DN A i n t o a nucleosome.INTRODUCTION

We have recently shown ( 2 , 3 ) t h a t t h e arginine-rich histone p a i r , H 3a n d H 4 , p l a y s a central r o l e in t h e o r g a n i z a t i o n o f t h e nucleosome. R ec on-s t i t u t e d DNA-histone c o m p l e x e s c o n t a i n i n g t h e H 3 - H 4 p a i r behave very muchl i k e native chromatin with respect t o many chemical p r o b e s . Such c o m p l e x e s ,when t r e a t e d with s t a p h y l o c o c c a l n u c l e a s e , DNAase I , and DNAase I I , g i v erise t o DN A f ra gm en ts o f discrete subnucleosome size similar t o thosep r o d u c e d when chromatin i s d i g e s t e d . The hi stones o f these complexes ar ealso r e l a t i v e l y resistant t o attack b y t r y p s i n and c h y m o t r y p s i n . Al l o fthese effects d e p e n d upon t h e p res enc e o f both H3 and H 4 ; all c o m p l e x e scontaining both histones behave like c h r o m a t i n , whi l e all those c o m p l e x e sl a c k i n g either H 3 or H 4 d o not.

The four "core" hi stones o f the nu c l e os ome ( H 2 A , H 2 B , H 3 an d H 4 )distort t h e local conformation o f DN A t o which t h e y are b o u n d . Germonde t a l . ( 1 ) have s hown t h a t when an e q u i m o l a r mixture o f these histonesi s reconstituted with relaxed closed circular D N A , a n d t h e complex t r e a t e d

X ) I n f o r m a t i o n R e t r i e v a l L i m i t e d 1 F a l c o n b e r g C o u r t L o n d o n Wl V 5FG E n g l a n d 1 1 5 9


2/23

N u c l e i c A c i d s R e s e a r c hwith n i c k i n g - c l o s i n g e n z y m e , t h e s u b s e q u e n t l y isolated DNA i s s u p e r c o i l e d ,i . e . i t s t o p o l o g i c a l winding number h a s been c h a n g e d . I n t h i s p a p e r , werepeat t h e e x p e r i m e n t o f Germond e t a l . ( 1 ) u s i n g only H 3 a n d H 4 , a n d showt h a t t h e arginine-rich histone p a i r i s c a p a b l e o f producing t h e samee f f e c t a s an e q u a l weight o f t h e f u l l c o m p l e m e n t o f h i s t o n e s . All c o m -b i n a t i o n s o f h i s t o n e s l a c k i n g e i t h e r H 3 o r H 4 have n o detectable e f f e c t i nt h i s s u p e r c o i l i n g a s s a y . O u r r e s u l t s o n c e again s u p p o r t t h e i d e a t h a t H 3and H 4 are o f c e n t r a l importance i n nucleosome o r g a n i z a t i o n .

I n t h e c o u r s e o f t h i s w o r k , we have a l s o e x a m i n e d t h e possiblerelationship between t h e conformation o f naturally s up er co il ed DN A andt h e conformation o f D R A i n t h e n u c l e o s o m e . Although i t has b ee n a ss um edt h a t native supercoiled DN A ( s u c h a s t h a t i s o l a t e d from t h e S V 4 0 minichromo-s o m e ) i s i n a state s u i t a b l e f o r t h e r e a d y formation o f n uc le os om es wh enhistones a r e a d d e d , t h a t i s n o t t h e c a s e . C a l c u l a t i o n s s u g g e s t t h a t t h eequilibrium conformation o f s u p e r c o i l e d DNA i s quite d i f f e r e n t from t h a trequired f o r a reasonable nucleosome m o d e l , a n d t h a t t h e formation o f nucleo-somes from s u p e r c o i l e d DNA r e q u i r e s a l a r g e a m o u n t o f additional f r e e e n e r g y .T h e estimated interaction f r e e e n e r g y o f o n e o r t w o p a i r s o f histones withint h e nucleosome a p p e a r s sufficient t o provide t h e n e ce s sa ry d ri vi ng energyf o r t h i s folding o f t h e D N A .MATERIALS AND METHODS

PreSarion o f C o l E l D N A , Histones a n d Histone-DNA C o m p l e x e s . Col E lp l a s m i d DN A was p r e p a r e d f r o m E . coli strain A 7 4 5 met t h y ( C o l E l ) a c c o r d i n gt o s l i g h t modifications o f p r e v i o u s l y p u b l i s h e d p r o c e d u r e s ( 4 , 5 ) . R e l a x e dc l o s e d - c i r c u l a r Col E l DN A was obtained b y treating 1 m g o f s u p e r c o i l e d DNAi n 6 m l o f buffer A ( 2 0 0 mM N a C l , 2 0 mM T r i s * H C l ( pH 8 .0 ) , 0 . 2 5 mM E D T A , 5 %( v / v ) g l y c e r o l ) with 1 0 0 0 t o 1 5 0 0 units o f n i c k i n g c l o s i n g extract ( N C E ,s e e b e l o w ) a t 3 7 C f o r 3 0 m i n . A f t e r the addition o f 1 % sodium d o d e c y l -s u l f a t e , t h e DN A wa s p u r i f i e d b y ethidium bromide-CsCl d e n s i t y e q u i l i b r i u mc e n t r i f u g a t i o n . - The r e l a x e d c l o s e d c i r c u l a r DNA was c o l l e c t e d from t h eg r a d i e n t s , t h e ethidium b r o m i d e r e m o v e d b y extraction with n - b u t a n o l , t h es a m p l e was d i a l y z e d a g a i n s t 5 0 m M N a C l , 1 0 mM T r i s - H C l ( p H 8 . 0 ) and 1 mM E D T A ,a n d t h e DN A p u r i f i e d further b y p h e n o l - c h l o r o f o r m - i s o a m y l alcohol ( 4 8 : 2 4 : 1 ,v / v ) extraction and e t h a n o l p r e c i p i t a t i o n . All DN A s a m p l e s w e r e r e s u s -p e n d e d a n d s t o r e d in 5 0 mM N a C l , 1 0 m M T r i s - H C l ( p H 8 . 0 ) , a n d 1 mM EDTA a t4 0 C .

T o t a l h i s t o n e s were extracted with 0 . 4 N H 2 S o 4 from chromatin p r e p a r e dfrom duck e r y t h r o c y t e s ( 2 ) . Histones H 2 A , H2B a n d H 4 were p u r i f i e d from

1 1 6 0


3/23

N u c l e i c A c i d s R e s e a r c hs to ck s ol uti on s o f total hist ones according t o previously pub lishedmodifications ( 2 ) o f t h e method o f Van der Westhuyzen e t a l . ( 6 ) . PurifiedH 3 was obtained b y s o m e modifications o f t h e p r o c e d u r e o f R u i z - C a r r i l l o andAllfrey ( 7 ) t o be published elsewhere ( C a m e r i n i - O t e r o , Simon and F el s e nfe l d,manuscript i n p r e p a r a t i o n ) . The four histones H 2 A , H 2 B , H 3 a n d H 4 andt h e pairs o f slightly ly sine- rich histones ( H 2 A / H 2 B ) and argi ni ne-ri chhistones ( H 3 / H 4 ) were prepared by t h e following sequential " s t r i p p i n g "procedure. F i r s t , t h e ly sine- rich histones ( H I and H 5 ) were dissociatedf r o m chromatin by dialyzing chromatin against 0 . 7 M NaCl, 5 mM Tris- H Cl( p H 8 . 0 ) , 0 . 5 mM EDTA at 4 0 C and removing t h e " s t r i p p e d " p r o d u c t byexclusion column chromatography on B io -G el A -5 M ( B i o - R a d ) . Part o f t h i sproduct was extracted with 0 . 4 N H 2 S O 4 a t 4 0 0 t o yi el d the f o u r histonesH 2 A / H 2 B / H 3 / H 4 . The slightly ly sine- rich hi sto ne s we re then obtained b yd i s s o c i a t i n g t h e remainder o f t h e 0 . 7 M NaCl " s t r i p p e d " product i n 1 . 2 5 MN a C l , 5 mM T r i s H C l ( p H 8 . 0 ) , 0 . 5 mM EDTA a t 4C and again separating t h eproducts by exclusion column chromatography. The arginine- rich histonesl e f t on t h e DNA were then extracted with 0 . 4 N H 2so4 a t 4 0 C . Furtherd e t a i l s o f t h i s purification procedure will be published elsewhere ( S i m o n ,Camerini-Otero a n d F e l s e n f e l d , manuscript i n p r e p a r a t i o n ) . The purity o fa l l t h e histones was routinely monitored b y electrophoresis on 1 5 % poly -acrylamide sodium d o d e c y l s u l f a t e stacking g e l s according t o a methodd e s c r i b e d previously ( 2 ) . The individually purified h is to ne s h ad < 1 % im-p u r i t i e s , with t h e exception o f H 2 A , which had < 5 % contamination withnonhistone proteins. T h e histone pairs had < 5 % a n d < 1 % impurities f o rt h e ( H 2 A / H 2 B ) and ( H 3 / H 4 ) p a i r s r e s p e c t i v e l y .

Histone-DNA complexes ( r e c o n s t i t u t e s ) w e r e p r e p a r e d b y m i x i n g hi stonesand Col E l DN A i n t h e presence o f 5 0 mM N a C l , 5 0 mM 2 - m e r c a p t o e t h a n o l ,1 0 mM T r i s . H C l ( p H 8 . 0 ) a n d 1 m M E D T A ; u s u a l l y t h e DN A concentration wa s5 0 p g / m l , a n d unless otherwise s p e c i f i e d , 0 . 2 5 gm o f each histone w a sadded per gram o f D N A . Hi stone concentrations w e r e determined as de s c ri be dp r e v i o u s l y ( 2 ) . T he se s ol uti on s we r e d i a l y z e d f o r 2 to 1 2 hr at 4 0 Cagainst 5 M u r e a , 2 M N a C l , 5 mM Tris-HCl ( p H 8 . 0 ) , 0 . 5 el E D T A , a n d thens u b j e c t e d t o step g r a d i e n t d i a l y s i s . The steps w e r e as describ ed p r e v i o u s l y( 2 ) except t h a t all the d i a l y s i s steps included 5 m M Tris.HCl ( p H 8 . 0 ) , 0.5m M E D T A . At t h e end of t h i s reconstitution t h e protein t o DN A ratio wasexactly equal t o t h e in put r atio ( 8 ) . F u rthermore none o f the histoneswas lost selectively and all o f the protein was b ound t o t h e D N A . Bindingof t h e histones was measured using an assay that detects f r e e histone after

1 1 6 1


4/23

N u c l e i c A c i d s R e s e a r c ht h e histone-DNA complex has b ee n b oun d t o DEAE-c el l u l os e ( 2 ) .

For some experiments an alternate reconstitution procedure was used:Histones and Col E l DN A were mixed i n t h e same solvent and a t t h e sameconcentrations a s a b o v e , and then dialyzed f o r 2 t o 1 2 h r a t 4 0 C against2 M NaCl, 5 m M T ris .H Cl ( p H 8 . 0 ) , 0 . 5 mM EDTA. After 9 0 m in the sampleswere then dialyzed against 5 m M T ris -H Cl ( p H 8 . 0 ) , 0 . 5 mM EDTA.

S eq ue n ti al a dd i ti o n o f histones an d staphylococcal nuclease dig estiono f reconstitutes. For these experiments histone-DNA c o m p l e x e s were pre-pared b y t h e reconstitution procedure described above with t h e followingexceptions: 1 ) Du ck DNA was used ( p r e p a r e d a s describ ed previously ( 2 ) )and 2 ) the concentration o f t h e DN A was approximately 2 5 0 p g / m l . Otherhistones were a d d e d t o t h e s e reconstituted complexes in t h e followingmanner. At the end o f t h e reconstitution procedure the s ol vent compositionwas 5 mM Tris-HCl ( p H 8 . 0 ) , 0 . 5 mM E D T A ; t o t h e complexes, dissolved int h i s solvent, an equal volume o f histones i n the same solvent was adde ddropwise a t r o o m temperature with continuous stirring. A volume o f approxi-mately 3 ml was added over a period o f 3 m i n . I n one experiment s hownbel ow ( F i g u r e l e ) histones were added dropwise t o p rotei n-free D N A . Thesef i n a l complexes were t h e n d i g e s t e d with staphylococcal nuclease, t h e DN Apurified, a n d t h e samples examined on 6 % p ol yac rylami de gels a s describedpreviously ( 2 ) .

Preparation o f Ni c ki ng-Cl osi ng Extract. Ni c ki ng-c l osi ng extracts( N C E ) were prepared from both d u c k erythrocyte a n d reticulocyte nuclei.Reticulocytes were o bta in ed f ro m t h e bl ood o f ducks treated with I - a c e t y l -2 - p h e n y l h y d r a z i n e a c c o r d i n g t o a schedule d e s c r i b e d elsewhere ( Z a s l o f f andF e l s e n f e l d , manuscript i n p r e p a r a t i o n ) . Fresh cells were washed four timeswith PB S b u f f e r b y c e n t r i f u g a t i o n a n d resuspension; t h e y were then r esu s -p e n d e d in 0 . 2 5 M sucrose, 1 0 m M Tri s -HC l ( p H 8 . 0 ) , and 1 m M magnesiumacetate a n d washed t w i c e . The c e l l s w e r e washed f i v e times in t h e s a m es u c ros e solution and 0 . 5 % ( v / v ) Triton X - 1 0 0 . The r e s u l t i n g nuclei w e r ethen washed a n additional three times in t h e ab sence o f T r i t o n . Thenuclei were swollen b y w a s h i n g t h e m i n 1 0 m M i T r i s . H C l ( p H 8 . 0 ) , 0 . 5 m MEDTA. In all c a s e s , a Dounce homogenizer ( l o o s e p e s t l e ) was used f o rresuspension. The n u c l e i , swollen t o about five t o ten time s the ir originalv o l u m e , were then mixed with an equal volume o f 300 mM phosphate bu ffe r ( p H7 . 5 ) , homogenized with a Dounce homog enizer and centrifuged a t 25,000 xgf o r 1 5 m i n . The supernatant i s the ni c ki ng-c l osi ng extract.

The activity o f t h e ni c ki ng-c l osi ng e xtr ac ts wa s monitored by measuring

1 1 6 2


5/23

N u c l e i c A c i d s R e s e a r c h

t h e conversion o f supercoiled Col E l DN A t o t h e r e la xe d c l os e d- c ir c ul a rf o r m a s d e m o n s t r a t e d b y agarose g e l e l e c t r o p h o r e s i s ( s e e b e l o w ) . I f aunit o f r e l a x i n g activity i s defined a s t h e amou nt r e q u i r e d t o completelyrelax 1 v g o f supercoiled Col E l DNA i n 3 0 m in a t 3 7 0 C i n buffer A ( s e ea b o v e ) , t h e specific activities o f different extracts varied from 2 5 0 t o1 2 0 0 units p e r mg o f extract protein ( 8 ) . Extracts f r o m r e ti cu lo c yt esuniformly gave t h e higher s p ec if ic a ct iv it ie s . The extracts were allmonitored f o r t h e presence o f histones a s demonstrated b y SDS-gel electro-p h o r e s i s ( s e e a b o v e ) : t h e e xt ra ct s c o nt ai ne d l e s s than 2 p g o f eachhistone per mg o f e xtr ac t p ro te in .

These extracts are easy t o prepare, have a ct iv iti e s c o mp a ra b le t ot h o s e o f other cells ( 9 - 1 3 ) , d o not require divalent i o n s , and are a l s o r e -m a r k a b l y f r e e o f proteases a n d nucleases a s shown by t h e f ol lo wi ng e xp er i-ments. Free histones i n water, chromatin, o r reconstitutes were incubatedf o r 1 h a t 3 7 0 C with 1 0 p g o f extract protein per p g o f t ota l h is to n e,a n d after t h e incubation t h e histones were examined by S D S g e l electro-p h o r e s i s ( s e e a b o v e ) . Under these c o n d i t i o n s t h e r e was neither anyd e t e c t a b l e diminution i n t h e amount o f protein i n t h e histone bands norany evidence o f h is to n e d e gr ad ati on p r o d u c t s . T h e absence o f appreciablenuclease activity was es ta bl is hed b y ca rr yin g o u t t h e unwinding reactionsi n t h e absence o f EDTA a n d i n t h e presence o f 1 m M magnesiu m a n d / o rcalcium i o n s ; u n d e r these conditions none o f t h e supercoiled DN A wasconverted t o e i t h e r t h e nicked o r linear f o r m s o f C o l E l D N A .

Conditions f o r nicking and c l o s i n g DN A a n d agarose g e l electrophoresis.Three t o 5 p l o f NC E c on tai ni ng approximately 3 t o 5 j g o f extract protein( 3 t o 5 u n i t s ) were added t o 2 0 0 t o 4 0 0 ng o f DNA ( e i t h e r f r e e o r c o m p l e x -e d with h i s t o n e s ) i n 2 0 p l o f buffer A . A f t e r 3 0 m i n . a t 3 7 0 C t h e reactionwas s t o p p e d with sodium d o d e c y l s u l f a t e ( 1 % ) . Five p l o f a mixture contain-i n g 5 0 % s u c r o s e , 0 . 0 2 % b r o m p h e n o l b l u e , a n d 1 mM EDTA ( p H 7 . 0 ) were a d d e dt o e a c h s a m p l e a n d t h e s a m p l e s were i n c u b a t e d at 4 5 C f o r 1 5 m i n ; t h es a m p l e s were t h e n l o a d e d onto a n agarose g e l . U p to 1 2 s a m p l e s at a t i m ewere electrophoresed i n a 6 X 1 2 0 X 1 6 0 m m s l a b ( E - C A p p a r a t u s C o r p . ,Model 4 7 4 ) o f 1 . 0 % agarose ( M i l e s C o . ) with the f o l l o w i n g b u f f e r : 4 0 mMTris b a s e , 3 0 mM N a H 2 P 0 4 , 1 m M EDTA ( p H 7 . 9 ) . E l e c t r o p h o r e s i s was per-formed at 1 8 - 2 0 C f o r 1 2 t o 1 4 hr a t 3 V / c m . After el ectrophoresi s t h eg e l s were stained in t h e dark f o r 4 t o 6 h r in e le ctr o ph o re si s b uf f ercontaining 1 p g / m l o f ethidium brom ide, and destained f o r 2 t o 4 h r i ne l e c t r o p h o r e s i s b uffer o r water. The slabs were p ho to gr ap he d u si ng t w o

1 1 6 3


6/23

N u c l e i c A c i d s R e s e a r c hshort wave ultraviolet l a m p s ( T y p e S - 6 8 , Ultravi ol et Products, I n c . ) an dPolaroid Type 1 0 5 f i l m , with a Wratten 2 3 A filter over t h e c ame ra l e n s .RESULTS

Sequential additions o f hist ones . We have s hown that dig estionwithin t h e n uc le os om e b y staphylococcal nuclease gives ri se t o a largearray o f DN A f ra gm en ts o f well-defined size ( 2 , 1 4 ) . O nc e t h e digestionl i m i t i s reached t h e double-stranded DN A f ra gm en ts range i n size between1 5 8 a n d 3 8 base pairs; t h e fragments are separated in size by approximately( b u t not e x a c t l y ) 1 0 base pairs ( ( 2 ) ; Figure 1 , s l o t s a and g ) . Most ofthese DNA d i g e s t f r a g m e n t s can b e generated i n t h e absence o f t h e lysine-r ic h h is to ne s, H l and H 5 ( 2 , 1 4 ) . B y examining t h e DN A o f staphylococcaln uc le as e d ig es ts o f reconstitutes in vo lv in g m os t combinations o f histoneswe were able t o show that t h e arginine-rich histones are b oth n ec es sa ry an dsufficient t o protect d is cr ete DN A f ragm en ts f ro m dig estion ( ( 2 ) ; Figure1 , slot f ) . We have reported t h a t wh en a rg in in e -r ic h histones, or event o t a l h i s t o n e s , are added directly t o DN A a t low io nic s tr en gth , t h e DN At h a t i s protected f ro m d ig es ti on i s polydisperse. We s ho w her e that thedirect addition o f histones can result i n d is cr ete f ra gm en ts only when t h eDN A has b ee n a pp ro pr ia te ly " p r i m e d " . Figure 1 shows the results o f theseexperiments.

The arginine-rich hi stones w er e r e co n st it ut ed onto DN A by our stan-d a r d r e co n s ti tu ti o n p r oc e du r e ( s e e Materials and M e t h o d s ) ; a similarreconstitute with t h e slightly ly sine- rich histones was also prepared.T o both o f t h e s e reconstitutes t h e complementary p a i r o f histones wasa d d e d dropwise i n low ionic strength ( s e e Materials a n d M e t h o d s ) ; thesecomplexes were d i g e s t e d t o a l i m i t with s t a p h y l o c o c c a l nuclease a n d t h eDN A extracted from them examined b y p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s .S l o t s a and g o f Figure 1 s how t h e DN A o f chromatin limit d i g e s t marke rs .When a reconstitute o f H 3 / H 4 and DN A i s d i g e s t e d a n d t h e DN A i s e x a m i n e d ,t h e pattern shown in s l o t f i s seen. When the H 2 A / H 2 B p a i r i s added tot h i s reconstitute additional discrete fragments o f DN A a r e p r o t e c t e d fromdigestion ( s l o t s b a n d c ) . On t h e o t h e r h a n d , adding t h e arginine-richhistone pair ( H 3 / H 4 ) d i r e c t l y to a reconstitute containing th e s l i g h t l yl y si n e- r ic h h is to n es results in a complex t h a t fails t o pr otect an ydiscrete DN A fragments from digestion ( s l o t d ) . Slot e shows t h e re s u lto f adding t h e H 3 / H 4 pair directly t o DNA.

We have previously shown by examination o f the products o f digestiono f H 3 / H 4 reconstitutes that t h i s histone pair can interact with a n d org anize

1 1 6 4


7/23


A:..:A B C D E F G

Figure 1 . Sequential addition o f histones. The DN A extracted from staph-y l o c o c c a l nuclease d i g e s t s o f s e v e r a l s a m p l e s was e l e c t r o p h o r e s e d on a 6 %p o l y a c r y l a m i d e s l a b g e l . S l o t s a a n d g are markers from a l i m i t d i g e s t o fc h r o m a t i n ; t h e t o p band c o r r e s p o n d s t o a f r a g m e n t 1 5 7 bas e pairs l o n g , t h el o w e s t distinct b a n d t o a f r a g m e n t 3 8 base pairs l o n g . S l o t s b and c aretwo d i f f e r e n t H 3 / H 4 / D N A reconstitutes t o which H 2 A / H 2 B were a d d e d ; d , anH 2 A / H 2 B / D N A reconstitute t o which H 3 / H 4 was a d d e d ; e , DN A t o which H 3 / H 4was a d d e d ; and f the H 3 / H 4 / D N A reconstitute used i n b but without theadditional H 2 A / H 2 B . All histones were a t an input ratio o f 0 . 2 5 g pergram o f D N A .DN A stretches at least as l o n g as the 1 4 0 base p a i r nucleosome " c o r e "We proposed t h a t t h i s " a r g i n i n e - r i c h histone k e r n e l " was th e substrate o nwhich t h e s l i g h t l y - l y s i n e rich histones H 2 A / H 2 B c o m p l e t e t h e nucleosome.This proposal i s supported by t h e above results.

1 1 6 5


8/23

N u c l e i c A c i d s R e s e a r c hConversion o f supercoiled DN A t o r ela xed covalently c l o sed circular

D N A . Our endonuclease-free a n d p r o t e a s e - f r e e n i c k i n g - c l o s i n g ext ra ct( N C E ) i s similar i n activity t o extracts t h a t have been isolated p r e v i o u s l yfrom other e u k a r y o t e s ( 9 - 1 3 ) . T h e e f f e c t o f t h e n i c k i n g - c l o s i n g e x t r a c t o nsupercoiled DN A i s shown i n Figure 2 . S u p e r c o i l e d DN A has a h i g h electro-phoretic m o b i l i t y , while c o n t a m i n a t i n g nicked circular DN A f o r m s a s l ow ermoving band ( ( 1 , 1 5 ) ; Figure 2 , s l o t a ) . N i c k i n g - c l o s i n g extract c o nv ert st h e f a s t moving band o f superhelical DN A t o a series o f bands o f electro-phoretic mobility comparable t o t h a t o f nicked circular DN A ( ( 1 , 1 5 ) ;Figure 2 , slot b ) . These bands represent a Boltzmann d i s t r i b u t i o n , aboutt h e relaxed s t a t e , o f c o v a l e n t l y c l o s e d circular DN A molecules with di ffer-e n t topological w i n d i n g , o r l i n k i n g , numbers ( s e e b e l o w ) ( 1 6 , 1 7 ) . Partiallysupercoiled ( o r r e l a x e d ) molecules h a v e intermediate mobilities and are alsoseen a s a series o f b a n d s each corresponding i n principle t o a di fferentvalue o f t h e l i n k i n g number ( F i g u r e 2 , slots h and j ) , though t h e resolu-tion o f t h e bands v ar ie s wi th l i n k i n g numb er a n d with conditions o f electro-phoresis.

Treatment o f reconstitutes with nicking-closing extract. Germond e ta l . ( 1 ) have shown t h a t t h e f o u r histones H 2 A , H 2 B , H 3 and H 4 , when r e c o n -stituted onto closed circular D N A , deform the DNA i n a m a n n e r topologicallyequivalent t o t h e induction o f s l i g h t l y m o r e than o n e s u p e r h e l i c a l turnf o r each nucleosome ob served i n t h e electron m i c r o s c o p e . H o w e v e r , undert h e reconstitution conditions used b y these w o r k e r s , t h i s e f f i c i e n c y o finduction could o n l y b e ac hie ve d wh en two grams o f t h e f o u r histones w e r eadded f o r each gram o f D N A ; t h i s i s twice t h e ratio o r d i n a r i l y found i nt h e nucleosome.

I n t h i s s t u d y we confirm these r e s u l t s ; h o w e v e r , u s i n g o u r re c onsti -tution m e t h o d s , i t i s possible t o induce a b o u t o n e s u p e r h e l i c a l turn foreach a d d e d n u c l e o s o m e - e q u i v a l e n t o f t h e f o u r histones ( 1 g o f histonesper gram o f D N A ) . We have also used these methods t o ex ami ne t h e effecto f individual histones and combinations o f h i s t o n e s o n s u p e r c o i l i n g .B r i e f l y , t h e experimental scheme i s a s f o l l o w s : Histones are reconstitutedonto covalently closed circular DNA t h a t has previously b een relaxed bytreatment with nicking-closing e n z y m e . After reconstitution t h e complexi s t h e n treated with ni c ki ng-c l osi ng enzyme, deproteinized, and t h e DNAelectrophoresed i n a gel system that separates covalently closed circularspecies according t o linking number, a s describ ed above.

When a reconstitute o f 1 g o f t h e four histones (isolated a s a

1 1 6 6


9/23


a b c d e f g h i j k I

Figure 2 . Gel electrophoresis o f histone- Col El DN A c omp l e x e s u ntreatedan d tr eated to t h e limit with nicking - closing e x t r a c t s . Slots a and bcontain supercoiled Col E l DN A before and after t r e a t m e n t with nicking -closing e x t r a c t . Slots c through I are paired samples, b e f o r e and aftertr e at m e n t , of reconstitutes o n t o relaxed DNA. Slots c and d , all fourh is to ne s, p ur if ie d as a group o f four; e an d f , th e four histones, p u ri fi edas pairs; g an d h , hi stones H 3 and H 4 , p u ri fi ed as a pair; i an d j , hi stonesH3 an d H 4 , purified a s single histones; and k and 1, hi stones H 2 A and H 2 B ,p u ri fi ed as a pair. Al l hi stones were re c onstituted at an input ratio of0. 25 g per gram of DNA. In this and the following figures, the samples inslots a a n d b are a c o n tr o l demonstrating the presence of enzyme activity.T he 'relaxed' DN A in slot b i s n o t the same s amp l e u s e d in the recon-stitution experiments; s m a l l d i f f e r e n c e s b e t w e e n the patterns i n s l o t b a n dslot c ( f o r example) a r i s e f r o m v a r i a t i o n b e t w e e n ' r e l a x e d ' DN A samples,an d n o t f r o m the e f f e c t s o f hi s t o n e s .mixture of the four histones, see Materials and Methods) per gram o frelaxed Co l E l DN A i s deproteinized and t h e DNA i s examined b y electro-phoresis the p a t t e r n shown i n F i gu re 2 , slot c i s obtained. Figure 2 ,slot d shows the p a t t e r n o f t h e DN A from the same reconstitute i f th esample i s treated with nicking - closing enzyme b e f o r e bei ng deproteinized.The DN A has an electrophoretic mobi l ity similar to that o f n ative , f ul lys u p e rc oi l e d Col El D N A ; as G e r m o n d e t al. ( 1 ) reported, the presence of thehi stones induces a de formati on topolog ically e qu i val ent to supercoiling ( s e eDiscussion for an ex p l anati on of this " i n d u c t i o n " ) . I n our ex p e ri me nt thisdeformation has b e e n accomplished with one nucleosome- eq uivalent o f hi s to n e s .Figure 2 , slots e and f sh ow a similar experi ment in whi c h the DN A f r o m a

1 1 6 7

. . . . ; . : ! : ` r >. . x ' r :, s-


10/23

N u c l e i c A c i d s R e s e a r c hreconstitute o f a mixture o f t h e f o u r h i s t o n e s , isolated a s the p a i r s H2A/H2Ba n d H 3 / H 4 ( s e e Materials a n d M e t h o d s ) , i s e xa mi ne d b ef or e and after treat-ment with nicking-closing e x t r a c t . T h e r e s u l t s are t h e same.

We next e x a m i n e d t h e e f f e c t o f various o t h e r combinations o f histoneson s u p e r c o i l i n g . The H 3 / H 4 histone pair ( i s o l a t e d as a p a i r ) was re c on-stituted with relaxed C o l E l DN A at 0 . 5 g protein/g D N A , and treated withN C E . The resulting DN A ( b e f o r e a n d after NCE t r e a t m e n t ) i s shown i n F i g u r e2 , s l o t s g and h . I t i s clear t h a t a t t h i s protein t o DN A r a t i o , approxi-mately that found i n n ati ve n uc le os om es f o r this pair o f histones, asignificant amount o f s u p e r c o i l i n g has been i n d u c e d , but n o t as much a st h a t achieved with a f u l l c o m p l e m e n t o f histones ( c f . F i g u r e 2 , slots d an df ) . I n Figure 2 , s l o t s i a n d j , ar e shown the results o f a si mila r experi-ment i n which H 3 a n d H 4 were p u r i f i e d s e p a r a t e l y , rather than a s a p a i r .The s m a l l difference between t h e am o u n t of s u p e r c o i l i n g in slots h an d j i sneither reproducible n o r s i g n i f i c a n t . In c o n t r a s t , when the s l i g h t l ylysine-rich histone p a i r H 2 A / H 2 B , p u r i f i e d as a p a i r , wa s u s ed in ther e c o n s t i t u t e s , n o s u p e r c o i l i n g was induced ( F i g u r e 2 , slots k and 1 ) .

Other combinations t h a t d i d n ot i n c l u d e t h e H 3 / H 4 p a i r also f a i l e d toi nd u ce s up er c oi li ng . Some o f t h e s e e x p e r i m e n t s a r e shown i n F i g u r e 3 . Th et r i p l e t s o f p u r i f i e d histones H 2 A / H 2 B / H 3 ( F i g u r e 3 , s l o t s e and f ) an dH 2 A / H 2 B / H 4 ( F i g u r e 3 , s l o t s g and h ) and t h e s i n g l e a r g i n i n e - r i c h hi stonesH 3 ( F i g u r e 3 , s l o t s i - Z ) a n d H 4 ( n o t s h o w n ) d i d n ot alter t h e finaltopological conformation o f r el ax ed c lo se d circular D N A . In t h e ca se ofreconstitutes with t h e single p u r i f i e d histones H 3 o r H 4 , e xp er im en ts we recarried o u t a t protein t o DNA weight ratios o f 0 . 2 5 , 0 . 5 a n d 1 . 0 ; t h er e s u l t s were all t h e same a s i n Figure 3 , s l o t s j a n d D. As a c o n t r o l , amixture o f t h e f o u r individually purified his to ne s wa s reconstituted ontoD N A ; full supercoiling was induced ( F i g u r e 3 , slots c and d ) .

I n o r d e r t o e sti ma te m o re accurately t h e amount o f supercoiling t h a tcan b e induced b y a give n am oun t o f histone, reconstitutions were carriedo u t a t varying protein t o DNA r a t i o s , and t h e c om pl ex es e xa mi ne d a sa b o v e . With equimolar mixtures o f t h e f o u r histones, full supercoilingoccurred a t between 0 . 8 g a n d 1 . 0 g o f t o t a l added histones p e r gram o fD N A . " F u l l s u p e r c o i l i n g " i s used here t o denote degrees o f supercoilingt h a t are electrophoretically indistinguishable from native supercoiledD N A . Since t h e resolution o f t h i s g e l system i s l e s s than optimal f o rh i g h l y s up er co il ed m o le cu le s ( 1 8 ) , i t may b e t h a t t h e actual supercoilingat t h e e n d point i s slightly l e s s t h a n t h a t found i n native C o l E l D N A . I t

1 1 6 8


11/23


a bc d e f h i jk I

F i g u r e 3 . Gel e l e c t r o p h o r e s i s pattern o f histone- Col E l DN A c o m p l e x e s .S l o t s a a n d b , s u p e r c o i l e d DNA b efore and after NCE treatment. S l o t s ct h r o u g h I a r e p a i r e d s a m p l e s , before a n d after t r eat m ent w i t h i N C E , o freconstitutes o n t o re l ax e d D i N A . S l o t s reconstituted with: c and d allfour h i s t o n e s , all p u r i f i e d a s i ndi vi du al h i s t o n e s ; e a n d J f , histones H 2 A ,H2 B a n d H 3 , p u r i f i e d a s s i n g l e h i s t o n e s ; g and h , histones H 2 A , H 2 B a n d H 4 ,p u r i f i e d as s i n g l e h i s t o n e s ; i and j, histone H 3 ; a n d k a n d 1 histone H 3 ata n i n p u t ratio o f 0 . 5 g per gram o f D N A . All h i s t o n e s , e x c e p t where n o t e d ,were at a n i n p u t ratio o f 0.25 g per gram o f D N A .i s p o s s i b l e i n p r i n c i p l e t o a n a l y z e t h e . e l e c t r o p h o r e t i c band p a t t e r n at inter-m e d i a t e p o i n t s i n a titration o f DN A w i t h h i s t o n e s , and t o deduce t h es t o i c h i o m e t r y m o r e p r e c i s e l y i n t h a t w a y . Such p r e c i s i o n i s p r o b a b l y notj u s t i f i a b l e u n t i l i t c a n be s hown t h a t every h i s t o n e molecule i s c o r r e c t l yrecomb ined with D N A .

A s i m i l a r titration with t h e histone p a i r H 3 / H 4 i s s h o w n i n F i g u r e 4 .Here a g a i n full s u p e r c o i l i n g occurred at s omewhere between 0 . 8 g and 1 . 0 go f a d d e d h i s t o n e s per gram o f re l ax e d G o l E l D N A . I t i s h i g h l y u n l i k e l yt h a t a n y o f t h e s u p e r c o i l i n g i s d u e to t h e g r a t u i t o u s a d d i t i o n o f hi stonesp r e s e n t i n t h e n i c k i n g - c l o s i n g e x t r a c t : w e estimate t h a t t h e a d d i t i o n o fNCE c o n t r i b u t e s t o each r e a c t i o n a t m o s t 0.02 g o f each hi stone per gram o fDNA.

E x p e r i m e n t s s i m i l a r t o t h o s e presented above c a n b e c arri ed o ut u s i n gr e c o n s t i t u t e s o f histones with u n r e l a x e d , s u p e r c o i l e d , D N A . When suchr e c o n s t i t u t e s , c a r r y i n g a mixture o f the f o u r histones a t 1 g total histonesper g r a m o f D N A , a r e tr ea te d w it h N C E , t h e d e p r o t e i n i z e d DN A has t h e s a m e

1 1 6 9


12/23


a b c d e f g h i j k I

Figure 4 . T h e e f f e c t o f varying h i s t o n e s H 3 a n d H 4 on t h e induction o fs u p e r c o i l s . S l o t a a n d b , s u p e r c o i l e d DN A b ef or e a n d after NCE treatment.S l o t s c t h r o u g h I ar e p a i r e d s a m p l e s , before and after treatment with N C E ,o f reconstitutes with v a r y i n g a m o u n t s o f H 3 a n d H 4 , p u r i f i e d a s t h e p a i r ,onto r e l a x e d C o l E l D N A . F o r t h e p a i r , t h e i n p u t a m o u n t per gram o f DNAw a s : c and d , 0 . 4 g ; e a n d f , 0 . 5 g ; g a n d h , 0 . 6 g ; i a n d j , 0 . 8 g ; andk a n d 1 , 1 . 0 g .e l e c t r o p h o r e t i c m o b i l i t y as t h e s t a r t i n g D N A . These results are similar t othose r e p o r t e d b y Germond et a l . ( 1 ) , e x c e p t t h a t t h e e f f i c i e n c y o f p ro-tection ( p e r gram o f h i s t o n e ) i s a b o u t twice a s g r e a t i n our e x p e r i m e n t s .When s u c h reconstitutions ar e carried o ut with other combinations o f h i s t o n e s ,we f i n d again t h a t t h e H 3 / H 4 p a i r i s r e q u i r e d to " p r o t e c t " s u p e r c o i l s . I nt h e a b s e n c e o f o t h e r h i s t o n e s , t h e DNA i s f u l l y p r o t e c t e d a g a i n s t e l e c t r o -p h o r e t i c m o b i l i t y c h a n g e s a t a protein t o DN A w e i g h t ratio o f a p p r o x i m a t e l y0 . 8 t o 1 . 0 . T he se r es ul ts a r e summarized i n T a b l e 1 . A l s o shown in Table 1a r e t h e results o f e x p e r i m e n t s c a r r i e d o u t with s a m p l e s p r e p a r e d with ad i f f e r e n t reconstitution procedure t h a t did not include u r e a , a n d involvedo n l y o n e g r a d i e n t dialysis s t e p ( s e e M a t e r i a l s and Methods). The resultso b t a i n e d with t h i s method o f r e c o n s t i t u t i o n a r e s i m i l a r t o t h o s e o b t a i n e d byour usual m e t h o d s . Both f o r m s o f r e c o n s t i t u t e s "protect" supercoils witht h e same e f f i c i e n c y . H o w e v e r , r e c o n s t i t u t e s f o r m e d without urea r e q u i r e da b o u t 2 5 % more protein p e r u n i t r elax ed DN A t o achieve a given effect i n t h esupercoil induction e x p e r i m e n t s .

1 1 7 0


13/23

N u c l e i c A c i d s R e s e a r c hTable 1

Results o f treating reconstitutes with nicking-closing extractHistones i n Method o f Reconstitution

R e c o n s t i t u t e a A ( u r e a , N a C l ) b B ( N a C l ) bType o f DN A Type o f DN A

I I r I I rH 2 A / H 2 B / H 3 / H 4 ( S ) ++ ++H 2 A / H 2 B / H 3 / H 4 ( P ) ++ ++ ++ ++H 2 A / H 2 B / H 3 / H 4 ( S P ) ++H 3 / H 4 ( S ) +H 3 / H 4 ( P ) + + + +H 2 A / H 2 B ( S )H 2 A / H 2 B ( P ) - - -H 2 A / H 2 B / H 3 ( P )H 2 A / H 2 B / H 4 ( P )H 3 ( P ) cH 4 ( P ) c

++ = c o m p l e t e protection o f supercoils a n d complete induction o fs u p e r c o i l s for reconstitutes onto supercoiled ( I ) a n d r e l a x e d ( I r ) DN Arespectively.+ = r o u g h l y on e h a l f protection a n d i n d u c t i o n o f supercoils ( c f .F i g . 2 )

* = reconstitutes d o n e a t concentrations o f DNA o f both 5 0 a n d 2 0 0p g / m l , o t h e r w i s e reconstitutes were o n l y d o n e a t DN A concentrations o f 5 0p g / m l .( S ) Purified b y " s t r i p p i n g " o f h i s t o n e s .( P ) I n d i v i d u a l l y purified h i s t o n e s .( S P ) Four histone mixture o f t h e two p u r i f i e d " s t r i p p e d " p a i r s .a

I n t h e experiments r ep re se nte d h er e a l l t h e histones w e r e at ani n p u t o f 0.25 g o f each histone per gram o f D N A . For reconstitutesi n v o l v i n g d i f f e r e n t amounts o f H 3 and H 4 s e e F i g u r e 2 .b For different methods o f reconstitution s ee Materials and Methods.c Experiments were performed with 0 . 2 5 g , 0 . 5 g and 1 . 0 g o f eachb i s t o n e per gram o f DNA.

DISCUSSIONThe " i n d u c t i o n " and " p r o t e c t i o n " o f s u p e r c o i l s . A c o v a l e n t l y c l o sed

d o u b l e - s t r a n d e d DN A molecule i s characterized b y i t s t o p o l o g i c a l w i n d i n gn u m b e r , a ( 1 9 ) also termed t h e l i n k i n g n u m b e r , Lk ( 2 0 , 2 1 ) . This q u a n t i t y i s

1 1 7 1


14/23

N u c l e i c A c i d s R e s e a r c han integer and i s invariant with respect to all c h a n g e s that k e e p the co-valent b o n d s o f the b a c k b o n e intact.

Fuller ( 2 1 ) has shown how t o relate Lk t o t h e geometry o f s u p e r h e l i c e s :T h e l i n k i n g number can b e e x p r e s s e d as t h e s u m o f two other q u a n t i t i e s , th ewrithing n u m b e r , W , and th e total twist n u m b e r , T w :

a - Lk = W + Tw ( 1 )W i s determined ( 2 1 , 2 2 ) b y t h e space cu rv e formed b y t h e axis o f t h e dou bl e -stranded h e l i x . For purposes o f t h i s discussion we consider o n l y thedeviations, ALk and A T w , o f these p ar am ete rs f ro m their values i n re l ax e dDNA. Since W X Q f o r relaxed c o v a l e n t l y closed circular D N A , ALk = W + ATw.The exact c o n f o r m a t i o n assumed b y the relaxed circular DN A d oub le h el ix whi c hwe t a k e a s our r e f e r e n c e point varies with s a l t a n d temperature. The di s -crete b a n d s o f t h e g e l e l e c t r o p h o r e t i c patterns c o r r e s p o n d to t h e i n t e g r a lvalues o f Lk ( 1 5 - 1 7 ) . H o w e v e r , t h e actual m o b i l i t y o f each b and i s deter-mi ned b y t h e average s h a p e o f t h e mol ecu lar s p e c i e s with t h a t l i n k i n g nu m b er .Fully r el ax ed c ir cu la r D N A , with W \ ) 0 , h a s t h e smallest m o b i l i t y .

Our experimental results ( F i g s . 2 , 3 a n d 4 ) c a n be e x p l a i n e d in t er m so f these parameters. When histones bi nd to a re l ax e d c o v a l e n t l y c l o sed DN Ac i r c l e , t h e deformations t h e y in duce i n DN A s h a p e and twist will c ontri buteboth t o c h a n g e s i n W and T w . Unless these f o r t u i t o u s l y b a l a n c e , the regionso f DN A t h a t are histone-free m u s t e x p e r i e n c e compensatory d e f o r m a t i o n s thatk e e p t h e l i n k i n g number constant. As Germond et a l . ( 1 ) f i r s t p o i n t e d o u t ,treatment o f t h e complex with n i c k i n g - c l o s i n g enzyme r em ov es t h e s e e n e r -g e t i c a l l y unfavorable d e f o r m a t i o n s i n t h e regions o f DN A not constrained b yh i s t o n e s , resulting i n a molecule with ALk equal t o t h e sum o f t h e changesi n W and Tw induced by t h e histones. I t i s not possible, using t h i s kind o fe x p e r i m e n t , t o separate th e contributions t o ALk made by twisting o f t h edouble helix from those made b y bending o f t h e helix a x i s ( 1 ) . An y dec reas ei n t h e value o f L k from i t s value f o r relaxed circular DN A i s seen a s an" i n d u c t i o n " o f supercoils. The r e s u l t s shown i n Figures 2 , 3 and 4 a r econsistent with t h e series o f events postulated a b o v e . I t i s easy t o applya similar line o f reasoning t o explain t h e "protection" o f s u p e r c o i l s .

Supercoiling and folding i n t h e nucleosome. Crick ( 2 2 ) h a s recentlypointed o u t some o f t h e pitfalls i n attempting t o calculate t h e linkingnu mbe r o f a known structure. Conversely, one must be cautious i n inferringt h e details o f DN A folding from measurements o f t h e linking number. I tmight seem attractive t o s u p p o s e , f o r e x a m p l e , t h a t a closed supercoiledS V 4 0 DNA molecule, a s isolated from t h e c e l l , has a c onformation similar1 1 7 2


15/23

N u c l e i c A c i d s R e s e a r c ht o t h e o n e i t assumes i n t h e n u c l e o s o m e . A s we will s h o w , h o w e v e r , t h i si s n o t t h e c a s e ; t h e t w o DN A s tr uc tu re s m us t d i f f e r m a r k e d l y i n c on-f o r m a t i o n a n d e n e r g y .

Models o f DNA folding i n t h e nucleosome ( 2 3 , 2 4 ) a l l assume t h a t t h eDN A i s wrapped a r o u n d t h e histone c o r e . F o r purposes o f our discussion,we will assume t h a t 1 4 0 base pairs o f DNA are tightly bound t o t h e c o r e ,a s suggested b y previous investigations ( 2 5 - 2 7 ) . We t a k e t h e d i a m e t e r o ft h i s DN A c o i l a s 1 0 0 t o 1 1 0 A ( 2 8 - 3 0 ) . We assume t h a t t h e DN A i s u n i f o r m l ywound a r o u n d a c y l i n d e r o f length equal t o t h e 1 1 0 X d i a m e t e r , s o t h a t1 4 0 base pairs make a b o u t 1 . 4 t u r n s a r o u n d t h e c y l i n d e r . T h i s g i v e s acoil with a pitch o f 8 2 A a n d a pitch a n g l e o f a b o u t 1 2 0 . Such a stru ctu reh a s a p a c k i n g ratio o f 4 . 3 . T h e conclusions we will r each bel ow d o no td e p e n d s tr on gl y up on t h e numerical d e t a i l s o f o u r m o d e l . In f a c t , our m o d e lh a s t h e mi ni mu m uniform DN A curvature p o s s i b l e , consistent wi th n uc le os om ed i m e n s i o n s .

What energy i s r e q u i r e d t o b e n d DNA i n t o t h i s c o n f o r m a t i o n ? Weassume a free energy o f b e n d i n g o f t h e form ( 3 1 )

A G ( 1 / 2 ) B k L , ( 2 )Bwhere B i s a b e n d i n g f o r c e c o n s t a n t , k i s t h e c u r v a t u r e ( t h e inverse o f t h er a d i u s o f c u r v a t u r e ) o f t h e b e n d , and L i s t h e t o t a l l e n g t h o f DN A b e i n gc o n s i d e r e d ( h e r e , 4 7 6 A ) . The cu rvature o f t h e s u p e r h e l i x f o r m e d b y t h eDNA i n t h i s structure i s a b o u t ( 1 / 5 5 ) A 1 ' . The value o f t h e constant Bi s e q u a l t o R T t i m e s t h e persistence length o f DNA i n Angstrom units ( 3 1 -3 3 ) . I f we t a k e t h e persistence length a s 6 0 0 X ( 3 4 , 3 5 ) , B h a s t h e value3 6 0 K c a l - X / r a d - m o l e a t T = 2 9 8 0 K . Alternatively, B can b e d ete rm in ed( 3 2 ) from t h e temperature d e p e n d e n c e o f t h e p e r s i s t e n c e l e n g t h , which g i v e sa value o f about 2 6 0 K c a l - X / r a d 2 - m o l e . Using t h e s e values o f B , we estimatet h e b e n d i n g energy o f DN A p e r mol e o f nucleosome a s 2 0 t o 2 8 K c a l . Note t h a twe have not considered possible c on tr ib utio ns f ro m DN A t w i s t i n g , which couldmake t h e t o t a l free energy o f deformation even l a r g e r .

H o w d o e s t h i s value compare t o the f r e e energy o f s u p e r h e l i x formationi n histone-free D N A ? The free energy AG required t o convert a covalentlyc l o s e d , r el ax ed c ir cu la r DN A t o a molecule with AL k t o p o l o g i c a l t u r n s , hast h e f o r m

AG = A ( A L k ) 2 ( 3 )5where A i s estimated t o have t h e v al ue 0 .12 0 K c a l / m o l e fo r S V 4 0 DN A ( 1 6 , 1 7 , 5 8 ) .Continuing t h e use o f S V 4 0 DN A f o r illustrative purposes, we calculate t h e

1 1 7 3


16/23

N u c l e i c A c i d s R e s e a r c hfree e ne rgy o f supercoiling for the DN A iso lated f r o m m i n i c h r o m o s o m e s , whichhas been shown ( 1 8 ) t o have A L k = - 2 6 i n i t s " n a t i v e " s u p e r c o i l e d state.We find AG = 8 1 Kcal per mol e o f D N A . T h i s ener gy m ust be c o m p a r e d t ot h e bending energy o f 4 0 0 t o 5 6 0 Kcal required t o form 2 0 nucleosomes,t h e approximate number seen i n S V 4 0 mi ni chromos omes in vivo ( 1 , 3 6 , 3 7 ) .

The validity o f t h i s comparison o f c ou rs e d e p e n d s upon t h e correctnesso f equation ( 2 ) f o r A G . Since t h i s equation was originally derived forsmall deformations ( t h o s e with r a d i u s o f c u rvatu re o f t h e order o f a per-sistence l e n g t h ) i t i s possible that t h e value o f 21- 28 K c al per mole o fnucleosome or 400-560 Kcal per mole o f S V 4 0 minichromosome i s a n ov er -estimate o f A G B . I n d e e d , i f minimization o f b e n d i n g energy w e r e a n im-portant consideration i n the formati on o f n u c l e o s o m e s , i t m i g h t b e m o r eadvantageous t o bend t h e DN A b y f o r m i n g about a d oze n kink s ( 3 8 , 3 9 ) , at an**estimated c o s t o f 1 - 2 Kcal per kink i n l o s t energ y o f base s t a c k i n g . Aswe will s h o w , t h e ene rgy o f f o l d i n g derived f r o m histone-histone inter-actions i s probably l a r g e e n o u g h t o overbalance e v e n t h e l a r g e s t e n e r g i e so f b ending discussed h e r e , s o neither mechani s m o f b e n d i n g c a n b e eliminatedf o r energetic r ea s o n s . Whatever t h e b e n d i n g m e c h a n i s m , i t i s l i k e l y thata considerable am o u n t o f energy m u s t be introduced to f o l d supercoiledDNA i n t o nucleosomes: s u p e r c o i l e d DN A i s e n e r g e t i c a l l y o n l y s l i g h t l ym or e f avo rab le than r elaxed DN A f o r n u c l e o s o m e formation.

S u p e r c o i l e d DN A and nucleosome DNA di ffe r n o t o n l y in t h e i r conforma-tional e n e r g y , b u t also i n their s h a p e . Although t h e p rotei n-free S V 4 0 DN Awith ALk = - 2 6 could i n principle assume a left-handed toroidal conformation,most evidence suggests t h a t t h e structure actually ob served i n solution i st h e t o p o l o g i c a l l y e q u i v a l e n t , b u t c o n f o r m a t i o n a l l y distinct, right-handed,i n t e r w o u n d form ( 4 0 , 4 1 ; Camerini-Otero & Felsenfeld, manuscript i n pre-p a r a t i o n ) . T h e dimensions o f t h i s interwound superhelix can b e estimatedf r o m h y d r o d y n a m i c measurements and electron microscopy: th e structure i sr o d l i k e ( 4 0 , 4 1 ) with a large pitch and therefore little curvature ( s e eb e l o w ) . A s we will n ow s h o w , t h i s low curvature accounts for the low freeene rgy o f supercoiling relative t o t h a t required t o form the highly bentDN A o f t h e nucleosome.

The free energy of supercoiling c a n be dec omp os ed i n t o a contributionfrom b e n d i n g , and another from twisting ( 2 1 , 5 8 )

AG = A G B + A G T w ' ( 4 )where A G B i s given by equation ( 2 ) . I n a reg ular interwound superhelix o f1 1 7 4


17/23

N u c l e i c A c i d s R e s e a r c ht o t a l DNA contour l e n g t h L with N s u p e r h e l i c a l turns ( L / 2 l e n g t h and N/2t u r n s i n each direction along t h e s u p e r h e l i x ) , t h e twist r e l a t i v e t o t h erelaxed f or m i s given by ATw = ALk + N p / L , ( 2 1 , 2 2 ) , where ALk i s - 2 6 f o rS V 4 0 and p i s t h e pitch o f t h e superhelix. A G B can also be expressed i nterms o f N a n d p . T he c ur va tur e o f a helical curve o f pitch p an d radius r

2 2 2 2 2 2 2 2 2i s g i v e n b y ( 2 1 ) k = 4 i r / ( 4 i f f r + p 2 ) . Since L = N ( 4 ' r r + p ) , e q u a t i o n( 2 ) can b e written a s2 2 2 2 2 3A G B =2" BN ( L N p ) / L ( 5 )

The calculation o f t h e equilibrium d i m e n s i o n s o f such a uniform inter-wound s u p e r h e l i x i s not i n g e n e r a l s t r a i g h t f o r w a r d since t h e e q u i l i b r i u m , a sFuller ( 2 1 ) has pointed o u t , i s determined b y t h e balance between b e n d i n gan d twis tin g f o r c e s operating u n d e r additional ( a n d u n k n o w n ) steric c o n -s t r a i n t s . At e q u i l i b r i u m t h e n u m b e r o f t u r n s , N , will n o t i n g e n e r a l e q u a lt h e c h a n g e in l i n k i n g n u m b e r , A L k , and i n m o s t c a s e s t h e t o t a l energy o fsupercoiling ( A G s ) will contain both b e n d i n g and to rs io nal contributions.F o r t h e purposes o f our a r g u m e n t , we ar e o n l y i n t e r e s t e d i n e s t i m a t i n g t h ec o n f i g u r a t i o n i n which b e n d i n g makes the maxi mu m p o s s i b l e contribution tot h e f r e e e n e r g y , g i v e n a known value o f A G s . This s t r u c t u r e has t h e maximump o s s i b l e u n i f o r m cu rvature o f D N A , a n d c o r r e s p o n d s to t h e r e q u i r e m e n t t h a tAT w = 0 , s o t h a t a l l t h e s u p e r c o i l i n g energy i s derived f r o m b e n d i n g . [ I fsome o f t h e measured s u p e r c o i l f r e e energy d erive s fr om t o r s i o n , t h e c o n -tr ib uti on f ro m b e n d i n g must b e s m a l l e r , and t h e t o t a l curvature o f t h e DN Ai s also s m a l l e r . We neglect contributions from t h e l e n d s ' o f t h e rodli kes t r u c t u r e . ] When ATw = 0 , AGT will v a n i s h , and N p / L = 2 6 . F rom equation( 3 ) , we f i n d AG = 8 1 , and since L = 18270 X f o r S V 4 0 DN A ( c a l c u l a t e d froma molecular weight o f 3 . 6 x 1 0 d a l t o n s ( 4 2 ) ) , substitution i n equation ( 5 )g i v e s N X 3 0 , p X 5 0 0 A , and r X 5 0 . Thus t h e dimensions o f t h i s regularsuperhelix are very different from t h e o n e s req uired f o r nu c l eos omef o r m a t i o n . Even i f t h e left-handed to ro id al f or m o f S V 4 0 DN A wer e morestable t h a n t h e r ig ht -h an d ed i nt er wo un d f o r m , i t i s evid ent f rom sim ilararguments t h a t t h e DNA could n o t be highly c u r v e d . The arguments abouts h a p e d i f f e r e n c e are simply another way o f emphasizing t h e differences i nenergy o f curvature between DNA i n S V 4 0 s up er co il s an d DNA i n nucleosomes.

Finally, t h e an alys is given ab ove should alert u s t o t h e possibilityt h a t while certain h i s t o n e s might well be able t o induce a gentle fold i nt h e DN A ( s u c h as i s present in an S V 4 0 DN A superhelical t u r n ) they mightnot be able t o completelely fold t h e DN A i n t o a nucleosome. These t w o

1 1 7 5


18/23

N u c l e i c A c i d s R e s e a r c hprocesses are e n e r g e t i c a l l y d i s t i n c t : t h e f i r s t involves r e l a t i v e l yl i t t l e energy while t h e l a t t e r r e q u i r e s c o n s i d e r a b l y m o r e e n e r g y . A l s o ,since i t i s most l i k e l y t h a t t h e f i n a l f o l d i n g o f t h e DNA i n t o a nucleosomei s accompanied b y a further i n d u c t i o n o f s u p e r c o i l s , histones t h a t d o notc a r r y o u t t h i s f i n a l compaction m i g h t n o t i n d u c e s u p e r c o i l s as e f f i c i e n t l ya s those t h a t d o .

Hi stone-Hi stone interactions and nucleosome structure. Are lowerenergy k i n k s p r e f e r a b l e t o h i g h e r energy continuous d e f o r m a t i o n s o f D N A ?I s i t necessary t h a t n uc le os om e m od el s i n v o k e t h e l a r g e s t p o s s i b l e numbero f histone-histone i n t e r a c t i o n s ? In t h i s section we suggest t h a t i n f a c tm an y d if fer en t mo dels o f nucleosome structure can b e a c c o m m o d a t e d becauseo f t h e large energies a v a i l a b l e from interactions between h i s t o n e s .

I t i s n ow g e n e r a l l y a c c e p t e d ( 2 3 , 4 3 - 4 5 ) t h a t each histone mol ecu l econsists o f two k i n d s o f d o m a i n s : a h i g h l y b a s i c p o r t i o n that interactsprimarily with t h e D N A , a n d a h y d r o p h o b i c r e g i o n which i s t h e site o fhistone-histone interactions. We n o w as s u m e t h a t A G ' , t h e total f r e e energyo f f o l d i n g , can b e d e c o m p o s e d i n t o two p a r t s , t h a t due to t h e p r i n c i p a l l yi o n i c histone-DNA i n t e r a c t i o n s , A G ' , a n d t h a t d u e to p r i n c i p a l l y h y d r o p h o b i chistone-histone i n t e r a c t i o n s , A G ' , s o t h a t A G ' = A G O + A G ' . We furthera s s u m e t h a t a t p h y s i o l o g i c i o n i c s t r e n g t h s A G ! ( i n c o r p o r a t i n g t h e freeenergy o f approximately 2 0 s a l t l i n k a g e s p e r h i s t o n e ) i s mu ch l a r g e r thanA G O ( 4 6 , 4 7 ) .

We n e x t imagine t h a t we s t a r t with a folded nucleosome, and u n f o l d t h ep ar ti cl e wh il e s t i l l keeping t h e h is to ne s b oun d t o t h e i r initial sites o nt h e D N A . The histone-histone interaction f r e e energy lost during t h i sunfolding i s t h e f r e e energy we wish t o e s t i m a t e . Though we d o n o t k n ow t h enature o f t h e c o n t a c t s , we will suppose t h a t t h e forces hol di ng t h e nu c l eo-s o m e i n i t s folded form a r e derived f ro m t h e histone-histone hydrophobici n t e r a c t i o n s measured i n s o l u t i o n . I n t h e discussion t h a t follows we estimatet h e f r e e energy f o r a DNA-bound h is to ne c om pl ex undergoing a n internalr e a r r a n g e m e n t : ( u n f o l d e d nucleohistone c o m p l e x ) - + ( f o l d e d nucleosome). I nt h e unfolded c o m p l e x , s o m e histone dimer i nte ra ct io ns m a y already e x i s t .S i n c e we assume t h a t t h e DNA o f t h e unfolded structure i s completely u n b e n t ,neither t h e s e i n t e r a c t i o n s nor t h e i o n i c i n t e r a c t i o n s will contribute t o t h ef r e e energy o f f o l d i n g .

D ' A n n a and Isenberg ( 4 8 ) and others ( 4 9 , 5 0 ) have studied t h e pairwiseinteractions o f t h e four histones i n solution. They found t h a t f o u rp a i r s o f histones i n t e r a c t strongly a n d i n t h e following order o f strength

1 1 7 6


19/23

N u c l e i c A c i d s R e s e a r c h( 4 8 ) : H 3 / H 4 > H2B/H4 X H2A/H2B > H 3 / H 2 A . Using t h e data o f D'Anna andIsenberg ( 4 8 ) a n d Roark et a l . ( 5 0 ) one can estimate t h a t t h e unitaryf r e e energy ( 5 1 ) f o r p a i r formation ranges from - 1 6 Kcal to - 9 K c a l , o ron t h e average about - 1 2 Kcal per mole o f d i m e r . Since two monomersbound to DN A will have a more favorable free energy o f dimerization thanwhen f r e e in solution, t h i s energy represents t h e m i n i m u m energy availablef o r pair f o r m a t i o n .

Ignori ng nonideality, and assuming a t r u e equilibrium, there are a tl e a s t two f a c t o r s which will increase t h e favorable free energy o f i n te r -action o f t h e bound histone pair relative to t h a t f o r histones i n solution.F i r s t , t h e electrostatic repu l si on energy o f highly p o si ti ve l y c ha rg edhistone molecules interacting i n solution i s reduced when the histonesare bound to D N A . We estimate t h a t this contributes a t least - 5 Kcal/moleto AGH ( 5 2 , 5 3 ) . Most importantly, we m u s t consider those contributionsto A G H arising because t h e histones t h a t remain b o u n d to DN A upond i s s o c i a t i o n d o n o t regain their f ull translational and rotationalentropies as t h e y d o when t h e y di s s oc i ate in solution ( 5 4 ) . This a lsoi n c r e a s e s the available free e n e r g y o f association. T he c h a n g e in e n t r o p yf o r t h e formation of protein di me rs has b e e n ca lcu lated f o r other p u r p o s e sb y Doty and Myers ( 5 2 ) and Chothia and Janin ( 5 5 ) . I f , when b o u n d to theD N A , t h e hydrophobic portions o f t h e histones are as immob ile in themonomers as i n t h e d i m e r , t h i s contribution could be as large as - 2 5Kcal/mole o f d i m e r . Allowing f o r some i nc reas e d mob ility in the monomer,a n d f o r additional internal vibrational and rotati onal m o d e s in thed i m e r , would de c reas e the magnitu de of this number. It is not u n r e a s o n a b l eto suppose, however, that after taking account o f these t w o effects, theeffective A G H for d i m e r f or ma ti on w hi le the hi stones r emain DNA-bou ndcould b e at l e a s t - 2 0 Kcal/mole o f dimer.

We do n o t k n o w how many histone-histone c o nt a ct s are formed during thef o l d i n g reaction we have considered. I f several o f t h e D'Anna and Isenbergs t r o n g interactions are involved i n t h e folding reaction i t i s clear t h a tt h e energy derived f r o m t h e s e interactions ( w h e t h e r corrected ( - 2 0 K c a l /per mol e o f d i m e r ) or n o t ( - 1 2 Kcal/per mol e o f d i m e r ) ) can supply more thane n o u g h energy to d e f o r m t h e DN A ( 2 0 to 2 8 K c a l ) . More i n t e r e s t i n g l y , how-ever, our calculations show t h a t even i f m o s t of these s t r o n g interactionswere involved in f o r m i n g a s t r u c t u r e such as a c y c l i c heterotypic t e t r a m e r( 4 5 ) which did n o t fol d D N A , one or two histone pair i n te r ac ti o ns w ou l d b esufficient to stabilize t h e f ol de d n uc l eo so m e s t r u c t u r e re l ati ve to the

1 1 7 7


20/23

N u c l e i c A c i d s R e s e a r c hunfolded f o r m . I n o n e o f t h e s i m p l e s t versions o f s u c h a m o d e l t h i s f o l d i n gc o u l d b e i m a g i n e d t o arise from t h e dimerization o f t h e s e t w o u n f o l d e dh a l f - n u c l e o s o m e s . With t h e a b o v e mentioned corrections, t h e relevant his-tone interactions might be t h e relatively weak reaction 2 ( H 3 / H 4 ) - * ( H 3 / H 4 ) 2( - 4 K c a l / m o l e o f product i n free solution ( 5 0 ) ) . Our estimates suggestt h a t structures stabilized by only one o r t w o histone pair interactions areenergetically r e a s o n a b l e , and t h a t t h e y m ig ht in vo lve a delicate balancebetween t h e opposing f o r c e s o f p a i r formation a n d DNA b e n d i n g . Such asituation might b e a d v a n t a g e o u s f o r a biological system i n which nucleosomeu n f o l d i n g ( d u r i n g r e p l i c a t i o n , f o r e x a m p l e ) might o c c u r .

Reconstitutes o f t h e arginine-rich h i s t o n e s . T h e observation t h a t as t r o n g l y interacting tetramer o f t h e arginine-rich histones, H 3 a n d H 4 ,f o r m s i n s o l u t i o n ( 4 8 , 4 9 ) l e d Kornberg ( 2 4 ) t o suggest t h a t s u c h a tetramerh a s a central role i n nu c l eos ome s t r u c t u r e . Only r e c e n t l y , however, hasd i r e c t evidence f o r t h e structural importance o f t h e argi ni ne-ri ch histonesbecome a v a i l a b l e . We have s h o w n t h a t t h e a r g i n i n e - r i c h h i s t o n e s , whenreconstituted onto D N A , are unique i n t h e i r ability t o create structureswith many o f t h e features o f native c h r o m a t i n or nucleosomes. I n par-t i c u l a r , c o m p l e x e s o f DN A with H 3 a n d H 4 , when d i g e s t e d with a v a r i e t y o fnucleases o r proteases, y i e l d discrete DNA and p r o t e i n f r a g m e n t s similar t ot h o s e o b t a i n e d b y d i g e s t i o n o f c h r o m a t i n . T h e presence o f H 3 a n d H 4 i snecessary a n d s u f f i c i e n t t o obtain s uc h r es ul ts ( 2 , 3 ) .

We a l s o examined t h e kinetics o f digestion o f complexes o f DN A wi th H 3a n d H 4 , a n d f o u n d t h a t a t early t i m e s o f digestion with either staphylo-coccal nuclease o r DNAase I , fragments a s large a s 1 3 0 nucleotide pairs and1 8 0 nucleotides, r e s p e c t i v e l y , a r e produced a s transient i n t e r m e d i a t e s . O nt h e basis o f t h e s e f i n d i n g s we concluded t h a t m o s t o f t h e DN A o f t h enucleosome i s i n f a c t interacting with t h e s e histones ( 2 , 3 ) . We havep r o p o s e d t h a t t h e a r g i n i n e - r i c h histone-DNA c o m p l e x f o r m s a k e r n e l o n whicht h e slightly l y s i n e - r i c h histones ( H 2 A a n d H 2 B ) can c o m p l e t e t h e n u c l e o s o m e .This evidence i s supported by t h e results o f Boseley e t a l . ( 5 6 ) , who haveshown t h a t certain H 3 / H 4 / D N A c o m p l e x e s h a v e many o f t h e X-ray diffractionproperties o f whole c h r o m a t i n . The data i n Figure 1 are clearly consistentwith t h e important role we have suggested f o r t h e arginine-rich histones.

The results i n Figures 2 - 4 show t h a t only those reconstitutes c o n -taining both H 3 a n d H 4 deform relaxed circular DN A i n a way which leads toa change i n l i n k i n g number upon treatment with n i c k i n g - c l o s i n g e n z y m e . W ec a n n o t , o f c o u r s e , s how t h a t other combinations o f hi stones d o not f o r m

1 1 7 8


21/23

N u c l e i c A c i d s R e s e a r c hs i g n i f i c a n t structures t h a t d i s t o r t DN A l o c a l l y , b u t i f t h e y d o , t h e dis-tortion m u s t involve e q u a l a n d opposite c h a n g e s i n W a n d Tw which d o nota f f e c t L k .

We a l s o c a n n o t rule o u t t h e possibility t h a t other reconstitutionmethods would reveal presently u n detected c o nt ri b ut io n s t o structure fromhistone combinations l a c k i n g H 3 a n d H 4 . On t h e o t h e r h a n d , t h e urea -s a l t g r a d i e n t method o f reconstitution u s e d h er e wo rk s well when anucleosome-equivalent o f t h e f o u r histones ( H 2 A / H 2 B / H 3 / H 4 ) i s combinedwith a DNA fragment 1 4 0 base pairs l o n g . Particles f o r m e d in t h i s way ar eidentical t o native nucleosomes i n t h e i r n u c l e a s e digestion patterns a n dhave i d e n t i c a l sedimentation properties ( S i m o n , Camerini-Otero & F e l s e n f e l d ,manuscript i n p r e p a r a t i o n ) . Furthermore, t h i s method has been u s e d withp a r t i a l c o m p l e m e n t s o f histones t o p r o d u c e c o m p l e x e s which ar e indistin-g u i s h a b l e , b y a variety o f p r o b e s , from those o b t a i n e d b y s t r i p p i n g selectedhis to ne s f ro m whole chromatin ( 2 , 3 ) . I t should a l s o b e p o i n t e d out t h a tuse o f a quite different reconstitution method h a s n o effect upon t h eresults o f s u p e r c o i l i n g e x p e r i m e n t s ( T a b l e 1 ) .

The results shown in Figure 4 suggest t h a t H 3 a n d H 4 ar e as effectivef o r s u p e r c o i l i n d u c t i o n , o n a w e i g h t b a s i s , as a mixture o f t h e fourhistones ( 5 7 ) . A n u c l e o s o m e - e q u i v a l e n t o f H 3 a n d H4 ( 0 . 5 g per gram o fD N A ) i n d u c e s about h a l f a s mu ch s u p e r c o i l i n g as a nucleosome e q u i v a l e n t o ft h e four histones ( 1 g per gram o f D N A ) . I f we take i n t o a ccou nt t h ep o s s i b i l i t y t h a t n o t every histone m olec ule m ay r eco mb in e perfectly, t h i sr e s u l t must b e interpreted a s a l o w e r l i m i t t o t h e efficiency o f t h e H 3 / H 4p a i r i n i n d u c t i o n o f supercoils.

I t i s quite p o s s i b l e , h o w e v e r , t h a t eve n under t h e best o f circum-stances a n u c l e o s o m e - e q u i v a l e n t o f the a r gi ni ne -r ic h h is to n es ( 0 . 5 g p e rgram o f D N A ) c an no t in duc e full s up er co il in g, a nd t h a t only when t h enucleosome i s c om pl ete d b y t h e s li gh tl y l y si ne -r ic h h is to ne s wi ll t h e DNAb e c o m p l e t e l y supercoiled. I n a n y c a s e , t h e data on t h e induction andprotection o f s up er co il s b y t h e a r gi ni n e- r ic h h is to n es i s consistent witht h e r es ul ts o bta in ed with other techniques. The data taken a s a wholestrongly support t h e concept t h a t these hi sto ne s p la y t h e c en tr al r ol e i nnucleosome structure.ACKNOWLEDGMENTS

W e thank D r . Rob ert Bird f o r help i n preparing C o l E l DNA a n d D r .Richard Simon f o r v al ua b le d is cu ss io n s a n d a d v i c e . We are also indebted t oD r . James McGhee f o r several valuable suggestions and a critical reading o f

1 1 7 9


22/23

N u c l e i c A c i d s R e s e a r c ht h e manuscript a n d t o M r s . Barbara De Larc o f o r help i n p repari ng t h i smanuscript.REFEREN CES

Dedicated t o t h e me mory o f Jerome Vinograd, a goo d f riend a n d t e a c h e r .t A preliminary re p ort o f some o f these results was presented at a Sy mposiumo n Chromatin Structure and Function at the First International C ongre s s onC e l l B i o l o g y , Boston, Massachusetts, September 6 , 1 9 7 6 . Felsenfeld, G . ,Camerini-Otero, R . D . a n d Sollner-Webb, B . ( 1 9 7 7 ) i n " In te r na ti o na l C el lBiology 1 9 7 6 - 1 9 7 7 " . Rockefeller University Press, B . Brink ley and K .Porter ( e d s . ) , i n press.

I t i s o f course als o p os sib le that the kinking mechani s m i s i nvol ved i n DN Abending in s o l u t i o n , a n d t h a t t h e c onstant B o f equation ( 2 ) i t s e l f r e f l e c t st h e kinking process. Presently avai l abl e data do not p e rmit us to dis-t i n g u i s h k i n k i n g f r o m continuous b e n d i n g .1 G e r m o n d , J . E . , H i r t , B . , O u d e t , P . , G r o s s - B e l l a r d , M . a n d C h a m b o n , P .( 1 9 7 5 ) P r o c . N a t . A c a d . S c i . U.S.A. 7 2 , 1 8 4 3 - 1 8 4 7 .2 Camerini-Otero, R . D . , Sollner-Webb, B . and Felsenfeld, G . ( 1 9 7 6 )Cell 8 , 3 3 3 - 3 4 7 .3 S o l l n e r - W e b b , B . , C a m e r i n i - O t e r o , R . D . a n d F e l s e n f e l d , G . ( 1 9 7 6 )C e l l 9 , 1 7 9 - 1 9 3 .4 C l e w e l l , D . B . and Helinski, D . R . ( 1 9 6 9 ) Proc. Nat. Acad. S c i . U.S.A.6 2 , 1159-1166.5 S a k a k i b a r a , Y . and T o m i z a w a , J . ( 1 9 7 4 ) Proc. N a t . Acad. S c i . U.S.A.

7 1 , 802-806.6 Van d e r W e s t h u y z e n , D . R . , B o h m , E . L . and Vo n H o l t , C . ( 1 9 7 4 ) BiochimB i o p h y s . Acta 3 5 9 , 341-345.7 R u i z - C a r r i l l o , A . a n d A l l f r e y , V . G . ( 1 9 7 3 ) A r c h . Biochem. B i o p h y s .

1 5 4 , 1 8 5 - 1 9 1 .8 L o w r y , 0 . H . , R o s e b r o u g h , N . J . , F a r r , A . L . and R a n d a l l , R . J . ( 1 9 5 1 )

J . B i o l . C h e m . 1 9 3 , 2 6 5 - 2 7 5 .9 Champoux, J . J . a n d D u l b e c e o , R . ( 1 9 7 2 ) P r o c . N a t . A c a d . S c i . U.S.A.6 9 , 1 4 3 - 1 4 6 .1 0 Baase, W . A . a n d Wang, J . C . ( 1 9 7 4 ) Biochemistry 1 3 , 4299-4303.1 1 Keller, W . ( 1 9 7 5 ) P r o c . N a t . Acad. S c i . U.S.A. 7 2 , 2550-2554.1 2 Pulleyblank, D . E . and Morgan, A . R . ( 1 9 7 4 ) Biochemistry 1 4 , 5205-5 2 0 9 .1 3 V o s b e r g , H . P . , Grossman, L . I . and V i n o g r a d , J . ( 1 9 7 5 ) E u r . J . B i o c h e m .5 5 , 7 9 - 9 3 .1 4 A x e l , R . , M e l c h i o r , W . , S o l l n e r - W e b b , B . a n d F e l s e n f e l d , G . ( 1 9 7 4 )

Proc. N a t . A c a d . S c i . U.S.A. 7 1 , 4 1 0 1 - 4 1 0 5 .1 5 K e l l e r , W . a n d W e n d e l , I . ( 1 9 7 4 ) C o l d Spring Harbor S y m p . Quant.B i o l . 3 9 , 1 9 9 - 2 0 8 .1 6 Depew, R . E . a n d Wang, J . C . ( 1 9 7 5 ) P r o c . N a t . Acad. S c i . U.S.A. 7 2 ,4 2 7 5 - 4 2 7 9 .

1 7 Pulleyblank, D . E . , Shure, M . , Tang, D . , V i nograd, J . and Vosberg, H .P . ( 1 9 7 5 ) P r o c . N a t . A c a d . S c i . U.S.A. 7 2 4 2 8 0 - 4 2 8 4 .

1 8 Shure, M . and V i n o g r a d , J . ( 1 9 7 6 ) C el l 8 , 2 1 5 - 2 2 6 .1 9 Vinograd, J . and L e b o w i t z , J . ( 1 9 6 6 ) J . Gen. P h y s i o l . 4 9 , 1 0 3 - 1 2 5 .2 0 Glaubiger, D . and Hearst, J . E . ( 1 9 6 7 ) Biopoly mers 5 , 6 9 1 - 6 9 6 .2 1 Fuller, F . B . ( 1 9 7 1 ) Proc. N a t . A c a d . S c i . U.S.A. 6 8 , 8 1 5 - 8 1 9 .2 2 Crick, F . H . C . ( 1 9 7 6 ) Proc. N a t . Acad. S c i . U.S.A. 7 3 , 2 6 3 9 - 2 6 4 3 .2 3 Van Holde, K . E . , Sahasrabuddhe, C . G . and S h a w , B . R . ( 1 9 7 4 ) N u e l .Acids Res. 1 , 1 5 7 9 - 1 5 8 6 .2 4 K ornbe rg, R . ( 1 9 7 4 ) Science 1 8 4 , 8 6 8 - 8 7 1 .

1 1 8 0


23/23

N u c l e i c A c i d s R e s e a r c h2 5 Sollner-Webb, B . and Felsenfeld, G . ( 1 9 7 5 ) Biochemistry 1 4 , 2915-2920.2 6 Axel, R . ( 1 9 7 5 ) Biochemistry 1 4 , 29 21- 29 25 .2 7 S h a w , B . R . , Herman, T . M . , Kovacik, R . T . , Beaudreau, G . S . a n d VanHolde, K . E . ( 1 9 7 6 ) Proc. Nat. Aca d. S c i . U.S.A. 7 3 , 505-509.2 8 Baldwin, J . P . , Boseley, P . G . , Bradbury, E . M . and I b e l , K . ( 1 9 7 5 )Nature 2 5 3 , 245-249.2 9 Pardon, J . F . , Wo r cest er , D . L., Wooley, J . C . , Tatchell, K . , V a nHolde, K . E . and Richards, B . M . ( 1 9 7 5 ) Nucl. A c i ds R e s . 2 , 2 1 6 3 -2 1 7 6 .3 0 Bradbury, E . M., Hjelm, R . P . , Carpenter, B . G . , Baldwin, J . P . andHancock, R . ( 1 9 7 7 ) in Molecula r Biology of the Ma m m a l i a n G en eticApparatus, in p r e s s .3 1 Landau, L . D . an d Lifshitz, E . M . ( 1 9 5 8 ) Stati s ti c al Physics p p . 478-4 8 2 , Addison-Wesley, Reading, Mass.3 2 Gray, H . B . an d Hearst, J . E . ( 1 9 6 8 ) J . Mol. Biol. 3 5 , 1 1 1 - 1 2 9 .3 3 Schellman, J . A . ( 1 9 7 4 ) Biopoly mers 1 3 , 2 1 7 - 2 2 6 .3 4 Jolly, D . an d Eisenberg, H . ( 1 9 7 6 ) Biopoly mers 1 5 , 61-95.3 5 Godfrey, J . E . an d Eisenberg, H . ( 1 9 7 6 ) Biophys. C h e m . 5 , 301-318.3 6 Griffith, J . ( 1 9 7 5 ) S c i e n c e 1 8 7 , 1 2 0 2 -1 2 0 3 .3 7 Cremisi, C . , Pignatti, P. F . , Croissant, 0. a n d Yaniv, M. ( 1 9 7 6 ) J .Virol. 1 7 , 204-211.3 8 Crick, F . H . C. a n d K l u g , A. ( 1 9 7 5 ) N a t u r e 2 5 5 , 5 3 0 - 5 3 3 .3 9 Sobell, H. M . , T s a i , C . C . , Gilbert, S . G . , J a i n , S . C. a n d Sakore,T . D . ( 1 9 7 6 ) Proc. Nat. Ac ad . Sci. U .S .A . 7 3 , 3 0 6 8 - 3 0 7 2 .4 0 Gray, H . B . , Jr . ( 1 9 6 7 ) Biopoly mers 5 , 1 0 0 9-1 0 1 9.4 1 Upholt, W . B . , Gray, H. B . a n d Vinograd, J. ( 1 9 7 1 ) J. Mo l . B i o l . 6 1 ,2 1 - 3 8 .4 2 T a i , H . T . , S m i t h , C . A . , Sharp, P. A. a n d Vinograd, J. (1972) J .Virol. 9 , 3 1 7 - 3 2 5 .4 3 Bradbury, E . M. a n d Rattle, H. W. E. ( 1 9 7 2 ) E u r . J. B i o c h e m . 2 7 ,270-281.4 4 De Lange, R . J . an d S m i t h , E . L. ( 1 9 7 5 ) i n C i b a Sy mposium on Str u c tu r eand F u n c t i o n o f C h r o m a t i n 2 8 , 59-70.4 5 Weintraub, H . , Worcel, A. an d Alberts, B . ( 1 9 7 6 ) C e l l 9 , 4 09 - 4 1 7 .4 6 Bradbury, E . M., Cary, P . D . , Crane-Robinson, C . , Rattle, H. W. E . ,Boublik, M. a n d S a u t i e r e , P. ( 1 9 7 5 ) Biochemistry 1 4 , 1 8 7 6 - 1 8 8 5 .4 7 Record, M . T . , Lohman, T . M . , de Haseth, P . ( 1 9 7 6 ) J . Mol. Biol.1 0 7 , 145-158.4 8 D'Anna, J . A . and Isenberg, I . ( 1 9 7 4 ) Bio ch emist r y 13 , 4 9 9 2 - 4 9 9 7 .4 9 Kornberg, R . D . an d Th o m a s , J. 0. ( 1 9 7 4 ) S c i e n c e 1 8 4 , 8 6 5 - 8 6 8 .50 Roark, D . E . , Geoghegan, T . E . a n d K e l l e r , G . H . (1974) Biochem.Biophys. Res. Commun. 5 9 , 542-547.51 Kauzmann, W . ( 1 9 5 9 ) in Advances in P r o t e i n C h e m i s t r y 14, p. 3 5 ,Academic Press, New York.5 2 Doty, P . and Myers, G . E . ( 1 9 5 2 ) D i s c u s s i o n s F a r a d a y S o c . 1 3 , 5 1 - 5 8 .5 3 Tanford, C . ( 1 9 6 1 ) Physical C he m i s tr y o f M a c r o m o l e c u l e s . pp. 519-521John Wi l e y and Sons, N e w Y o r k .5 4 Page, M . I . an d J en c k s , W. P. ( 1 9 7 1 ) P r o c . N a t . Acad. S c i . U.S.A.6 8 , 1678-1683.5 5 Chothia, C . and Janin, J . ( 1 9 7 5 ) N a t u r e 2 56, 7 0 5 - 7 0 8 .56 Boseley, P . G . , Bradbury, E . M. , B u tl e r -B r o wn e , G . S. , C a r p e n t e r , B .G . and Stephens, R . M. ( 1 9 7 6 ) Eur. J. B i o c h e m . 6 2 , 2 1 - 3 1 .5 7 That H3 and H4 can induce s u p e r c o i l s in DN A h a s b e e n r e c e n t l y con-fi rme d b y M . Bina-Stein and R . Simpso n (personal c o m mu nication).58 Bauet, W . and Vinograd, J . ( 1 9 7 0 ) J . Mol. B i o l . 47, 419-435.

Documents

R.Daniel Camerini-Otero and Gary Felsenfeld- Supercoiling energy and nucleosome formation: the role of the arginine-rich histone kerne