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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

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.--,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 ular associations.This findinr was â decisive elemenr
identiryins the componcnts that arc involvcd iî the bsic towards th€ uncov€ring of the ii;octiôùâliry oflhe secondâry
cellulâr processes. Whcn it was not€d lhal somc elcrnents
wcr. in vârioùs conccntmlions, narurally or âniticially. duc DNA looDinsis nor restricredlo Fânsffiplion. ll i< âl$
to i rodùced genebc mutâtiods, it was css€ntiâlly dêscrip- involved in ieolierion. to initiâte it for the bacterialRox
tve. Thus. for DNA-protcin intetrationsinvolvcd in g€ne Dlâsmid{Muci.h€ri€t er a./. l98Er Viron g/ r'/ lqell or on
expr€ssion and thei. regulalion, the genersl idcâ w&s thâl itre commn ro sto-oir for rne / . .o/i F-factormd to limtr ,ts
the DroÈin had to be in sufficient âmount in th€ ccll to cn- numberto ône coiy (cf. zuma, ând Bastia2005).The ini-
surebinding to the corespondingsite and gencnte ûe pm- tiâror Droteinbridaci lh€s. loops. Hoqevêr. the cop) num_
c€ssof inlerest. ln bactcria for instânc€, it seem.d unimDor- brr odthe itercn+lassofplÀsmidsis generall) controlledb'
-hândcumnp-, i.e. intermolecularaçsociâlionsbridged b'
t nt to us€ strâins ovcrproducing a traft.ription factor
instead offte wild-typ€ slrain and physiologicâl ârnountsof the initiator-prot€in(Palând Chanoraj 1988;McEachemer
prorein. And b€câuseit is eâsier technically, multi-copy al- l9E9l.
Dlâsnidsare ofien usedin Dlaceof chromosomâlconsEucts. Int€moleculâr àssociations are âlso r€present€d by the
Sinc€ then. il has appeâred in sev€ral instaîces that the Dairins of chrcmosomesfor rhe @otdinatedresularionor
number of copies of a gene or of â protein wss eblc to 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 -
chùge lh€ regulationprocess- prinriig of rhe nalnmalian td2 Hta locus lt does so b!
One enmpl€ is pmvided by .he t. colt n'rlti-site /ac ionlro ing lhe targelof lhe enhâncershæd b) lhe r*o im_
repression. From th€ approach eûploy€d to demonsFat€th€ prinæd genesrlmugtr attemateD\A looping 4d meth'l-
fêÀsibiliù of DNA looping betwc€n /cc rÊplecror and two ;dôn ôfihe CrCF bindinq sne lMunell cr J. 20M1. lt also
/r. opeEton on thê sâme moleculc, it wes cld thâr DNA DaniciDâtes in chmmoso;al âçsociâlions(Ling er d/. 2006:
looping could t'€ concentralion-depend€nl (lkâmer el a/. zhg.o tt al.?.006\.
1987: Amoùyal 1991). When mediatedby /æ rcpr€ssor, The inrerDlat between DNA lmping. simple binding
D^_A læpina is lost with increased conceû'ariôns of reF and DNA pairinB i5 de!elop€d for lhesethrÊespecrficsitu-
ressor ând DNA ûrolecul€s to the benefit of other int€mct-
lions. independenlbinding to the op€ratorsor inlermolec-
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 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
T
CRP

mode
Concentr.tion Repregsion Ft 2 Tt. v..iod Ép,sc

n-
-"7n A ReP(+)
op.nro. inl.nctor oL
a.ir.d b.tra. ,.. '.nrts
.n.l . lrrgn !t qrryi.C lro
// DIA (+] sinple ,4. op.drûr 3itd (frr .ù

w luùr) rtih i..É.i!g .G


c.lrndon! ôr ÉpEls ...1
DNÂ{Ford colnml (A)

0 Cooperation
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
Rep (++) ciâtiùs. Th€thrrd olmn trni
A -
\vs DilA (+) î|odes
indeoende|lt mt6 fi€ 6p61et Epl6rd

-:_ -êNo
-sffi -s-g-
A A - * Rep (++)
Dt{A(r+)
Sharingof oîe node
betvreen
molecules
"U
inq DNA {Bl *ould occur.For incEâç€d concenEatioru of cell for ùe ouc.based Dlasmids Thur. the use of slrains
reircssor, ttre sheswould be indep.ndentlyoccupiedin â ovemrodùciricrfie reonssor and oI mulri-coP) plasmids
'Iandem' struaure (C), and for both incr€$€d rEprÊssor miclir hav€ conc€aledD\A loopins in rhe fomer ânificial
and DNA concentmtions,int€rmoleculù species{D) where /dc-resulâtion.To dêi€c1DNA iooPin& if il did Gcur. ùe
ih€ repressoris in sandwichbetu€€ntwo DNA molecul€s, assavilaa ro se caflled out as clos€ a5 possible to !fie ndive
conditions. with $e ihrce oDerÀtorson the chrcmosome (or
The Kram€r er dt (19t7) stùdy is the exacrtranslâlion ver) low copy plamids. al rnoso ând siù the repressor
ot rhisconc€ntralion sch€meandof oùer tniiciPatedloop producÊdby ùe chDmosomal /dct gene
te.lurÊs.The Erârdâriongel assrys' were performedso as The s€cond -clichê- was r€lârcd lo the suppo5€dl' too
rhe Dmo€rtiesof thÊ loops ùe dirÊcrl' r.sd on the gels low âffiniry ofthe s€€ondff) rites for them io be implied in
withouian) effon:conlinuityofDNA loopinS-ovcr â lùge rcorÊssion. ll waç relyina on a fâcl lnom for long and @n-
r.nce of dist nces. a ooposedto punc$al fonndion of firmed bv s€venl qrcuDs:lhe associationof the reprcssor
"*ie"tome-tree srrucluresat 210 bp or oiher punctual "irh a Ésnent carrvini all thrÉeoperaùors is hardl, stron-
sùuctur€s,concenEalioneffect on DNA loop fonnarion. eer tlouriola or lesslthô with a hâgmenlcarr)ing the sin-
identification ol intermolecularadducts,phasingst shon ;le O, oDeEtor(Pfahl er d/. l97c; O Connan er d/ 1980)
distânc€ a! a oons€quence of DN\ loopin&ifnor a pmof b\A sudercoilingbmke lhis cliché" v) work of trârsi-
Èlælron micmscoDi \râs rescrvedto ihe obs€r ion of lion betweenChemistry and DNA looping (Amouyal ând
lareesizr loopsanàio thedernonst arionofù oplimal dis' Buc 1987)hâd mademe â*âre lhat D\A is sup€rcoiledin E
"cycliz'
lâni€ for "DNA cyclisation-âround50o bP{ùe col'. lf D\A loooinq wa fe&rible on â linear remplale.
tion_aDDroâch is detâilcdin Amouyalls9l). D\A suDer$ilina co;ld D€duô h, or on rhe conhr) arsisr
TT; studycteely showedlhat DNA loopingis fâvocd n. Thus.Êom ù;beginning of m) worl on DI\A loopin8in
D vito by ihe corcentmtionsthst do dot saturatethe op€r- l9E4-1q85.a secondsteDMs DlannedçiLh â supercoilêd
âtors rÀ,iihreDrêssôrln ihe wt. stnin, th€re is oûly one remplâte(cf. Klëmer ?r d/ 1988). In facr. /u vl.o. DNA
coDyof DNA r'er cell, rhe cbomosomc.ùrd d|€ cell prcd- loo;ins is st onclv stâbilized b! DNA supêrcoiling:lhe
ucé only l0 cnpiesof t€Fâmêricrepresrôr(Gilben and haliliie oflh€ o/-'ol-OJ comple).on â plàsmidis 2t h in-
Muller'tiill 1966)-The concentrÂlionof reprcssorcan be stead of 7 min on a linear template (Whitson er d/. 1987)
incrcased lo-fold in â /dclvst$in (CâlosândMiller lc8 l) On a otasmid,Éle s€condq operalorsdo$ Iâcl dissoci-
and aboul l00-fold in a /dcl' strain(M0ilcr-Hill €, d/. arion i0o-fotd accordinÊlo Whjrsôn Pr d/ (1q87). ftom 7'
1968)-Th€ numbe.otoperator copieswas20 in the H€mt fold ro J2-fold a.cordinqto Eismannand MÛller'Hill (1990).
ùd Bennet(1984) studyând canreaah500_700copiesp€r 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
t Th€ DÉlinD.ry d.t sth rhè ELrdatiù s€l drqinats wc @_ inc of the O/-O, rEsion by variouschemicalâgenB shows
nu.'€ied b tE Bslin Sunme Schml ù DNA sdrdE in 1986 iût oJ is only Gcupi.d when DNA is 5upercoiled(Boro-

r06
L.vt. rw\ od.nNù,. I(t ). l

Fi& I R.plic.rion ofô. ii.m}


A .Lr olDLmid& Pr*l À: cù4!
Controlof plaEmid
copynumbor @irve bindiq of lhe 'nitratd rô
Roglic.tionInitirûon the ft.mns for æphcalio. inititiid
l{€gaiivecoikol Gencol cs) Below.ponrcurù
Porltivscontrol 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
lnitlaùol ofoÉ F factor*th DN looprng
for lniuiion of its op) nùmbd to
O llonomel

t Di|nêr

R6KTit€rcns
lrrcC
/ r'j--v'-?-\
t__ -,, -
R6Korlo :K otiF

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 This 's an over-all effect rçsulting from altema.ive looF
cGworlers hâve deduced thar OJ binds thc r.prersor ,, ins between eilher o/ and 02. or 01 and OJ. This conc€n'
rivo oniy l0-fold less tiShtl) th8n O/, whercby i, vtlo in' triion effecris moE cleârly followed $hen onlj one loop is
deD€ndeitlyon a linear template,Ol binds ûe npressoraL Dr€s€nl,eilher ùe O/-O2 loop or ùe O/-OJ looPùd shen
teâsr 100-fold less tighlly lPfaÏl "r a./. lc79l Wi er e/ o/ ônly t*o opemtors atÊ prcsenl and th€ rhird one s tnac-
lcSla). DNA sup€rcoilinsmatcs DNA loop fomat'on so
asv thar the Éûuiftm€nl for relatile onenhtion of two fac In a siluation wher! only lhe O? aûd O.1 op€latols âi€
o;Ébrs that ii obvious betw€en 153 and 168 bp on a pressnt on the chmmosome, tbe r€pression enhâncemenl of
lin€ar or rÊlâxedDNA lemplâte.is no longer legible on a O? versusO/. falls fiom e l0/l I.s-fold effectto none (l l/
suD€rcoiled one. hstead. loopsârÊ formed for all distÂnces | .5 fold effect) with an increasing aûount of tetEmers Êoln
ând only rheir stâbilii), insteadof their existence.is now 50 to 90o nlnolecules (Oehlû et al. tqq),1994). The influ-
s€nsitive to helical ôrientâtion of the sites (X-ramer er d/. enc€ of O? is also lost with elevated reprÊssor concentrâ_
1988). tions and wh€n 02 or 03 r.place O/ ott the promolcr
ln sDite of thes€ r€sults. the clichés took some lime ao roehl€r ?t d/. 1994).On ùe @ntrâD, qhèn lh€ dimer reP
disapæ:r sbce all the ld. rÊgùlations p€rfoEn€d before irês rhe natral teù-ane.ic reDressor.i.e. in lhe absenceof
1990 still included rhe Ol site âs â crlpric sit€ thought D\A looDinr"rh€reis no influenceofO? 41400bp ofâ first
ina.tive, or wer€ run with stains ovearoducing dle re' operatorbveilæping the promote( and lhis situat;onis nol
pcs{or and plesnids sith a hiSh level of copies. *hich concentralion{eocndent.
iôhêwhâr ærtùt6s the int€mrctation of thcir dalâ The ()/-oJ interætion is less simDl€ rhan the Ot'o2
Finâlly, th€ inactivation of ea.h sile by mutagen€sis, int€.action. tnd€€d, the ptotêin r€sponsible lor âctivalion of
includine tbe OJ site under conditioûs Às clos€ &s Dossible the /d. oDeron,th€ cR? pmlein. binds a site cenerÊd âr
to ùe w:. ones. with tlrc chmmosomal repressor aène and -ô1.5 clôaeto ùe OJ sit C82). ls CRP r€rponsiblefor ùe
rhe rbre€ siles oD thÊ chromolorne, deternined lhe contri- r.oressionobservedfrom oJ, $hether by competingwilh
burion of eâch opemror \ire to rÊpression (Oehl6 el a/ re;ressor for bindina ât lhe \ame locaiion or by âssinins
1990, 1994). Ih€ pictùr€ of /ac rÊpressionwas definilel) rcbressor binding tF;ea a"a Huason lee6: Penos and
môdified. h âpDcârÈdthal repression of lhe læ op€mn re_ st€irz 1996)?
lied on th€ ùË operâûors and not exclusivelyon O/. This In lhe ircz consûùctsof Amouyal and von Wilckeo-
conùibutiotr deDdds on the intracellulâr €oncenûalion of Bersmann l t992),ther€is no sirefor CRPbindingandâni-
/ac rcDr€ssor.wh€n the three ooerators âr€ on the chromo_ fici;l lacrlltsiks hav€b€enusedeitheron thepromler. âl
some. rpDressionenhance.ent;t o/ by bolh 02 and OJ. the Oi locstion.or uDstrEai:,atthe 03 location.The lacwM
dettr; Ëom 72-fold to 48-fold and 6-fold *h€n lhe sitcs do not allow itpEssion by th€mselv€swhen located
number of lac reoressor tetraners incr€es€s Èom l0 io 50 eirheron the DromolBor utslream.Howeler.whentwo
ând q)0, r€spectùly. In fac,t, reprËssion issued Fom o,i- /dcnM op.É6r siiesarein ilæa o1Ot ândOJ. lhe Fgal-
02 ot OI-OJ oôoperation is replaccd by rept€ssion fiom actosidaseâctivity is rËpEssedfi-om E' ùo ls-fol4 depên-
independentbindingâr Oi, O? and Or. dineon ùËir rclâtiveoricntation ândon whether!h€rePree
This view is supponed by the rcsult5 obrâined wilh the sor-e€n€and oDeldot sites are c-âniedby lhe chmmosome
siosl€ oDeÉto.s Ând âlso b\ rhe usê of reDressor mutants or s--coovrlas;ids. This siBnificânllevel of coopemtion
û'aiæ;liners unable ro forin le ramers ind ùoinduæ DNA wa! reoipauceawi|h two o.l;peralors (oehler e, d,/. 1994)
loooine(Alb€nipr dl. l9q l: Chakeriane, al. l99l).Thusar Funheimore, rhemoærarionr€sul!;ng tïomtheexchdgeof
reËrivèlv bw r€Dre5sorconcentration(200 subunhs of O/ b! OJ in lhe oriÊinâllyO/-O2 construcr (Oehlere, d/
either di;êr or teiimerl. lhe dimer induc€sdl€ sâmelevel 1994;is âeainan indlcdiônthatDNA loopinainlolvesoJ
of reor€ssion ftorn ihe three oD€râtors lha'| Ih€ tc6am€r withourù; n€€dfor CRP.Thu!,unassisted DNA loopingis
fiom ùc single o/ opemtortoéhler e/ a./. t990: o€hler pr a tru€ comDonentof the nâtùrâlly obsewêdrepressionbe-
a/. 1c9.1).This is the indicationùar. *hen th€ reprcssorrs tweenO/ ;nd oJ. Th€ concentmtion€ffect h also consis-
unâble to folm DNA loops, it batdly biods 02 and oJ al renrçith D\A loopinasincetheeffectofOJ with respe.tlo
low' n€âr-.o-physiological intrâcellulat repr€ssor concentra- 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 In case of lhe ,. .o/t F-factor, handcùffing is replaced
proc€ss,leâdingto ar âdditionallevel of Eprcsion (Oehler by DNA loopina b€rween lhe ongin ofreplicalion and ir.{,
et al. 1994t. al âbout 1.5 kb (ri& 3B). DNA looping inhibib strands€p-
Note that in the artificial regulation descaibedin amtion required for iniiiation of rEp'icâlior at the onein
Amouyalandvon Wilcken-Bergmânn (1992)or Perezel .1/. (Zz man ând Bastia 2005). This allows the c€llto linir the
(2000), there is no n€€d for âssistânce,excepi for nâluml numberofcopies to â singleone in caseofthe F-faclor-
sup€rcoilin&whelhe. two "poor" /rc op€ratorsites côop' Thus, the iniriaror is boih involv€d in the positive and
erare(Amouyalandvon Wilcken-B€rgmann 1992)or two the neeative codtrol of replicâtion, like th€ fâns.riplional
proteinswith good affinity for their sires.inte.nct weâkl!, activatorând repressorprotein,AraC (Schleif2003).And in
lik€ the two galacioserepressordime6 (Perezer c./-2000). a similar wây, conformational ûansitions modiry the fùnc-
Accordi.g fo a recent work by Zhane et al. (2m6\, tion of the protein. Additionally ùoa r€p€rtoire of mulatioff
DNA looping b€iweentwo /ac operâtorss€pfl{€d by 100 for âll kûown initiator.. thrÈe structures have beel| solved:
bp would rÊquirethe nucleoidHU protein.It shouldb€ the iteron-R€pE monomer conplex from F-factor (Komori
noteddlat interpetation of the dataobtainedwith Hu-def- et al. t999). the din€rizatioû domâin of pPSl0 RepA
ici€ntsûainsis uneasybecâu.se ofvariousmutarions gene- (Oiraldo ard Fernandez-Tr€sgùefÎês2004) and lhe il€ron-Pi
Er€db) lhebacrerium lo compensare for ihis deficienc).
as monom€r comple)( (Swan er dl. 2006). ThÈ provides a
pointedout in Perezer dl (2000) and in Arlouyal (2005). *eallh of information and a glob.l view of how the initiâtor
Second,DNA sup€.coilingis sufficienrto €xplâinDNA
looping with two /ac operâtorsâs abovedelailed(s€€also In the solutioq lhe initialor €xisls predominanlly undêr
Pmhil ed Nelson2006). th€ d;mericform deprivedofiûitiâtioû actiliry In this fom,
il binds porly rh€ iterons ând cannot activât€ rcplicalion
2. RÊPUCAIIOI{ OF THE ITERON4LASS OF fiom the ite.ons. It d, however, bind ân inven€d.epeat, in
PLASIIIOS AND CONTROLOF THEIRCOPY which tbe two half-sitesofthe iterod âr€ in oppositedirc-
iIUIBÊR IN BACÎERIA tions. when lhis inv€rted rcp€at is presenl, âdjac€nt to lhe
;t€rcnsofth€ origin ofreplication, the initiator is âlso âble
Al an inlehâtional confer€nc€wher€I wâs pr€s€ntingth€ to repress ils oùrl synthesis (see fo. example replicâtion
fils1 da(aoDDNA l@pins with the /ac repressor,sp€cialists conlrol region of the pPslo plâsinid! Ginldo ând Femsn-
ofplâsmid replicationcameto m€andmad€the remârkthat dez-Treseu€res 2004).
lhe concentràtion scheme displayedin Fig. 3 c-oulds well A chàperone system gcnenlly mediates th€ conveB'ot
b€ applied io the control of plâsmidcopy number(ct Pal of the inâctiv€ dimeric foIm into an active one for initialion
md Ctattoraj 1988;Nordstrom1990). (Zzaman et al.20O4l. the active fonn is a monomer actu-
In facr the Dunberof Dlasnids,lhcse€xtrâ-€hromoso- ally olisonenzed by its binding to th€ iterons (Germino and
msl genericelemenEin bacleriâ-is tepr siihin precise Ba$ia 1983).
limit! in lhe cell.Therea.elor exÂmpleI to 2 Pl plÀsmids Th€ two foms ôf the protein. monomeric d dimeric.
p€r c€ll (ct DÀse, a/. 2005), abour l0 R6K plâsmids(cf. are involv€d in DNA looping or handcuffing. Co mry to
Muckergeêe, a/. 1988),aboutI 5 colEI plâsm'ds(ct TwigS the &ans€ripiional repressors, tbe mommer has th€ p4ùli-
and Shenâtt 1980)a'd only one F-faclor {cf. Zzâmm and arity ro bind the teo half-siresoflh€ iteronboth throughlhe
Bastia2005)in E. coli, l5 to | 8 pPSl0plâsmids in Psetdô N-|Éminal ed th€ C-terminaldomains.a: obsen€d for rhe
ûonas ætusinosa (cî. Gi.aldo and Femandez-TrÊsslelres F RepE-ire.oû (KoNri et a/. I 999) and R6K PÈiteron crys-
2004). lals {Swan er dr. 2006).
The us€of multiple shoft repets of DNA (of abour20 For some pl&smids. such as the R6K family. this is mr
pb), or itemns,is a major wây of controllingthe nùmberof the end of ihe siory a.d replicâtion pmvides an example of
plasmidsin Crm-negârive bâcr€ria.This fâmily includes th€ glmnastics that the same moleculârel€menlsmn p€F
th€ R6K, PI, F andpsclo plasmids, amongoth€rs.Tbese fom to âchi€vedifferenl soal! in the cell rhh ân ecooom)
iterons ar€ prEsental the r€plicârio't origin. Their .eplic- of meôm (cf Schleif20ol). Indeed,fôr lhe R6K plasmids
âtion pr€sentssom€analogywith thc tnnscripfionâlrÊprÊs- cârrying the disbnr T s€l of iterons, replication from the sol€
orisin ofrcplicalion is not eflicienr.Replicalion.ËquiB lhê
To controlthe numberofrheseplasmidsonc€a specific dis-tanty itéronsto procced.From lhis poinl of vièw. the I
numb€rof copieshâsbeenreached,the bactêriumprÊvenis irerons arc also rEDlicâlion eûhânc€rs.
Vor€ prtciscÛ. ttle replicâtionproceedstrom two ori-
Replica.ionis inhibitedby plasmidpairingar the origin sins, û and 0. tÏe I elementcontainsan amy ol *vetr :l
of rcplicaiio', called "handcuffing"(Pal ând Cha$oBj bp rep€arsrhât can bind the inirialor protein.The r origin
1988;McEâchem et a/. 1989;Fig.38) asdelccledby ligâ- cônuinr a bindingsile for the iniliator. bul il binds the prot-
tion €xpenments(Kunnimâllaiyaâner a/. 2005), the actual ein poorly by iéIÎ tnitialion at this origin can onl] start
basisfor th€ lC rechniqu€widely us€din eukaryoticcells when th€ seven 1 ilemns, about 4 kb away (the largest dis-
to det€ctDNA loopingor pairing(s€esectionl). tance obseFed ôr ethâncer âction in t. cor), âr€ physicÂlly
DNA pairingis bridgedby â k€ycompoûe ofnplica- lir*ed ro the û sire thmugh tite initiato. (FiF 3A). This
tion- lhe iditidor brotein. The iniriator is inde€dfirsr re- côntact stabilizes lhe protein al the r origin. In the same wây,
quired for initiation ofreplicariôn at the ircrons,by cooper- the Ê origin containsa half-itemnonly invôlved in iniliation
ative bindingto th€ itcrons(ri& 3A). Hândcufiingpre- when aathe sam€ 1ime, lhe initiator occupies the T ilerons,
ventsnew mundsofplasmid r€plicationândis li'ked to the about 1.2 kb awây (Muckherjee"/ al 1988; Mimn e, al
incr€asinsconcenùâtionof initialor with the number of 1992:Swanel aI2006).
olâsmids:Thus, th€ numberof Pi dim€rs,rh€ initiâlorol
R6K rÊplicâtion,can reâchabout 6000 mol€culesp€r cell BY DNA
3, CONTROLOF GET{OIIC IT|iPRII{TIT{G
(Filutovicze, al. 1986). LOOPI'{GOR CHROflOSOITiEPAlRll{G lN
For some plasmidsof lhe iteron ianily, such as th€ fitAit[ALs
R6K andPl plâsmids,thereis anothers€tofDNA repeats,
ât \ômedistârcefiom lh€ originof Rplicârion.in a region F.om lh€ above êxamDlesrelaled to bacteri4 it is clear thâl
ùat cùses incompatibility,i.e. the impossibility for two when DNA looping is possible /r crr, mediated by a protein,
goups of plasmidsto co-existin the sam€cell. This distant rraln-associatiods arE also Dossiblewith the sane molecuhr
region is nâmedi/,c for Pl, ard Tfor R6K. tn fârr, the elements wten severâl DNÀ molecules are presed, in order
bacteriumapplieslhe sâm€proc€dur€,handcufrn& when to achieve the sane biolosical process. Recent findinss r€l-
ânotherplâsmidcarryingthe samereplicario.elements is ated ûo genomic imprinting of the r.i,al]]'û]'lian I8l2 HIg
inlroduced andinùrÊâses thè globalnunberof pl6rDids;r 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

t0E
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 resuldion of ditrercnl cenesând loops at the mammâlian
fourd as oân ofthe 5'-HS4 insularor elem€tu ofthe chicken tsh Htq locw, eNl'ô to tonFot rheir Senomicimprintins in
Fslobin locN. sepùsring the active globio domsin Êom rhi. *ar ts€eforexamole.Reik and Waher2001)
the h€ærochromâlicômain. Thê s'-Hy elem€m prolecls This. dre insulin ùowth Èclor. lGF2. is ê rÊSulatorof
qen€ expression agâinst enhancers wheo plac€d b€lween lelal growth. The con€spondingeene is imprinted.i.e ii is
scne and eîhancer. as well as asÂinsl position êtr cts on
rhe -chmmosome onh ;xDressedfi'om on€ of the two parcntalalleles.|ts ex-
the tsee for examplé Bù'gess-Beusse Pt a/ preisioi is coordinaadwilh lhal o1ânotherimprinled8en€.
2002). CTCF is requirEd for the €nhanccr blocking activity Hi9. fTe t*o senes âte locsled s0 kb apan on mous€ chc
(Bell et ol- 1999'r. mosôm€7 (Fi; 4A). On lhe mâl€mâlchromosome.H/9 is
This activity is â reneBl f€slureof ùe CTCF prolein expressed whereâs /&/2 is repress€d Lhmughou develoÈ
ihâr is hist'ly ;nservèd In higher eukaryolcs. from Dtos- m;nr. lhe ooDosireis found on fte pa|€malallel€ ln mous€
orr'la lo humahs { Moone et a/. 2005). neonarallivè: râtlscriplionofihê rqo Senesis regulâtêdbv
Thê ll zinc fineersoflhe prolein Âllow iis bindingto â an enhâncei located do*nstr€ah offll9 (Fig. 4A), âbout l0
multiDlicitt ofD\À krsels (cf. Ohlsson3r a.L20Ol) CTCF kb from 919 ând about 120 kb from 1&2. A clusler of fou'
bindsrcpe;lÊdsitesin ;hishly cooperativ€mânnertParlrsr bindinc sites for CTCF is locâted between th€ two genes' in
,/ 2{XXr-ln the akenc€ ofDNA. it cân âlso dimerizewitb ùe lCn imprintingconFol regionlhal regulateslhe methyl-
help oftûe zinc fingers or lhe C-tedinal pan offÊ pmtein arionofdilïerÊnt sitrs wilhin th€ clusterand is locarcd2 tb
.Pânret al.2m4\. upstr€an of â,19. CTCF carttot bind to its sites wh€d they
lD rnous€eD'throidcells, the CTcF-binditg sitêspârti-
ciDatc in sDariaiinteractionsbetw€m the activ€ ÈSlobin cx rle maæmal inherited chromosome,th€ ditrerer!
gËncsand. âbour 50 kb amy. th€ LCR rÈquir€dfor their tiallv merh!târedresion. DMRI, also cônrainsbindingsites
hieh levelexDr.ssion. This rEsuhsin whd was lÊimedby for aTcF. ir DhysicàllyinrcrÀclswith the unmerhylaGdlcR
rtrËaurtronair Aclive ChmmâtinHub wiih sevemlâctive lhouqh CTCF ând porsibly other proteins(Knluti ?r al.
looDsa.tins in conc€rtover 200 kb (Tolhuiser d/ 2002). 200o-),a\ dêtectedby ùe lC technique(Dekkerer 4/ 2002).
Th; cTcF bindins siresseemto snuciurean inâcrile chrû. The ICR also ohvsic{llv int€mctswilh th€ maFii arlâch-
malin ore-franew;rk. âs wa! found fi'om ônâlysisofihc 0_ mem resion VÀJù bdled nen ùolhe DMRI dom.;n. l&2
globin locusin prceedtor c€lls, wher€lh€ globin senesare is rappfo in a 20 kb loop (Fi& 4B). This is sêeminglysùÊ
norvetacriv€{SDlint€r?/ a/.2006). fici;fto orevenl ûe enhanc€r to âctivate th€ mat€mal /&2
îtCn atsoi"em' to organirÊspatiallythe coordinated 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$l A.t'à.nr oa!a'
ùlc l|, FilinFvr q ld|||trd D, Pùr-r.rr E, Ctô Q, s|ritn Sr, eær l/.!l S. 6?&S?49
Mùrù.[ À G|tt À B.rtlùbr 14 AEold & i.rl U.I-lttif} S.hLll RF {2OOl)ArrC F@in: a lovè-hde relatoÉhD ,rdJqF 2s, :?4232
Do n oth.d n zbù J. n nlrir, & Lrrdr@ v (2û5) crcF is Scur DF. Ire TM, Rd JÀ w.rmrn Û Elron li, Rr.hov. f, ( l99ri îÉ
(jo@Éd ûd lrnsdt /d to huDæ rri dfers dhlE Udbng of rbe Dnrwy elr..*.nbly o, hdnoÈteÊ Àl rcPæss' drffi n to ifu
tb* iÉllas E)tt8o Rù\ 6. 165-t70 anNieùrld 32.61194t39
M.€. P.Bd R lvlyltl B, Ev.r.rt & Gùb MP,CLnbd P ( l98l | 11É Solirurù Cq Lhl MD,Iot. T, l4 G& rbv.ll t (2m5) Intrc|'c
Sv,tO2ù9 ED.d t6 ô sking cfiæt d g@ .xF6id borhi. Svro od nosomal æ'ditr bdvg allman*ly dprcs! læi NdE af,5,617_
dù6 cùlmic Mbirot! lr'!.&o !ët& R.t@t 9.û114.xa
Moriær L D.nd.æ[ q llrûed ( ( 1939)Plmficdio !n d.ltlm_ Srli.i.r [. Earlt H, Xd6 J, hian nJ, Klds P, C|6.n F, Gjjd \
rq ofdÉ rf.oR r!îN ofr'. .,l lhe EMBoJMtô,32r]ir d.lrr w {2mO CICF ndlc lonc-tge choûtu loping d'd ldl
MrdL.iÉ S, Erird E, a.tih D (1933)E âÉdei. tn'æûm n hislon. Dodif.did lærs Gd6 dtt DzxloPM b,23$'
i. thhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhh
plsùt R6K inwlB â DNA lmp ûniared Ùr iniùd F@in ael 9.
l7J-333 s*rn Ml( A.s. q ltvtÉ c (2006) Crvsbl stuctùæ of ininthlÈû s_
Md.'-tli! B, CnDo l. Cilù. W (l%3) Mulùs $,. nlrê rR ræ Dld of planù R6K. /dzodir4! oI tb Nahotol Ac.dcùr'ol|1tu! LiSl
t stæ IMrÊt ô[ thè N.ttml ,14'deûr .t &1æs lN1a9, 1259'
Tolhli! B, P.hh RJ, SdirE t, G'Ù H t;, tr. r'.. W (2004 replng dl
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