Genes, Genomesa,td Genomics aznt ourots"i,onr**"

Transition from DNA Looping to Simple Binding or DNA

Pairing in GeneRegulation and Replication:
A Matter of Numbersfor tbe Cell

MichèleÂmouyal

-.
.--,s;;'"ffJi:i:#Jl'i"ij.'
ABSTRACT
RèpÈssionof ùe l. .dli læros opercnis æhi.ved rhoughDNA loôpingerd rh@ ope6to6 at the phlsiolo8icâl .epressr @ncenràrion.
ln smins ole.prod$ing the rcpese. or ûth plænids wi(h ! hieh opy nmbê.. lhe coilpe.drile modeof.epresion is naked b) orh.r
môdes 1, erd. trten Éveral DNA molæuls æ pÉsem DNA loop fomation is Fgta.ed br intê|mlsule dscialions slill mediared
b) the lac leprcssr ln b€r|ria such associarion\ kmm æ 'hùd@fling' ùd redialal bI tlæ innidor prctcin. &e obsèûed in
;Dlicarion of rne ilmn-clds of plavnids Wh€nno<terôlemours of ininalor ând its bindinglo the Eplicalion ongin (æhievedin ente
iftnrn.es. b) DNA lmpin8) allow Eplicarion ro prlcréd. high ænertrations prevenrÈpli@rio. od l@d to hsndcùfnt8 thal ænÈlls rhc
nùnbe. ofpldmids. ID principl€.wh€n DNA looping is f6ible. DNA poidns is âie Possibleifnorc thù one DNÀ moleÙle is Pl*nr
in rhe etl ln eut3ryotes,rhe actionofrh€ C.TCFprcr.in is paniolùly rcpèentalile ofthis situâtio.. nris key @nF)nènr of elemnrs
ùal insulde geneèxp6sio. from th€ slmuding 8Énomiceffects in von€brales.âle æts 6 ù oryani2rr of higb€rcnlcr chremann
slfuc{lm al lhe Èglobin ed /g,?H/9 lei- Ar this lsdet l()s, qlaF @ntrclsEenonicimpnnlit8 bl DN^ lmpin8 Reent dlia sug8sl
rhargcmmicimprinrin8dd momÀllelicexpNion niphl sie be6n1rcll.dlhruugh.hromoemc Pairing

X.,\Tonh: cl Cl'. lâ.1o* op€o oligomsutiot plMid eplicarioo

CONTENTS

I. FROMT1I[ NIIMBEROF L{C OPERITIOR SITESAND REPRI]SSOR MOLECULES TO DNA LOOPINCAND TO MTII Il'
oPERATORREPRESSTON Ot THE E. COI1LlCOPERON.......-............. 105
2. RÊPLICATION OF THE ITERON{I-ASSOF PLASMIDSAND CONTROLOF'HI]IR COPYNIJMBT]R IN BAC'I'ERIA".' IO8
J CONTROI- OF CCNOMICIMPRINTINCBY DNAT-OOPING OR CHROMOSOMf, PA]RINGIN MAMMAI,S ' .,'.'-."".. ' IO8

Molsular biolos, has been firsl and nonnally interEsted in
identiryins the componcnts that arc involvcd iî the bsic
cellulâr processes. Whcn it was not€d lhal somc elcrnents
wcr. in vârioùs conccntmlions, narurally or âniticially. duc
to i rodùced genebc mutâtiods, it was css€ntiâlly dêscrip-
tve. Thus. for DNA-protcin intetrationsinvolvcd in g€ne
expr€ssion and thei. regulalion, the genersl idcâ w&s thâl
the DroÈin had to be in sufficient âmount in th€ ccll to cn-
surebinding to the corespondingsite and gencnte ûe pm-
c€ssof inlerest. ln bactcria for instânc€, it seem.d unimDor-
t nt to us€ strâins ovcrproducing a traft.ription factor
instead offte wild-typ€ slrain and physiologicâl ârnountsof
prorein. And b€câuseit is eâsier technically, multi-copy
Dlâsnidsare ofien usedin Dlaceof chromosomâlconsEucts.
Sinc€ then. il has appeâred in sev€ral instaîces that the
number of copies of a gene or of â protein wss eblc to
chùge lh€ regulationprocess-
One enmpl€ is pmvided by .he t. colt n'rlti-site /ac
repression. From th€ approach eûploy€d to demonsFat€th€
fêÀsibiliù of DNA looping betwc€n /cc rÊplecror and two
/r. opeEton on thê sâme moleculc, it wes cld thâr DNA
looping could t'€ concentralion-depend€nl (lkâmer el a/.
1987: Amoùyal 1991). When mediatedby /æ rcpr€ssor,
D^_A læpina is lost with increased conceû'ariôns of reF
ressor ând DNA ûrolecul€s to the benefit of other int€mct-
lions. independenlbinding to the op€ratorsor inlermolec-
ular associations.This findinr was â decisive elemenr
towards th€ uncov€ring of the ii;octiôùâliry oflhe secondâry
DNA looDinsis nor restricredlo Fânsffiplion. ll i< âl$
involved in ieolierion. to initiâte it for the bacterialRox
Dlâsmid{Muci.h€ri€t er a./. l98Er Viron g/ r'/ lqell or on
itre commn ro sto-oir for rne / . .o/i F-factormd to limtr ,ts
numberto ône coiy (cf. zuma, ând Bastia2005).The ini-
tiâror Droteinbridaci lh€s. loops. Hoqevêr. the cop) num_
brr odthe itercn+lassofplÀsmidsis generall) controlledb'
-hândcumnp-, i.e. intermolecularaçsociâlionsbridged b'
the initiator-prot€in(Palând Chanoraj 1988;McEachemer
al- l9E9l.
Int€moleculâr àssociations are âlso r€present€d by the
Dairins of chrcmosomesfor rhe @otdinatedresularionor
i h e i r ; e n e s .l h c C T C F D r o t e i ni 5 i n l o l \ e d i n Ê e r c m r cr m -
prinriig of rhe nalnmalian td2 Hta locus lt does so b!
ionlro ing lhe targelof lhe enhâncershæd b) lhe r*o im_
prinæd genesrlmugtr attemateD\A looping 4d meth'l-
;dôn ôfihe CrCF bindinq sne lMunell cr J. 20M1. lt also
DaniciDâtes in chmmoso;al âçsociâlions(Ling er d/. 2006:
zhg.o tt al.?.006\.
The inrerDlat between DNA lmping. simple binding
and DNA pairinB i5 de!elop€d for lhesethrÊespecrficsitu-
I. FROI THÊiIUiIBER OF LACOPERAÎOR plâyed some coopemtivit). lt is closer lo â ldc .egulation
SITESA}ID REPRESSOROLËCULËS 10 DT{A sinc€ a /d. p.omoter controlling /acz expr€s,lion .Êplaced
LOOPIIIGAND TO i'ULIIOPERATOR the r/p pmnot€r-op€ralor on â plâsmid. However, lhe /dc02
REPRESSDI{OF THÊE. COL'LACOPÊROI{ and /æOJ sites \rer€ noi rÉmoved, since lhey w€rÊ lhouShl
to b€ inactive. Tbe /nc promoter was surroûnded by two
It has beo knom since the origin of rnol€.ular biology "ideâI" la? opentor shes with a ten-fold incrêaçêd atrniiy
(Jacob ând Monod 196l) that the /ac op€rDn is repress€d tor the r€pressor.as compârEd!o lhe /.rr r/ site (Sadler el a/.
fiom a 2l bp operator sequence,O./. locât€d on th€ promo- l98J). tT€ studywô câried out in€ /r'c,1"strainproduc'n8
ter rÊgion and ceniered at I I I with r€sp€ci io ihe tr|ns.rip- €v€. mo.€ reDr€ssôr thân the /der? strâin. about 100-fold
tion siân. A proteiî. Lâcl. is responsiblefor ùis repressjon morethanthe *1 strain(Câlosand Mill€r I 98| ).
when it binds the operator (Gilb€n e, dl 1966, in a hypo' Two 'clichés" would hav€ to be overcom€ lo show the
thesisformulatedby the forme. ones).A simplified or8ùi- contribution of OJ ând O,? to /dc rep.ession under ûâtivc
zalion ofthe /dc op€ron is shown in flg. r. conditions. They aæ complementâry becâuse of their rela-
In lhe s€venties, two homologous sequences,O2 c€n- tion to the law of març-action. First, ten copies of rep.essor
tered at +412 and OJ, c€nt€r€d at -82, werc discovered are not sufficienl lo hâve the secondâry sites occupied.
(Reaikotre, a/. 1974:Gilb.net al.1975t. Thes€ ten copies aæ the number of tetramers p.oduced by
During nenrly 20 years, these siles hâve bêen r€garded the lr, cell (Cilb€n and Mûlle.-Hill 1966).Thus. lhe con-
âs cryptic sites, like the two promoterlike elements, P2 a'd cênt.âtion of reoressor in lh€ cell has to b€ increased ùo
Pl. ofrhe /& operonIXiong er r/. Icgl ). In facr.repression force the oc.upancy of the sit€s. Second, th€ affinity of 02
in the âbsênc€ofthe s€côndar)op€rârorsis efricient due lo for rhe repressor, wilhout sp€âking of that of OJ, is loo
the strons amniD olreprcser for O,l (K l0''M . âcco'- weak to allow their occupùcy. Thus, their alfinity has to be
ding ro winter €t a./. l98l). Thus it was not clerr why rep- increasedto forc€ rhe otrupânc,
ression had to be assisted. Moreover. the constitutive mut- In anv casê.in 198.t-1985.wh€thetfor lhe naturalaEb-
alions *erê all mappedin OI and f,oneexclGively in the inoæ and galactose regulations, or for the artificial /4. re8-
O? or OJ rcgions. ulations (fof lâck of tuodionâl secondary operatoF), two
This ooint wâs coroborât€d b! tbe wea* afiinilies of opêrâ1orsites either separatedby llo-llsbp (lrÂni ?r dl
the repressor for 02 and OJ. ()2 has a -Io-lold r€duced af- 1983;HeFin and Bennet 1984)or 220-240bp (Dunn "r dr.
finiry mmpared ro O,l (Pfahl et al. 1979; wiîrel et al. 1984: Besse et at. 1986), wer€ rcquired to hav€ tull repress-
l98la) ând OJ an âi leasr 100-foldreducedaflinity (Pfâhl sion. How they coop€r.te for repressionat these dislanc€s in
et al. 1979].Wiîter et û1. l9Ela). A fragmenl catrying all f,. !oli. hâd to b€ exolaiæd.
three op€lalols has nrly the sârn€âmnity fo.lhe repress- DNA l@ping sanelaied by the simùltat€ous binding of
sor than a hâgrnenl c{rryins the sôle 01 op€ràtor (Pfahl e/ the .epressor to lhe lwo distant sites was one possible model,
d/. 1979:O Cormanet a/- 1980). bur wÀ resÂrdedas mosl unlilely before 1986.The oppos-
Fo.lhese r€asons.lhe Dossibleconùibution ofo2 or OJ hion to this model wâs strong. as R. Scfile;t recalls h
wàs disrârd€duntil the nexrdecade.and a first decisiveim- (Schl€if2003).
puls€ wift ihe dis€overy ofthe eukaryotic cnhânce6 (MG In fac! conceming lâcl specificâllt som€ early ù vir.o
reaue/ a/. l98li Banedi er a/. 1981).Soon aft€r, in I9E3- data ôlher s€€medto favor othe. models, such as sliding of
-phyriologicâl O/ sire
19t4. somewhst analoSous s€quenc€s were found nec€s- rhe reprcssoralong DNA uniil the
sffy for repr€ssion of two prckaryotic operoos, th€ arâbin- twinl€r ?r al. l98lb). Orher /, t/rro preliminât) dâr.ain
os€ and galactose op€rons, in addition to a fi$t operclor 1985 indicat€d that the reprEssr-op€mtor complex was not
sequencelocaæd in rhe vicinity of the pmmoter (Imoi e, d. relained by niùocellulos€ ir filær binding âssâyswn€n 220
l9El: Dlm"rdl. 1984). bp s€psratedthe rwo opelators. A nuclmsome-qle sùuctuF
As a .çsul! two groups in 1984-1985 tried ùoestablish a whcre DN.A is $rapped iwice around Lâcl. could explain
mùlti-site .epression in an artificiâl /ac systens of eJçres- rhis unusuâl silùation (B€sse el a/. 1986). Olh€r nuclec 'reP'
sion lâcking lhe nâtive 1dc operon (Henin er a/. 1984; som€-tlpe models we.e proposed later on, such as the
8e:!€ e, al l9E6). These works lumnârize the el€nents ressosome" thal would form between the lwo aal op€mtors
that *eR supposedto bê nec.ssaryfor multi-siterEprcssion sepaiâted by ll4 bp, the Aal repressor and vanoùs olhêr
h the mid-€ishti€s. proteins, th€ RNA polymerase, the CRP or HU pmteins (s€€
Since nulti-sil€ repression .|l/asobs€rved in tnc a@ opc- lor e\ânple Kuhnk€", d/ 1989:Al; et dl. 1996).
rcn wilh â control €lernentwirhin â strùc1ùlâlg.R rclhin- Thus D\A loopins for /ac repr€ssronspecificâllyand
dinS of /æO2, bùt not in the tac op€mn, lhe gercmic and lor oùer resuldionsin senelâl.wâs not obviousand its f€â-
biochemical orgânizâtion of rhe gar contml region s€cmed sibility had lo be demonstrôtÊd. The /dc reprcssor was ù
of first importanoe. For this reason, the artificial rcgLrlarion ideal pDtein for lhat pùrpos€ tecaus€ of its strùstur€. This
built by Herin and Bennet 1984 mimick / thc ga, opemn t€traffer is organized inio two dime6 that bind DNA, as
wirh a hybrid locarp ptotn'otet corftolling aala.rokinase det€rnined by vârious techniques (Kâniâ Ând Bmwn 1976:
o'cofrnan er a/. 1980;culâd and Maurizot 1981).ll could
. ln the native /ac operon, /acol overlaps wirh tbe prû then in principle inducc DNA looping when mix€d with â
moÈr. ln rhe gal opero4 the galo and gal0e opêûotrs ftâgm€nt cârryirg two /ac op€râtor sites.
suround the pro.noter, allowing the RNA polymetas€ In fact, according to the concentrations of repr€ssot and
lo sil on it in the presenceofthe rêpressor Thùs, a l,'p DNA, variou! conplexes, apân th€ loop. could be predicled
pmmoie. was flank€d by two /acot operarors. The for a mixrureof repressorând fragmentcârryingrwo distânl
/oco] op€Étor wâs employed as a s€quenc€ with suf- operâiorshes(Kraner ?, cl. lq8TlAmoulal l99lr Fi&2).
fici€nt aflinity to ênsure op€raror occupâncy. in a w) Sùfiiciently low conc€nô_atiônsof DNA and rep.essor
sinilat .ô aaloe ^nd galoi when binding the gdl rep- were €xp€cted to favor the siûple binding (A) to on€ oPer-
ato. site. tf fl€xibility of DNA allowed it, sinultaneous bin-
. The comtructs were cani€d by a 2otopy plasmid. ding to the secondoperaror site alld looping ofth€ interven-
. An effect was only_pbserved when ûe reprÊssor ùas
provided by a laclw slrain, produciog l0-fold morË ' "ln
repressorthanthe nalive sEain(Mùller-Hill el a/. 1968). 1984.t @ csÛdins sslely tlÉ fomation of DNA-proler l@ps
beuF I wr.d to Drek th. s@l6c inle@tion betlÉ rhe cRP ælr-
It was the first report lhat two /rc op€rttom call c.op€r- laror dd RNA pdFæ ,'r dE læ pmmoler(s omlusio. ôr a'lu$l
ate in repression. How€v€r. interpr€tation of the dstâ is andBùc 1937)by ltrtis €eqindts Ths ndl makna lmps b.t1"6
sonewhâr complicâled by the interference with tp .epres- ( RP&d R\A polymæ I lJæpGed lhb poj(t o rh. lr Ep|N in
I 985, fôrgdring th. lie'iio. slç fo. *ôich M mt Éluind The miriâl
The anificial /ac reaulationbuilt by Muller-Hill's gmup pm|d1, a wll æ I bnsp6ni6 |o the lac t@6so( s *dely spsd
-
ùâs i'rsoired b! llenin and Bennel\ wort and also d;s- b€làren s?s èv6 qrn.d

105
 DN^|q+|tldDNAp.ûi8'nÙÉ<.1|Mi.h.h\m!r|
promoter
T
CRP
Concentr.tion Repregsion
n-
-"7n A ReP(+)
// DIA (+]
w luùr) rtih i..É.i!g .G
0 Cooperation
Rep (++)
A -
\vs DilA (+)
-:_ -êNo
-sffi -s-g-
A A - * Rep (++)
Dt{A(r+)
"U
inq DNA {Bl *ould occur.For incEâç€d concenEatioru of
reircssor, ttre sheswould be indep.ndentlyoccupiedin â
'Iandem' struaure (C), and for both incr€$€d rEprÊssor
and DNA concentmtions,int€rmoleculù species{D) where
ih€ repressoris in sandwichbetu€€ntwo DNA molecul€s,
The Kram€r er dt (19t7) stùdy is the exacrtranslâlion
ot rhisconc€ntralion sch€meandof oùer tniiciPatedloop
te.lurÊs.The Erârdâriongel assrys' were performedso as
rhe Dmo€rtiesof thÊ loops ùe dirÊcrl' r.sd on the gels
withouian) effon:conlinuityofDNA loopinS-ovcr â lùge
r.nce of dist nces. a ooposedto punc$al fonndion of
"*ie"tome-tree srrucluresat 210 bp or oiher punctual
sùuctur€s,concenEalioneffect on DNA loop fonnarion.
identification ol intermolecularadducts,phasingst shon
distânc€ a! a oons€quence of DN\ loopin&ifnor a pmof
Èlælron micmscoDi \râs rescrvedto ihe obs€r ion of
lareesizr loopsanàio thedernonst arionofù oplimal dis'
"cycliz'
lâni€ for "DNA cyclisation-âround50o bP{ùe
tion_aDDroâch is detâilcdin Amouyalls9l).
TT; studycteely showedlhat DNA loopingis fâvocd
D vito by ihe corcentmtionsthst do dot saturatethe op€r-
âtors rÀ,iihreDrêssôrln ihe wt. stnin, th€re is oûly one
coDyof DNA r'er cell, rhe cbomosomc.ùrd d|€ cell prcd-
ucé only l0 cnpiesof t€Fâmêricrepresrôr(Gilben and
Muller'tiill 1966)-The concentrÂlionof reprcssorcan be
incrcased lo-fold in â /dclvst$in (CâlosândMiller lc8 l)
and aboul l00-fold in a /dcl' strain(M0ilcr-Hill €, d/.
1968)-Th€ numbe.otoperator copieswas20 in the H€mt
ùd Bennet(1984) studyând canreaah500_700copiesp€r
t Th€ DÉlinD.ry d.t sth rhè ELrdatiù s€l drqinats wc @_
nu.'€ied b tE Bslin Sunme Schml ù DNA sdrdE in 1986
r06
Fi& I S.h.mli. mp ol lt. 6- .di r.c-
ro.. op.Éi (rot b r.l.I opsrb
srt6 ù blÉ. struclonl 8aB oi th€ opê
M f ycnoq ponor.r ESrd n 3Én.
brndrngsrtéfd |j1ecRP dlivalor n ad
mode
Ft 2 Tt. v..iod Ép,sc
op.nro. inl.nctor oL
a.ir.d b.tra. ,.. '.nrts
.n.l . lrrgn !t qrryi.C lro
sinple ,4. op.drûr 3itd (frr .ù
c.lrndon! ôr ÉpEls ...1
DNÂ{Ford colnml (A)
Afdrg to only oe op.rdù
sirei(a) sihultsnæt biful'n3
ofùe rêpts to th. t*ô oÈ'
6tt tit6 with lo.ging of {h.
inlePding oNA (C) t"n
dm bind'.8lû tlE tK op.-
nb.snÊsoftn sæ Êtg-
ot
Superposition ngt. (D) intmolæulùâsc
ciâtiùs. Th€thrrd olmn trni
î|odes
indeoende|lt mt6 fi€ 6p61et Epl6rd
Sharingof oîe node
betvreen
molecules
cell for ùe ouc.based Dlasmids Thur. the use of slrains
ovemrodùciricrfie reonssor and oI mulri-coP) plasmids
miclir hav€ conc€aledD\A loopins in rhe fomer ânificial
/dc-resulâtion.To dêi€c1DNA iooPin& if il did Gcur. ùe
assavilaa ro se caflled out as clos€ a5 possible to !fie ndive
conditions. with $e ihrce oDerÀtorson the chrcmosome (or
ver) low copy plamids. al rnoso ând siù the repressor
producÊdby ùe chDmosomal /dct gene
The s€cond -clichê- was r€lârcd lo the suppo5€dl' too
low âffiniry ofthe s€€ondff) rites for them io be implied in
rcorÊssion. ll waç relyina on a fâcl lnom for long and @n-
firmed bv s€venl qrcuDs:lhe associationof the reprcssor
"irh a Ésnent carrvini all thrÉeoperaùors is hardl, stron-
eer tlouriola or lesslthô with a hâgmenlcarr)ing the sin-
;le O, oDeEtor(Pfahl er d/. l97c; O Connan er d/ 1980)
b\A sudercoilingbmke lhis cliché" v) work of trârsi-
lion betweenChemistry and DNA looping (Amouyal ând
Buc 1987)hâd mademe â*âre lhat D\A is sup€rcoiledin E
col'. lf D\A loooinq wa fe&rible on â linear remplale.
D\A suDer$ilina co;ld D€duô h, or on rhe conhr) arsisr
n. Thus.Êom ù;beginning of m) worl on DI\A loopin8in
l9E4-1q85.a secondsteDMs DlannedçiLh â supercoilêd
remplâte(cf. Klëmer ?r d/ 1988). In facr. /u vl.o. DNA
loo;ins is st onclv stâbilized b! DNA supêrcoiling:lhe
haliliie oflh€ o/-'ol-OJ comple).on â plàsmidis 2t h in-
stead of 7 min on a linear template (Whitson er d/. 1987)
On a otasmid,Éle s€condq operalorsdo$ Iâcl dissoci-
arion i0o-fotd accordinÊlo Whjrsôn Pr d/ (1q87). ftom 7'
fold ro J2-fold a.cordinqto Eismannand MÛller'Hill (1990).
whên comDùi's the ùlf-lives of $e Ol'O2'Ol a'd Ol
comol."esi oirc"r - .'o aDprcâches hâve ako unrav€ll€d
ù€ influenceof DNA sup€rcoiling /, vivo D\A fooFrilc
inc of the O/-O, rEsion by variouschemicalâgenB shows
iût oJ is only Gcupi.d when DNA is 5upercoiled(Boro-
 L.vt. rw\ od.nNù,.
A .Lr olDLmid& Pr*l À: cù4!
Controlof plaEmid
Roglic.tionInitirûon
Porltivscontrol
lnitlaùol
O llonomel
t Di|nêr
R6KTit€rcns
t__ -,, -
R6Korlo
wiec er d,r. 1987. see also Flashn€r and Gralla 1988 for lhe
i, vilro Ol-O2 oc.up ncy). From th€se assays,Grnlla and
cGworlers hâve deduced thar OJ binds thc r.prersor ,,
rivo oniy l0-fold less tiShtl) th8n O/, whercby i, vtlo in'
deD€ndeitlyon a linear template,Ol binds ûe npressoraL
teâsr 100-fold less tighlly lPfaÏl "r a./. lc79l Wi er e/ o/
lcSla). DNA sup€rcoilinsmatcs DNA loop fomat'on so
asv thar the Éûuiftm€nl for relatile onenhtion of two fac
o;Ébrs that ii obvious betw€en 153 and 168 bp on a
lin€ar or rÊlâxedDNA lemplâte.is no longer legible on a
suD€rcoiled one. hstead. loopsârÊ formed for all distÂnces
ând only rheir stâbilii), insteadof their existence.is now
s€nsitive to helical ôrientâtion of the sites (X-ramer er d/.
1988).
ln sDite of thes€ r€sults. the clichés took some lime ao
disapæ:r sbce all the ld. rÊgùlations p€rfoEn€d before
1990 still included rhe Ol site âs â crlpric sit€ thought
ina.tive, or wer€ run with stains ovearoducing dle re'
pcs{or and plesnids sith a hiSh level of copies. *hich
iôhêwhâr ærtùt6s the int€mrctation of thcir dalâ
Finâlly, th€ inactivation of ea.h sile by mutagen€sis,
includine tbe OJ site under conditioûs Às clos€ &s Dossible
to ùe w:. ones. with tlrc chmmosomal repressor aène and
rhe rbre€ siles oD thÊ chromolorne, deternined lhe contri-
burion of eâch opemror \ire to rÊpression (Oehl6 el a/
1990, 1994). Ih€ pictùr€ of /ac rÊpressionwas definilel)
môdified. h âpDcârÈdthal repression of lhe læ op€mn re_
lied on th€ ùË operâûors and not exclusivelyon O/. This
conùibutiotr deDdds on the intracellulâr €oncenûalion of
/ac rcDr€ssor.wh€n the three ooerators âr€ on the chromo_
some. rpDressionenhance.ent;t o/ by bolh 02 and OJ.
dettr; Ëom 72-fold to 48-fold and 6-fold *h€n lhe
number of lac reoressor tetraners incr€es€s Èom l0 io 50
ând q)0, r€spectùly. In fac,t, reprËssion issued Fom o,i-
02 ot OI-OJ oôoperation is replaccd by rept€ssion fiom
independentbindingâr Oi, O? and Or.
This view is supponed by the rcsult5 obrâined wilh the
siosl€ oDeÉto.s Ând âlso b\ rhe usê of reDressor mutants
û'aiæ;liners unable ro forin le ramers ind ùoinduæ DNA
loooine(Alb€nipr dl. l9q l: Chakeriane, al. l99l).Thusar
reËrivèlv bw r€Dre5sorconcentration(200 subunhs of
either di;êr or teiimerl. lhe dimer induc€sdl€ sâmelevel
of reor€ssion ftorn ihe three oD€râtors lha'| Ih€ tc6am€r
fiom ùc single o/ opemtortoéhler e/ a./. t990: o€hler pr
a/. 1c9.1).This is the indicationùar. *hen th€ reprcssorrs
unâble to folm DNA loops, it batdly biods 02 and oJ al
low' n€âr-.o-physiological intrâcellulat repr€ssor concentra-
I(t ). l
Fi& I R.plic.rion ofô. ii.m}
copynumbor @irve bindiq of lhe 'nitratd rô
the ft.mns for æphcalio. inititiid
l{€gaiivecoikol Gencol cs) Below.ponrcurù
srrù€tionof th€ R6K pl6m|d uln
DNA læprng lù mûrakù of R6K
at lhe r (or P) onsin. P.!.1 Bl
''hùdcùmn8 for lhe conhl of
ùe Dlomid mpy nùnbd (g6Fal
æ) B€lN, pan'culù srtuario
ofoÉ F factor*th DN looprng
for lniuiion of its op) nùmbd to
lrrcC
/ r'j--v'-?-\
:K otiF
This 's an over-all effect rçsulting from altema.ive looF
ins between eilher o/ and 02. or 01 and OJ. This conc€n'
triion effecris moE cleârly followed $hen onlj one loop is
Dr€s€nl,eilher ùe O/-O2 loop or ùe O/-OJ looPùd shen
ônly t*o opemtors atÊ prcsenl and th€ rhird one s tnac-
In a siluation wher! only lhe O? aûd O.1 op€latols âi€
pressnt on the chmmosome, tbe r€pression enhâncemenl of
O? versusO/. falls fiom e l0/l I.s-fold effectto none (l l/
| .5 fold effect) with an increasing aûount of tetEmers Êoln
50 to 90o nlnolecules (Oehlû et al. tqq),1994). The influ-
enc€ of O? is also lost with elevated reprÊssor concentrâ_
tions and wh€n 02 or 03 r.place O/ ott the promolcr
roehl€r ?t d/. 1994).On ùe @ntrâD, qhèn lh€ dimer reP
irês rhe natral teù-ane.ic reDressor.i.e. in lhe absenceof
D\A looDinr"rh€reis no influenceofO? 41400bp ofâ first
operatorbveilæping the promote( and lhis situat;onis nol
concentralion{eocndent.
The ()/-oJ interætion is less simDl€ rhan the Ot'o2
int€.action. tnd€€d, the ptotêin r€sponsible lor âctivalion of
the /d. oDeron,th€ cR? pmlein. binds a site cenerÊd âr
-ô1.5 clôaeto ùe OJ sit C82). ls CRP r€rponsiblefor ùe
r.oressionobservedfrom oJ, $hether by competingwilh
re;ressor for bindina ât lhe \ame locaiion or by âssinins
rcbressor binding tF;ea a"a Huason lee6: Penos and
st€irz 1996)?
In lhe ircz consûùctsof Amouyal and von Wilckeo-
Bersmann l t992),ther€is no sirefor CRPbindingandâni-
fici;l lacrlltsiks hav€b€enusedeitheron thepromler. âl
the Oi locstion.or uDstrEai:,atthe 03 location.The lacwM
sitcs do not allow itpEssion by th€mselv€swhen located
eirheron the DromolBor utslream.Howeler.whentwo
/dcnM op.É6r siiesarein ilæa o1Ot ândOJ. lhe Fgal-
actosidaseâctivity is rËpEssedfi-om E' ùo ls-fol4 depên-
dineon ùËir rclâtiveoricntation ândon whether!h€rePree
sor-e€n€and oDeldot sites are c-âniedby lhe chmmosome
or s--coovrlas;ids. This siBnificânllevel of coopemtion
wa! reoipauceawi|h two o.l;peralors (oehler e, d,/. 1994)
Funheimore, rhemoærarionr€sul!;ng tïomtheexchdgeof
O/ b! OJ in lhe oriÊinâllyO/-O2 construcr (Oehlere, d/
1994;is âeainan indlcdiônthatDNA loopinainlolvesoJ
withourù; n€€dfor CRP.Thu!,unassisted DNA loopingis
a tru€ comDonentof the nâtùrâlly obsewêdrepressionbe-
tweenO/ ;nd oJ. Th€ concentmtion€ffect h also consis-
renrçith D\A loopinasincetheeffectofOJ with respe.tlo
o/ frtts fto'n .28-fôldios.ô-foldfiom 200to J600subunits
t0?
 Dr-'a lqiq ùd DNA Fi;nÊ i! rh. c.ll Michal. ÂDryl
of telramer.Wiih the dimer.this effecrEtnains in th€ lower
tange.The4 DNA loopingcompeteswith the activation
proc€ss,leâdingto ar âdditionallevel of Eprcsion (Oehler
et al. 1994t.
Note that in the artificial regulation descaibedin
Amouyalandvon Wilcken-Bergmânn (1992)or Perezel .1/.
(2000), there is no n€€d for âssistânce,excepi for nâluml
sup€rcoilin&whelhe. two "poor" /rc op€ratorsites côop'
erare(Amouyalandvon Wilcken-B€rgmann 1992)or two
proteinswith good affinity for their sires.inte.nct weâkl!,
lik€ the two galacioserepressordime6 (Perezer c./-2000).
Accordi.g fo a recent work by Zhane et al. (2m6\,
DNA looping b€iweentwo /ac operâtorss€pfl{€d by 100
bp would rÊquirethe nucleoidHU protein.It shouldb€
noteddlat interpetation of the dataobtainedwith Hu-def-
ici€ntsûainsis uneasybecâu.se ofvariousmutarions gene-
Er€db) lhebacrerium lo compensare for ihis deficienc).
as
pointedout in Perezer dl (2000) and in Arlouyal (2005).
Second,DNA sup€.coilingis sufficienrto €xplâinDNA
looping with two /ac operâtorsâs abovedelailed(s€€also
Pmhil ed Nelson2006).
2. RÊPUCAIIOI{ OF THE ITERON4LASS OF
PLASIIIOS AND CONTROLOF THEIRCOPY
iIUIBÊR IN BACÎERIA
Al an inlehâtional confer€nc€wher€I wâs pr€s€ntingth€
fils1 da(aoDDNA l@pins with the /ac repressor,sp€cialists
ofplâsmid replicationcameto m€andmad€the remârkthat
lhe concentràtion scheme displayedin Fig. 3 c-oulds well
b€ applied io the control of plâsmidcopy number(ct Pal
md Ctattoraj 1988;Nordstrom1990).
In facr the Dunberof Dlasnids,lhcse€xtrâ-€hromoso-
msl genericelemenEin bacleriâ-is tepr siihin precise
limit! in lhe cell.Therea.elor exÂmpleI to 2 Pl plÀsmids
p€r c€ll (ct DÀse, a/. 2005), abour l0 R6K plâsmids(cf.
Muckergeêe, a/. 1988),aboutI 5 colEI plâsm'ds(ct TwigS
and Shenâtt 1980)a'd only one F-faclor {cf. Zzâmm and
Bastia2005)in E. coli, l5 to | 8 pPSl0plâsmids in Psetdô
ûonas ætusinosa (cî. Gi.aldo and Femandez-TrÊsslelres
2004).
The us€of multiple shoft repets of DNA (of abour20
pb), or itemns,is a major wây of controllingthe nùmberof
plasmidsin Crm-negârive bâcr€ria.This fâmily includes
th€ R6K, PI, F andpsclo plasmids, amongoth€rs.Tbese
iterons ar€ prEsental the r€plicârio't origin. Their .eplic-
âtion pr€sentssom€analogywith thc tnnscripfionâlrÊprÊs-
To controlthe numberofrheseplasmidsonc€a specific
numb€rof copieshâsbeenreached,the bactêriumprÊvenis
Replica.ionis inhibitedby plasmidpairingar the origin
of rcplicaiio', called "handcuffing"(Pal ând Cha$oBj
1988;McEâchem et a/. 1989;Fig.38) asdelccledby ligâ-
tion €xpenments(Kunnimâllaiyaâner a/. 2005), the actual
basisfor th€ lC rechniqu€widely us€din eukaryoticcells
to det€ctDNA loopingor pairing(s€esectionl).
DNA pairingis bridgedby â k€ycompoûe ofnplica-
tion- lhe iditidor brotein. The iniriator is inde€dfirsr re-
quired for initiation ofreplicariôn at the ircrons,by cooper-
ative bindingto th€ itcrons(ri& 3A). Hândcufiingpre-
ventsnew mundsofplasmid r€plicationândis li'ked to the
incr€asinsconcenùâtionof initialor with the number of
olâsmids:Thus, th€ numberof Pi dim€rs,rh€ initiâlorol
R6K rÊplicâtion,can reâchabout 6000 mol€culesp€r cell
(Filutovicze, al. 1986).
For some plasmidsof lhe iteron ianily, such as th€
R6K andPl plâsmids,thereis anothers€tofDNA repeats,
ât \ômedistârcefiom lh€ originof Rplicârion.in a region
ùat cùses incompatibility,i.e. the impossibility for two
goups of plasmidsto co-existin the sam€cell. This distant
region is nâmedi/,c for Pl, ard Tfor R6K. tn fârr, the
bacteriumapplieslhe sâm€proc€dur€,handcufrn& when
ânotherplâsmidcarryingthe samereplicario.elements is
inlroduced andinùrÊâses thè globalnunberof pl6rDids;r
t0E
In case of lhe ,. .o/t F-factor, handcùffing is replaced
by DNA loopina b€rween lhe ongin ofreplicalion and ir.{,
al âbout 1.5 kb (ri& 3B). DNA looping inhibib strands€p-
amtion required for iniiiation of rEp'icâlior at the onein
(Zz man ând Bastia 2005). This allows the c€llto linir the
numberofcopies to â singleone in caseofthe F-faclor-
Thus, the iniriaror is boih involv€d in the positive and
the neeative codtrol of replicâtion, like th€ fâns.riplional
activatorând repressorprotein,AraC (Schleif2003).And in
a similar wây, conformational ûansitions modiry the fùnc-
tion of the protein. Additionally ùoa r€p€rtoire of mulatioff
for âll kûown initiator.. thrÈe structures have beel| solved:
the iteron-R€pE monomer conplex from F-factor (Komori
et al. t999). the din€rizatioû domâin of pPSl0 RepA
(Oiraldo ard Fernandez-Tr€sgùefÎês2004) and lhe il€ron-Pi
monom€r comple)( (Swan er dl. 2006). ThÈ provides a
*eallh of information and a glob.l view of how the initiâtor
In the solutioq lhe initialor €xisls predominanlly undêr
th€ d;mericform deprivedofiûitiâtioû actiliry In this fom,
il binds porly rh€ iterons ând cannot activât€ rcplicalion
fiom the ite.ons. It d, however, bind ân inven€d.epeat, in
which tbe two half-sitesofthe iterod âr€ in oppositedirc-
tions. when lhis inv€rted rcp€at is presenl, âdjac€nt to lhe
;t€rcnsofth€ origin ofreplication, the initiator is âlso âble
to repress ils oùrl synthesis (see fo. example replicâtion
conlrol region of the pPslo plâsinid! Ginldo ând Femsn-
dez-Treseu€res 2004).
A chàperone system gcnenlly mediates th€ conveB'ot
of the inâctiv€ dimeric foIm into an active one for initialion
(Zzaman et al.20O4l. the active fonn is a monomer actu-
ally olisonenzed by its binding to th€ iterons (Germino and
Ba$ia 1983).
Th€ two foms ôf the protein. monomeric d dimeric.
are involv€d in DNA looping or handcuffing. Co mry to
the &ans€ripiional repressors, tbe mommer has th€ p4ùli-
arity ro bind the teo half-siresoflh€ iteronboth throughlhe
N-|Éminal ed th€ C-terminaldomains.a: obsen€d for rhe
F RepE-ire.oû (KoNri et a/. I 999) and R6K PÈiteron crys-
lals {Swan er dr. 2006).
For some pl&smids. such as the R6K family. this is mr
the end of ihe siory a.d replicâtion pmvides an example of
th€ glmnastics that the same moleculârel€menlsmn p€F
fom to âchi€vedifferenl soal! in the cell rhh ân ecooom)
of meôm (cf Schleif20ol). Indeed,fôr lhe R6K plasmids
cârrying the disbnr T s€l of iterons, replication from the sol€
orisin ofrcplicalion is not eflicienr.Replicalion.ËquiB lhê
dis-tanty itéronsto procced.From lhis poinl of vièw. the I
irerons arc also rEDlicâlion eûhânc€rs.
Vor€ prtciscÛ. ttle replicâtionproceedstrom two ori-
sins, û and 0. tÏe I elementcontainsan amy ol *vetr :l
bp rep€arsrhât can bind the inirialor protein.The r origin
cônuinr a bindingsile for the iniliator. bul il binds the prot-
ein poorly by iéIÎ tnitialion at this origin can onl] start
when th€ seven 1 ilemns, about 4 kb away (the largest dis-
tance obseFed ôr ethâncer âction in t. cor), âr€ physicÂlly
lir*ed ro the û sire thmugh tite initiato. (FiF 3A). This
côntact stabilizes lhe protein al the r origin. In the same wây,
the Ê origin containsa half-itemnonly invôlved in iniliation
when aathe sam€ 1ime, lhe initiator occupies the T ilerons,
about 1.2 kb awây (Muckherjee"/ al 1988; Mimn e, al
1992:Swanel aI2006).
BY DNA
3, CONTROLOF GET{OIIC IT|iPRII{TIT{G
LOOPI'{GOR CHROflOSOITiEPAlRll{G lN
fitAit[ALs
F.om lh€ above êxamDlesrelaled to bacteri4 it is clear thâl
when DNA looping is possible /r crr, mediated by a protein,
rraln-associatiods arE also Dossiblewith the sane molecuhr
elements wten severâl DNÀ molecules are presed, in order
to achieve the sane biolosical process. Recent findinss r€l-
ated ûo genomic imprinting of the r.i,al]]'û]'lian I8l2 HIg
l ô c u (a n dC I C I d i c a l et h a ll l , i si ' r o l r r s l r i c l e dl u r . . , ' / i
 6e' ti.1,æt c"d &nam6 ltt l. l

l*rlxul
B c
{ù/"

?r"

w2
l|atemalallolo P.ternalallela
fig'.P...|Âsih'li6e{tvEwoflh€doerg/}'/,|sThc@wsind'€l.lh€plmorgsofttElwsæ./gem$eI€n'r/l9mtn.nd4in
y€io* Thê drM slrar€dby th. rm sq6 @ iûticed b, a cd E-ette, lh€ ,ipri.tsi 61rot cgiôn (rcR), In frediun blw. rh. difdol'sfly
;.drytstqt doddB, DMRo, DMR ldd DMR2. in tsù! ùe naFix srllclmoi ftgion. MARI, m dârt 6lùe P'r.l B th€ spânal@hltætw indûld bv
crc; (.d p6ôl! où* p-Li.t wh6 lt bi,ns io its ùnn tnyb€d sii6 d rhc rcR rcsid âtd m th. DMRI ,.gor\ ù rhe tutùmÂl alele & à @l(
p.,!t c on dF patsn l arr.te,
oc <rrwJ m oty onræt ara switcfi d rh. Hl9 aæ ù16@ thers,? sFe, trappcdin æ of rrr ro.Ds.is tqéeed
dE c.rcF siù6 æ;.tnyrdd TlE 6lsisls rddtirc ùe ndhyrsrion slatùs orDNA the ICR-DMRI-MÂRI inæraciion ûftne nd.ml .U.le. ir
qlæed h-! s ICR- DMfo, |llt.'rà.tion. britlin8 ÛE 6h{É clôs to tne /sA llmdç Ûd swahins 6 tn' /ee gdq stEr6 DNA nerlrylatid
æ rh. t/9 l'motd

The CICF (CCCTC-binding fâclor) proteit l/âs first
fourd as oân ofthe 5'-HS4 insularor elem€tu ofthe chicken
Fslobin locN. sepùsring the active globio domsin Êom
the h€ærochromâlicômain. Thê s'-Hy elem€m prolecls
qen€ expression agâinst enhancers wheo plac€d b€lween
scne and eîhancer. as well as asÂinsl position êtr cts on
rhe -chmmosome
the tsee for examplé Bù'gess-Beusse Pt a/
2002). CTCF is requirEd for the €nhanccr blocking activity
(Bell et ol- 1999'r.
This activity is â reneBl f€slureof ùe CTCF prolein
ihâr is hist'ly ;nservèd In higher eukaryolcs. from Dtos-
orr'la lo humahs { Moone et a/. 2005).
Thê ll zinc fineersoflhe prolein Âllow iis bindingto â
multiDlicitt ofD\À krsels (cf. Ohlsson3r a.L20Ol) CTCF
bindsrcpe;lÊdsitesin ;hishly cooperativ€mânnertParlrsr
,/ 2{XXr-ln the akenc€ ofDNA. it cân âlso dimerizewitb
help oftûe zinc fingers or lhe C-tedinal pan offÊ pmtein
.Pânret al.2m4\.
lD rnous€eD'throidcells, the CTcF-binditg sitêspârti-
ciDatc in sDariaiinteractionsbetw€m the activ€ ÈSlobin
gËncsand. âbour 50 kb amy. th€ LCR rÈquir€dfor their
hieh levelexDr.ssion. This rEsuhsin whd was lÊimedby
rtrËaurtronair Aclive ChmmâtinHub wiih sevemlâctive
looDsa.tins in conc€rtover 200 kb (Tolhuiser d/ 2002).
Th; cTcF bindins siresseemto snuciurean inâcrile chrû.
malin ore-franew;rk. âs wa! found fi'om ônâlysisofihc 0_
globin locusin prceedtor c€lls, wher€lh€ globin senesare
norvetacriv€{SDlint€r?/ a/.2006).
îtCn atsoi"em' to organirÊspatiallythe coordinated
resuldion of ditrercnl cenesând loops at the mammâlian
tsh Htq locw, eNl'ô to tonFot rheir Senomicimprintins in
rhi. *ar ts€eforexamole.Reik and Waher2001)
This. dre insulin ùowth Èclor. lGF2. is ê rÊSulatorof
lelal growth. The con€spondingeene is imprinted.i.e ii is
onh ;xDressedfi'om on€ of the two parcntalalleles.|ts ex-
preisioi is coordinaadwilh lhal o1ânotherimprinled8en€.
Hi9. fTe t*o senes âte locsled s0 kb apan on mous€ chc
mosôm€7 (Fi; 4A). On lhe mâl€mâlchromosome.H/9 is
expressed whereâs /&/2 is repress€d Lhmughou develoÈ
m;nr. lhe ooDosireis found on fte pa|€malallel€ ln mous€
neonarallivè: râtlscriplionofihê rqo Senesis regulâtêdbv
an enhâncei located do*nstr€ah offll9 (Fig. 4A), âbout l0
kb from 919 ând about 120 kb from 1&2. A clusler of fou'
bindinc sites for CTCF is locâted between th€ two genes' in
ùe lCn imprintingconFol regionlhal regulateslhe methyl-
arionofdilïerÊnt sitrs wilhin th€ clusterand is locarcd2 tb
upstr€an of â,19. CTCF carttot bind to its sites wh€d they
cx rle maæmal inherited chromosome,th€ ditrerer!
tiallv merh!târedresion. DMRI, also cônrainsbindingsites
for aTcF. ir DhysicàllyinrcrÀclswith the unmerhylaGdlcR
lhouqh CTCF ând porsibly other proteins(Knluti ?r al.
200o-),a\ dêtectedby ùe lC technique(Dekkerer 4/ 2002).
The ICR also ohvsic{llv int€mctswilh th€ maFii arlâch-
mem resion VÀJù bdled nen ùolhe DMRI dom.;n. l&2
is rappfo in a 20 kb loop (Fi& 4B). This is sêeminglysùÊ
fici;fto orevenl ûe enhanc€r to âctivate th€ mat€mal /&2
promoler (Kurukuti e, a/. 2006)-

t09
 DNA roa9i'{ Dd DNÀ piims ii rrr

PrevioEçll lhe sâne grouphâdshownlhal on the pater- protein(Donoho€e, ar. 2007).

nal chromosome,the methylatedICR physicâlly idemcts Anolher L/rrs-chrcmosomal regulation was prêviously
with one of the diferÊ'tiâlly rnêlhylâ1ed rÊgion,DMR2, of described by Spilianalis e/ a/. (2005). Thb group has found
td2 ll./wll et ol. 2004\. This brings th€ /e/2 promoter that th€ interferon'T pmnoter regioD on mous€ chroînsome
into coniactwiih the €nhancerin a ll0 kb DNA looD.thus l0 was j uxtâpos€dwith lhe locus control region ofthe idr€r-
l€âdingto ,eP e),pression (Fig.4C). As theenhancer is oc- 'eukine sen€s otr chmmosome l1 in rhymoc)'tes precursoE
cupi€delse*here,it cârnot activar€l'.19. Th€ pmteinsres- ofth€ imlnune sysl€m T-cells, both CD4-help€r T-cells and
oonsiblefo. the ICR-DMR2iflter.ction hav€not b€€nidcn- CD8-killer T-cells.It now aDD€ars thât this inlcr-ch.omoso-
tified yet. Somezinc-fin8erproteinsbind m€rhylatd DNA. lnâl âssociationis lost when naive T-cells differentiâteinlo
Someofthem cofocalize fi rhe Hlg/tgl2 tCR lFill;on el 1,,1 or TH2 cells.On the cont âry, in activatedTt2 cells.the
a/. 2006)-Thes€proteinshaveb€enfound to Àssistr€pr€ss- pmduction of interleukin is associat€d with the folding of
siotrof lr.l 9. ln ânalogywith CTCF,they might also bridge ûe interleukin gene into sv€râl loops with coordinaled €x-
the tcR-DMR2 inrerâction. pression,due ro a potein, SATBI, only found in thymo-
thus CTcF-m€dial€dDNA looping and methylation claes. A câgelike distibution of SATa I surounds the nuc-
jointly di.ect the €nhancerto a d;fferEntproErot€rt!'get on leus ând anchors th€ loops io tie chromosomal soaffold (Csi
th€ Datemalandîtaremal chromosomes. er d/. 2006). As th€ nâive T-c€lls eith€r do nol €xp.ess th€
SinceCTCFm€diâtes DNA loopin&n is norsurprising. cyokines or Fom onl) one allele undersperific condirions.
at l€astwith th€ vi€wDointoflhis review.thârCTCF ;s âlso Spilinatis ?, d/. (2005) sùssestrhat pâiring is âssociared
involvcd in intemôleoulôrâssociatiods. with silencingof the allele.
Accordingly,Ling e, (r/. (2006)haverec€nllyfoundthat Thus in mâminals. chromosomal associarior s€em io
the 1&2/}/.19bcus on nouse chromosome7 wasphysically promoLesilencing and monoâllelic expression.*hercby
linked to ânolherlocus, t/J ,r/rvt, on chrcmosomeI I via D\A l@pins is morÊùnbivalenr.This is âlso *har emerses
CTCF boundto the ImprintingConlrol Regioo.This alsoci- liom bâcte.ialreplicâtion,where handcufiingis exclEively
ation wâs foùnd by scrceningfor all CTcT-mediâledasso- devoled !o silencins ùd to lhe control of plasmid copy
ciarions by th€ 3C t.chnique (or a variârt) ând by fluo- rumbea whereby DNA loopins can be us€d for borh posi.
.es.€nr ir rrr! hybridizalion (FISH)- This is ân intriguing liv€ (cf R6K) and n€gative c-{'ntrol (cf. F-fâcùor) of replic-
fact becâùseonly the pâtemalallel€ of chromosomeI I is ation,Iike in o-ànscriptionâlregulation.
foùnd thoùgh this locus is nol imp.int€d. D€l€tion of lhe
halehal ICR câhcelledthe association.Tfte silencing of 4. OLIGONÊR|ZATION OF THE PROTEIN
CTCF by RNA int€rfe.Encedisruptedthe coordinârereg-
ulation of the two loci. only expr€ssionof tfi€ pat€mal S€vdâl p.oteins involved in DNA loopins âre nalurally oli-
Wsbl,Nfllocrs is alt€red.Thui it was conclud€dthai the soderized, sômetimes to a high degree.
âssociationand CTCF allow fd',r-regulation of the aÊnes Thânls to a leucine zipper, two subunits of/ac reprEssor
or âltemâtivelylhal the two chromosomes shareseparatelyinteractins side-by-side as a dim€r, can also int€.acl heâd to
the sameelenents côncentrrted!t a tnnsc.iption fâctory heâd as a tetlâmer wilh two binding sites for DNA (Alb€ni
(Hugùeset al. | 995i Chakalov^et al - 2U)5)- ?l d/. l99l; Chakaheriâne, a,I l99l).
Sincethe associationis restrict€dto th€ Datemâlallele Th€ deo .ÊprÊssor is a tripl€ dimêt with thr€e biodiog
of chronosomel I, it might be involv€din the impri inS sites fo. DNA (Monensên el a/. 1989), rcsultitg in doùble
pncessof ùÊ Ig2/H l9locus on chonosome 7 (Ling e, aI. loop formâlior on binding with nativ€ DNA (Amouyâl e, a/.
2006), o. in a tnnsicnt m€thylationand silencing of lhe 1989).
ltrô./ gene a! the lvsbl/Nfl lo.tts (Krueger ed Gbôrne The )cl r€oressor is a dimer caoable of tetmm€rizâlior
2æ61. and oclaineriâioo in d|e abGenc€of DNA (Senear el dl
similar dala hâv€ b€€nquali&tively ob!âinedby zhao 1993:B€ll el dl. 2000: B€ll ard L€wis 2001). The octïner-
er o/. (2m6) with a 3c-bâs€dtechniqtæ,exccpt thar rh€y iztion allows DNA looping b€twee. the O/ ând Oraîays
find I 14 inter-orintra-chrcmosomal $socistions insleadofofrcp€Âts(Dodd er a/. 2001).
3 for Ling s, d./.(2006).Up io foirr chromosomes converg€ The record of oligomerizâtion pertains to thê I 86CI rep
to tie ICR of lhe /&2 H/9 locus.Cl@l), rficirÊ:p€rimen- ressor that compris€s 14 sùbunits âs â h6ptame. of dimers
aîanged like a wùe€l widr seven possible binding sites lor
tal conditionsincr€as€the sensitivityof thc tÊchniq're.It is
also cl€ar thei the uncoveredinter.crionsare pr€fcrôntiâl]yDNA (Pinkett e, or. 2006). Electrcstâtic forces mther than
linted to imprinting. Th€y aæ âlso sp€ciffc of a certain soecificseouencedêteminanli âre resoonsihlefor this oli_
stageof gmwth ard dependon th€ reprogrâmningof lhe gbmerizatidn.This otiSomerizâlionexdlainswell rhe âll{r-
non€ occupancy of the thr€€ | 86cl rep€âls of the phaSe PR
l er-chmmosomalâssociationsmight prEsent some prcmot€r (IHd "r or. 1996; Pi'kett"wh€el', e/ a/. 2006). Th€ arrays
sinilarity with tmnsveciion. s phenomenmknos since ot sires a.e wrapp€d ffound this making the sutF
1954 in Drosophila. Transvectionis an altcrationof gene ùnit orgsnizâtion pârticularly suit€d to the tandem alrange-
€xpEssior thât dcp€ndsupon whether or not gÊne! are ment ofthe siles (cf. Amouyal el lrl 1998).For û!e l86cl
pair€d with 6eir homolôgs(Wù 1993; Doncan2002). It r€pressor (ând lô â less€r extenl, for th€ lcl .ep.essôr), it
ger€rÀllyinvolvcsth€ actionofcnharcersin t dÆ. s€€msthat rhe cell has s€l€cted both site and prolein orgânÈ
CTCF binding sitess€nsitivero DNA meûylatiof, have zâdor, in a way lhey ar€ best fitted for âdjâcent bioding.
âlsobeenfoundnearthe 3'end ofxisr, ihe geneon the X- Now. apân fiom açsistingrepressorbinding to DNA
chromosômerhat triggers lhe ina.tivation of a single X- rep€at!.oligomerizarionofthe prolein "h€n il pmvides an
ckornosomein X-x fernalemanmals (Chaoe, al 2002), al.€ady assembledprotein bndge, cm also siabilize lhe loop,
aswell a5at the boundâ.ies ofdomaidswhichescâp€ x-in- as is the cas€ for lie /ac reDEssor However. more lhan wirh
activation(Filipova s/ .r/. 2005).Th€ precisemle ofCTcF a long rangediiarc€ aclion, oligomerizâlionin lhe absencê
in both cas€sis not known. However,il recentlyappear€d of D\A seemsro b€ associaledwith lhe fa.ilitated r€(og-
fton ttro r€€entstudies(B^chetet al.2006: hr et al.20061 nirion of nultiple loci scâter€don a preci* potion ofthe
that the two X-chromosomesar€ ù"ârsiertly pair€d, $ g€nom€. as app€ars liom the /o.. deo or /E6( / rÊgulations.
assess€d by RNA"FISH analysis.This pâiring prÊcedesX- Ind€€d, if th€ two arnys of Or ând Or sit€s of ihe lphage,
chmmosomeinactivation.Binding competiiionassayswitl, are 2.3 kb disrânq the mosl dista sit€s for lhe lt6CI pro-
sub-fi'agments of the X ina.tivâtion centerregion.disrupts lein, F. ând Fr, âre "only" 300 bp distânt, 8nd betw€€n lhes€
X-inactivational|d confirms this associarion.ComDetition two sites. ihere arE at le€sr four olher operator sites, clos€ lo
with DNA fiasmentscootaioinsrhe CTCFbiûdnrgsit. also PL and Pi, 62 bp distânt. the lhee /dc operato.s are like-
disruptsX-iBctivqtion (Donoho€er a1.2007).Like for the wise scanerÊdover 500 bp ând the thr€€ d"o op€ralots ôver
/gz,l1l9 ICR associarions, this suggeslsa role tor CTCFâs 880 bo.
a bridgingprorei'r.aloo8w;lh cofacrùrssu.h æ the Yll In t. coli. the lârSesrd;sranc€involled ir DNA lopins

110
 a.E,c.ffiardctu tO), l

is that obs€rvedfo. replicationinitiatioû of plasmidR6K. lm. 2856-2361

the t oriein il€on and the $vcn Y ilcrons D.kLr J. &Dt K I).|&r Xl,l, X}.tar N (X02) CtD(Ûinech|r'lr|)'@
SDecificallv. enfomarid &taY 295,136lll I
b;dsed b] Pi, are sepÀmred by 4 kb (Fig .lÀ) wiÈhlhe D.dd IB. P.rliE Àt ttdi!&i. D, Es.n Ja (200I) (xrandizaiÙ of rcl
oresérsàreofknowiecge.il is dimcullro sp€cirylhe pre- EplNr 6 n .d.d fù.iÊdiE rw*iù ôf PRM !d eftci.i sd'n'nB
;isc shrctûe ofthe proteinbridgeresponsiblefor this long Â'on begFy Cæ d Ds'.l"æt |5,1013-1022
disranceaction.Ho*.vêr, DNA looPinBmigl'l requir€a ooaa n, rr- .n Oçcl oNe Unaoe bvrhe@liph.s! 136eÉ.3s bnding
so€.ific iditiâror imerfacê,differenl ftom lhe contâctsthat ûottincl Jùùn d Bralqiùn Clcddtvz1l. ttsl2'll54l)
arÊâlrlady lstovn betw€entwo initialor ûoleculcs (Sran Domùæ Mq Zb.{ Lt, X. N, Sù| Y' r- JT i2OCr?) lthdifioriû ôf !
et ol.2006). cTcF coitu Yvl ld ù. x ôtmme binarysqtch ,&t .rdr t'zl, 25.
llandcotfing se€msto requirerÊp€ateditemns, fiorn 3 etus i lt@lÀIn ÀNdl kerb- ol(;u'
(DSClol) lo 7 (R6K-t) repeÂts, as *tll À5stroflginErac- Dulot IW l20(Tl)Tdvedrù
rionsihar ar€ providedby a highly coop€rrtiveorSarizadon Ihnn Ît! tur! s, O!É.r g sbGl RF 0 934)Àn o!.dd , -:30 be F|u
of th€ oroùeinrr ûe iteronqwhh r laver of dimeric ptoreur rhn ù Eqùnd for qr6in of @r,rD op€M p@@r .ddidd of DNA
holdiîi aDaniqo lÂyelsof D\A-boùndnonome$(Tout- b€licll n8 bd€ lù. ôD-û a.d D.mû{s cvolic.llvhitdm Ear<jd
darian-anà Helins*i 1998iZzamanând aadia 2005:Flt. P@.arrys ôl tfu Ndtûel r.rti.Ày,lt.1cleL4r US431.5017-5m0
3B). Dl'û.m En .!d Mdlrda a ( l99O)/d. ieD(6s forc shbl. looF ' v'!D
wid sDèctLd d d ræ DNA@biniIg ![ llllæ nû]r.l opsàG ./d/zJ
REFERÊNCES ôt |rol ardû anlosl 213. 16 -n 5
Fitipor cN, cr.q ù|( MoE Jr Trung ff, E{ YJ' NFv.. DK Ts
E, M.lkt'llû E .ti- xO. rcr. <n t:oo:l e-od.t6 b.@ cbrMtl ftdt@
^lb.ô S 6k S, wt Mhrm-A.rrt r.r 4 Xnt
of le ÈpM itrvô16 r ldie lÈÈ ôr ii'mtùd od egæ bid (-IC[ srÈ lrr CpC ùdh)ldh dÛng
il99lJ DiÉ+Lhq Mbly
1 LJI A, ll-,1ù2
tÀ @ NN Binog,fl 3, 5742 eth d.wl@@ D.*tqtu
Fill'm G Aè'rû s. s.lotit S Y.ud. D. Prckton(ùoul E- lxlæ PA
^ri t crot tr[, adù}| s 0 996)Hitu.-fir. Feô HU a ! sæific rd' (2006)A &f,ily ôf h'|'M 2iÉ û.gs dû bnd mclùvltrêdDNA ad Ét's
diÉidEl ESù]rF Gfeiot 6L in Ep6id ôf 3t l,@ipad bv O^L
Gdta)llI,17+133 i''''MÀtnû ùrtt.and ùl Czlbld 8ioloqr'X, )69'lil
tlgtg MJ. E li-Li DR (1936)P6iriw Ù'd egl rc
Arùyrl r( a* ù (!9rD To@lolictl uùitding or tus !d *Èl pmc F udld n|. M. FlLn
h bbrRN^ p.|!re J@/./ l'*,L. tttu atologl l lsaJa rol. oaû ûitidd FtÉô d ù mgjf 01Êplicdi@ Pmeedryt oItl":u-
^s;l ir I l94l r nr Rmæ 6hl of ffiÉù Ût{A looplngxù ,ôttl ^cabtu d*t.M LISÀA3, 15-9(a9
nr.ùnd Y. OÉ!. JD I l9A0 Dd @h.rsm of r.'Nim !t â ditu in
DN onlddd adr,t " ?3, 126l_!264
û E .itr: @ osqnls ttq ùr .îm P@tt"g of tk NaûMl .4caleù, d S'1tu2' ItSÀ6
^losj M (2æ5) Oæ tlgùlaton d{-dtd.È
r:o'ttt6 R."t4 Btolo7. lla, t9
r*rU rt ma--A.tra.- A I lco2' R.Dts@ of th F dJr 'ar6? r.i.d M r'd rudta JM O96) DNA l@pingând,?. rcPEsr4AP intæ
oodd tr cooFr'm b.twEi M ùldrndl6lt Ù4odatiÉ h.ll-opcr* nù.,L,æ2?a. l9:lGl93l
h tut:tu.'Raùad"I hn nt /.r 'çc@ Jlsltr' {B-{17 G.mim.r, s.!ô D (1933)Iredriôô of th. pldsnd R6Kdc'd'd tqle
id innitdpreir sirh È bhdingtitesm DNA t--t'lf,4 l2rll4
aMy.r it P.; N, Ir.|||ld S (1993)Adjærn c..FrÛ@ or rdciN d Pe?a'l"s' d
DN^ k mi rErdbriw of lonenisIæ i@iE a o'4ta Rextu dz Grb..r w. MolÈ-Bir B 0%6) lslad of ûe r^ r.!|€9
tk NdtMI k<ert olkffi L'.\,156, | 391J393
l Aeùètu d.t l.P,É 3rlm, All'3trl ^ (19/l) Lâ.1* oFd s'{læ
^rdEl ùL M.riæ L xe E, f,ùtd K O9€9)sinab 6d &{bk Loot Û|b.n W 6dl| + Mtr- I M.!t
sii6 cdr 3a ùd rh. æ1'v.li@ôf rdrq(6d Ir std H Blurc (Ë.Ls)rz'st '3qd
fd;.M sùé &oR Eprês bNls 10ils l@d oFe
lredrnd. wltcr d. Got'tq, sdlia pp 193_206
Cinldo a, Ffl.d@rrrit.c f, (2OOa) Twdv v@ ôf rh' p?Slo rÊ
s..hr C?. c,rsi.n M! areB & AuSll g eF t ^E t' 8jl & f,anr
tu ùê divano of DNA ndie
f, (rlxl6) Tmsirl col@rizdiù of x-iEdit*i6 €G ÛûFÙ6 $e liM: GiclÈ d ù. noÈûlt trslûis
rtû i! i;oMining b.c.nd d@tds tldtl t.69-*1
i.i.jrim of X diwtio. ,{tu CAI Eiolos a,BlBg
f,.rri I n-dl S s.n.nÉ $ tl03l) Ûq6m ôf . Ègrolti 3m. È s.rin cI, 8..ùd GN (l93,r) RoL of DNA Egiors tlùlirg dE tvÈoprt
of ,s op.rat@tlaMd (;æ t2.
oÉ; bv Fn i. svao DNA !.qffi .s/t.z('9-roa vturû nî Es.i.r'dtu @û rl bstd
B.r ^c, w; ^q F.kif"ld G ( !999)TlE l(dên! cr€î ù 'qris! foÉihe
dJr'E btc&i.g diùty of rinetrar. iNldc c.]l 94.337_396 Euru. TÀ Pùùo À M.Mtrs J. fl@L P.Jrctlo! It|" Co'r Pn I lçr)
À *'n.!|lfu or nuo- na *n?\t6 tttsil Jtntd 4(clt s I
a.{ oe f|m L E &.hiH a Iâa M (260) Crvsùl Stu ofllle}
c{min l donù. Fovidx . dod.l 6r coopdeÉ qsaor .nc. SlNlffi 19.t945
rwts
btdin& C., l0l. 301{l I Iàni r'9. Ot@ t" Adrtr s (1931)À cffil €ldal sirÀin ' $ÙcÛtl
@ x@lor.moaI.a/, (?l/ J,, ?3-l_733
S.! Cr- l-i M (2ml ) Cllshl sEtm of tlE I ttt .$ C{midl dm_
.in @ Joûttd olMoLdnd Btolog!a11,ll?l-llx ù;.r' R Mûloi J ( leôl ' adEt. Égùler! É4hm'ru 'n th. s06is or
sd M, w wildd"a.rttn tn 4 MdLôll| 8(1936) SvdtÉic llr r4._ .'titins Jdttul 4 LhLdlrù Rtol!'g 3 313'356
nd d.ddé EsB@ ûNud ro. EFB' Ylhd trÛod'.êd u!dl@ ie.tr J. B.ûi DT (1916)nE ûD.ridtl Fp{est Frre ot ' ffic 6
T1eE4AO Jq'nl1' lin-r3al Gpl"r|s+g.lnttdæ chiJllâ æ s9iûize,Jin ùtffi Pæ'aitas {ttP
id|drtosÉù.ar Êfr ,æ @!m,!!ol€t
to|wi.l JÀ Zrùa I. s.*I)'Lht g G..L J! (19r, DNA tFoilbs Nd taûl /@&,ty of 5. EB IÂA 73,3529'3513
plmol6 fthaid of t b@rrcp6n@ h.D iD rÛcDNÀ .Ia@l dlel' Korori f,, M.eilr E aisûli Y, Ltni 14 w.d. c, Mili x ( lry) ctve
..ab/ Aiabat 196.l0lllr bi idG of . ÛÛr.rvdic i.oridid iÛri.ld prdeit bdrd ro DNA a
hÉ8.; a. Lndl C, Litt M, Mûld Y xtirlll|lD 1' shÈ 2.6A@ttniû. lir EwOJNTat ra,15q- 4aJ
Xnn.r B. Nld.r|.' i4 A.dti i4 nÀd t' rn W0cli.ûD'r!'r' À
s tt( w.. À r.b.nr.ld G 12002)th. i@lrid ofsG ûÙ.iod
ah@! dd dkklÉingcùMdin . lræeadùw d tk Ndaùl Aa*n, ol Mrs.Ftlil s (l9AD ræ Epl6s foms l@pé ùlh li@ DNA drvi'a
scrE i^,1 9 (s.ppl .tI l6al! 613? t*o eiflbb 9...d &. otdrG fltt titrrBoJoe1n|6.l4alrlcl
xnn.. !- ^r{rd iL Nddr.it À M.|lFai Ella$) DNA cuDos'l-
cn s t- cq lorrisùiero.n{ r (2@6)sarB I F.ùt86 d.!.rcv k4'd
ùæn!|idltly.diw clûûhÉn fd .ryôd.acsd oI cvlobG Ds cns*É th. FdB Equ@d{ of M,a. op.rd,F lû DNA loop rs'
mûù qtfi /d r4rs tr l'r6oJtunl1 \41'556
I€c À|tu ,(;#rs 34, 1273_1233 'M-
xffiC.O.n ECS{2Û06)R ising!b. cùlln on nLicbonlrxld
c.L r/|.. raç,rn t t çrt ) n" ora &qEe dû4. Énins ftm tu ill
n'niro *nih ltdly dr@6 DoNrÀ *qgû\ IhtaûÙ a"'t M at* r'.r* - r;æ* n.dlltlC
Kuhnb q Aù.€ C, friÈ |tt' Ahrig r 0939) RNApo!]tæ d 3t! e
Crdd lt3, 559-560 ttd wt' DNA hdrns ro rh€ctul r'sim of
Ct t ri.. ^È Iær VM, Mtllv SI, Bâ.r.û J|( lrr.ù it ' !où JI' ocs ùr'n rbul|@dt
if* bÈJ.&t.td.,l, e.rdc 4.Ù nPl vauJ'ùnna 12111155
M!.rt { rÉS091) Eviddæ rù leooÉ ziDFr notri. hæ E?*M
KuriE.Ltt !r S ldr. x& r.rôsùi SÀ ribrÙi@ M ('06) RoL of
Èd.tu Ik Jnrrol of Biolosiul Cknery't6, 1!7||311
ct r.bt" r- n nnnd tr Mikùtù JÀ o.bo.* cq Fr.s ? (2m5) R.Pl A diffi ir dDlins ( ha&rmnC) of pLtnid R6K! " 'l.t6 r"t'-l
di)o !.d llwiFid: 3haphgtlE lort .F olt[.8m ^/tu Xet_ ol Èad.nolos lAl, !1n'3745
(u;bd s. ÎÈ.d vIç L'tilbr q P'4rctd D' M!ÉI À zrto 7-
tM Gd66.669477
CLow' f,.rlr XD, Sp.F R , Drvilor ls' IÀ ,T (20@)CIcf r @_ rrù.6rov v tdr w, oùrr.t lt (2006)cTcF biiding .r rn' t'9 D-
pnDongcttul tlgtd 'rd|s ÈÈntd[ 'nnêntd hrgrradPr *muti
di{b. ljdsling 6.rn fd x-in&riv.lion clFie sdæ 195 345_14?
@.itor inr.rr.nôtt oEÙ_ iefotuio b r.sd dùd€ qÉ b t4l2 Ptu'cÀ'F: dttt \:awa
clbrn R Mflrùlt JC (l93il Læ RepM&.
,l.ad.tu df.9t.d t^,, 1lB, 1063{-10639
br didroi$ sùly. NElei. ædr Âalu4h 9.517t\tu ÀR
Di ||( rtlic4È.ir! ia B..uEy 41. Ftte nÀ È!D t?. trulotr J. Litrs J(). û i. ur JP,r,| rù. ab IIL Qlù xw ft.rrv ri'L uorr'
,irxx-' crcr .'"a'æ,nqgt*l):@lolodl'dd bdÉ /9,? n/o
(l.tlodi D( r2&5, Muh'pk h.trEctû( Gùll's Ù .h. @ml olPl
" nra ràs'm P6e.d'"2! ût tk \tdtùl/td'd.qot &tqrc\ rxl st]dïsbI NI SeIas 312,1&212

l
 Dr. raia.nd DNA piimg b rbe€ll MichèL ^lûy,,

M.Xxùd ii, aon MÀ Tm*.r PA, HdiËri o|t11939) NegltrE @iûol Maner Plltti.s 11(6à r\txl9u1
of pl6ni R6K retli@im: pcsible rk ôa idmkolù opt'nB or R.n n; W.l&r J 12001) Gdmi. impnnlu8 p€mbl $fl|læ @ ÉF
rettiÉid cisi6 Pu?srrrx dtlc Ndtohi 'l.d.ry aIS.@M t$a iô@ Ndtæ RùÈ* (;tûç:2,2l-32
Itdlolt ws. wbt.r rq |l..Lr cx (1971) The lMid oa lll. reFt$
Mim A, Mu.lda q Bdù D (199t) ^clivûo of dsd r('Ùcdion ùi- bindng aB it ûE /r. oFo P,@a.l ne\ ol tlt Ndttul Adt&ra of Sa
si6 '' ve b9 DNA loping 6 ev€aled by ! mvd mrûr aod ôfd mili- r&?5 l/S,,1tI, 2l l+2113
.rd ,.æin &f.dit. i. co.!€.n,vily âi â ditu flt t: 4x, tnfln ll, sldld J& S.oo. & A.E JL O93l) A p.rfætly synneÙr. Ln ot€dù ùnds
rh€ /@ EDG$ Ey tigitly Pær',"F ot thc Ndr$l A.t'à.nr oa!a'
ùlc l|, FilinFvr q ld|||trd D, Pùr-r.rr E, Ctô Q, s|ritn Sr, eær l/.!l S. 6?&S?49
Mùrù.[ À G|tt À B.rtlùbr 14 AEold & i.rl U.I-lttif} S.hLll RF {2OOl)ArrC F@in: a lovè-hde relatoÉhD ,rdJqF 2s, :?4232
Do n oth.d n zbù J. n nlrir, & Lrrdr@ v (2û5) crcF is Scur DF. Ire TM, Rd JÀ w.rmrn Û Elron li, Rr.hov. f, ( l99ri îÉ
(jo@Éd ûd lrnsdt /d to huDæ rri dfers dhlE Udbng of rbe Dnrwy elr..*.nbly o, hdnoÈteÊ Àl rcPæss' drffi n to ifu
tb* iÉllas E)tt8o Rù\ 6. 165-t70 anNieùrld 32.61194t39
M.€. P.Bd R lvlyltl B, Ev.r.rt & Gùb MP,CLnbd P ( l98l | 11É Solirurù Cq Lhl MD,Iot. T, l4 G& rbv.ll t (2m5) Intrc|'c
Sv,tO2ù9 ED.d t6 ô sking cfiæt d g@ .xF6id borhi. Svro od nosomal æ'ditr bdvg allman*ly dprcs! læi NdE af,5,617_
dù6 cùlmic Mbirot! lr'!.&o !ët& R.t@t 9.û114.xa
Moriær L D.nd.æ[ q llrûed ( ( 1939)Plmficdio !n d.ltlm_ Srli.i.r [. Earlt H, Xd6 J, hian nJ, Klds P, C|6.n F, Gjjd \
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