.I. Mol. Hiol. (1992) 225, 327-348

McrBC:

a Multisubunit GTP-dependent Restriction Endonuclease

Ellen Sutherland-t, Linda Coe and Elisabeth A. Raleigh

(Received 6 f4’epternher 1997: acwpted

1.3 Jnnrcary

1992)

YIcrK(l-mediated rest,riction of modified DNA has been studied extensively by genetic. methods, but little is known of its molecular action. \Ve have used overproducing plasmitl constructs t.o facilitate purification of the MrrB, and Mlcr(’ proteins, and report preliminar>charac?erizaGon of the activity of the complex. Both proteins are required for cleavage of dependent on GTP. AT1 appropriately modified DXA zn r?tro. in a reaction absolutely inhibits the reaction. The sequence and modification requirements for cleavage of the suhstrat’r reflect those seen in uir-o. The position of cleava,ge was examined at the nuc~leotidr level, rtsvealing that cleavage o(zcurs at multiple positions in a small region. Based upon these ohservat)ions, and upon cleavage of model oligonucleotide substrates, it is proposed that the recognition site for this enzyme consists of the motif R”C(N,, sO)Rm(“. with calravage occurring at multiple positions on both strands, between the modified (! residues. and relation between cleavage anti In subunit, composition, cofacator requirement’, recognition sit,e, McrBC does not fit int’o any of the classes (types I to TV) of restriction enzyme so far described. h’r!y~or~.s: rrstrirtion

enzymes; DXA modification; RglR: nucleotide-binding protein

1. Introduction Rrstric>tio,l systems elaborated by bacterial to monitor the origin of species allow bacteria invading I)r\‘A and determine its fate. Tn most cases so far described. endonucleases recognize specific unmodified nuclrotide sequences and cleave the I)SA into fragments (Bickle, 1987; Modrich 8 Roberts. 198%). &Then the target is a bact,eriophage genome. this destroys its biological activity and reduces plaqut, formation in plating tests. It has also hren suggested that limited cleavage of invading 1)X.\ could promote recombination between invading DX.4 and the recipient genome (Ledrrberg. S. (cited in Radding, 1973); see also Ii:ndlich 8: Linn. 1985: Price & Hirklr, 1986: Raleigh rt al.. 19X9/1). Typi(aally. (~11s expressing a restriction endonucaleasr also elaboratc~ a cognate modification rnt~tt~j-lw. Lvhich recognizes the same sequence as does the endonuclease and methylates a specific bast within that sequence. This modification rendrrs the I)N;\ resistant to cleavage by the

GTP-binding

protc~irl:

cognate endonnclease (Bicklr. 19)XT: Modrich Cy: Roberts. 198%). One example is known of a restrication endonuclease that recognizes a modified sequence: this enzyme (Dpnl and isoschizomers) cleaves only appropriatel\ modified I)NA (containing the sequence G”ATC’. where “A is S6-rnethyladenine) (Lacks R- (Greenberg. 1975, l!Ki). A single exception to the rule that phenotppic restriction is mediated by a seciuenc~e-s~)~:c.ifi(~endonucleasr has been described in a laboratorv situation. A “restriction” phenomenon c.an be mediated by uracil I)?iAglycosylase (L:np) in Esrhrrichia coli (Duncan. 1985). Vng, 1n combinat.ion with apyrimidinics (AT’) endonucleases. causes double-strand breaks in DSA if the DSA has uracil in both strands. I’ng-mediated base removal is followed by cleavage of the AP site. and such cleavage at closel;v spaced positions on the two strands yields doublttstraricl breaks. Uouble-st’randed phage SI 13 cant aining uravil in one st)rand onlv is apparently not restricted. but the ura~il-ct)nt,ainirlg st,rand is degraded (Kunkel et ccl.. 1987). No sequence sprcificity is associated with these events. and the (sonbination of I’ng and the AP endonuc+ase is not wf’c~r7wl to as ii “restriction endonuc~lrase”. even

thou$~ the biological ~Jhenotttenon c~losc~I~resettttjles c~lassical restrict ion. Itrstric+ion rtidotruc~leases described so far tiavcb heen divided into three (or four) classes. hased on subunit organization, cofactor requirements and the relationship between cleavage sit,e and recognit8ion site (t%ic*kle, 19X7: Nlodrieh & Roberts. 1982). The simplest class. type TT endonucleases. are comprised of single polypeptides and often act as homodimers. The (*(tgt~ate metIt)-lase acts indcpt>ndrnt ly of I hr rttdot~uc~lease and 1s encoded hy a separate yrnt~. ‘I’lir~ etidotiucleasr~s require Mgz+ for caleavagc. hut no other cofactor. and cleave the f>N:Z aI a n(aarh>. fjosition that is fixed with respect to I he rec~o~tiiiiott site. The methyladenine-requiring enzyme. I)!),/ I. is a metn her of this class. One enzyme otirn classifiecl \vit h these (Roberts. 1990). Eco5il. comprises a sin&, polq’peptide displaying both modification and caleavage activities. with cleavage stimulat~etl 1)~ S-atl~nos~lmothionit~~ (SNIt). Tt wa,s proposed that this enzyme he reclassified as t)ype I\’ (F+tru+tb et f/I.. 198X). Xlore complicat,ed are the type II I rest.riction rttdonuc~leasrs. which act) as complexes of two sulj-units, one responsible for site recognition and tnodfixation (the PuZodsubunit), and t,he other (R.es) for nu&ase action. The Res subunit can act ottly in association with the Mod suhunitI. and the c~omplrx eatr both modif:\ I)NA and cleave it. so that (‘lC’itV> and tnodification cotnpetr i/r vitro. 1)S.A c*leavage rrquirf:s hot h hlg*+ and ATP. is stitnulat.c~tl tJy SAN. and oc(~urs at a fixed position near the utttrtodified recognition site. Slodification reyuires only SAXI, the methyl donor. The analog ATT’-;+ will also support cleavage, The factors tnodulatinp the choice of action of t,he c~jmplex (cleavage WIX/(S modification) are not understood. These en~~~ttres can he purified in holoenzytne form. w&h t.hr sub units remaining associat’ed during purification. IWost complicated are the type 1 enzymes. These are (Aomposed of three subunits: a specificity subunit (S), a modification subunit (>I) and a rest)ric*t ion subunit. (R). A c:otnplex of all t)hree subunits is required for cleavage of IISA: this complex can also tnodify. (‘lravape requires an untnodifird I)SA site. Mg2+. ATT’ and SARI. occurs at a sit,e far distant (thousands of bases) from the recognition site. and the cleavage site appears to he chosen at random (hut see 1)iscussion). Regulat’iotr of the cahoic’cbof modification IWSI~S cleavage is c+omplex and has heen studied intensively (Kickle, 1987: Kellrher et rcl.. 1991). The ATT’ requirement for cleavage cannot he satisfied tty ATT’-y-S. These enzymes (‘att often he purified in holornzytne form. The McrK restrict,ion systetn (formerly known ss RglR: Raleigh et al. (1991)); was first discovered in 19!52 (Luria & Human, 1952) and was investigat,ed t Abbreviations used: SAM. S-adenosylmet’hione; hm(!, hydroxymethvlcytosine: Exo V. exonuclease V; JAB. Luria~Bertan~ media: PCR. polymerase chain reactiotl: bp, base-pair(s); PMSF. phenylmethylsulfonyl fluoride; DTT, dithiothreitol: kb, lo3 base-pairs; Cng. uracil lIN&elycosylase; AP, apyrirnidinic.

1)). getirtic~ ttic~thotls iti thci 1960s ((:“ot’Kc)l’c,till,h h Itrvt~l. I!Iil : ~:c~ot~~o~J~‘ltlos. l!467: trlc.?-tositirs (h”c*) atIt Il~rolr failure to ttic,tliii titrttier the h”‘f’ 1)~ ~lttc~os~~lat ioti due to phayr or host tnutation~ (H;tttttt;ttr c( Fukasa\va. 1963: Shedlovsk~~ & tirc~ntrc~r. l!Ni:i) ‘l’hc, tnriemotiic~ atlo~Jted for the system. IlKI (fijr 0.~1 t.ic*(, glucYJsrtess [)tti1gP). rrfiec+tecl this sit uatiott. In the mid- I9XOs. rrstrical iott of I )KA trttb1tt,vlat ccl hy seciLtt’ti(“‘-“Itt’(,iti(. cytfjsitte tnrt tiylnst~h I\ :*h cliscovered (Novt,r~\\‘t,itlttt,t, PI t/l.. t !L%i: ttitlttigh & \\‘itsoti. l!IX$) and desifftiate(l mcr rest ric.1 ion. li,r modified ~~~-tosint~ restric+iotJ. This r,/,~,~N-tlt~lJ(~tt(lt~tJi restric~tioti \vas shown to t)(l ~etietic~all~ idr~til ic2I with thr previously (It~sc~riht~d r~~li2~tlr~Jt~ticlf~til restriction (Raleigh rt 01.. 19X9h). I)~motistrat ioti of’ this restriction &&t expli~itit~tl pr(.vioiJs oIlsc~r\-;r~ tions that the ~,rctres for tttatt!. I)NA tno(liti~~atiotJ tnethvla,ses should not tJ(> c~lottt~cl itt sotne >tr;ritth (I~lnr~etittial P/ 01.. t!W: iii. ux k t3altlallf: t 9X3). ;ttrtl that IjN.4 frotn diverse orgatiistns c~)tiltl I)(> c4otit~l 0tllV with low f#ic*ietic\~ iltl(1 iii tJiase(1 fashiott (:(*I.. tiJr’exatn~Jle. 12’hittakei. rt rrl.. l!Wi: ~Voc~tl~~~c~kit I//.. 19X9). Thr ~~s(~ttesinvolvrtl (m~r/2(‘) \~(‘r(’ c~ltrtit~l (Kriiger rt nl.. 1991: Raleigh d r/l.. lMX!VJ: Itos:, it t/l.. 19)X7: Soztiatnattttati & I)h~trtrialiti~ttrrl. I!)XX) iItrt1 sequf~~~l (I)ila rt /I/.. l!NO: Ross ct /I(., 1!fX!fb). It bvas shown that t\vo gent’s wet’e rt~c~uirc~cltier roasttic.tion (Ilila & K,aleigh. I!NX: I)il;t vt (/I.. 1990: t:oss rt Cl/. t 9XUn) ;ltld t tlitt 1tJt> I\\ 0 EC’tlC’s (lirc~c~te(l espressiotr of thretb I)rott‘itls (I)il;r /,I I//. t !4!40: Kriigrr et r/l.. 19!11: Ross 4 f/l.. l!LY9fr). .\ 1Jossiltle (:‘l’I’-tjitiditi~ ttiotif was itlr~ti1ifietl iti t tJr>atriitic~ ;tc,itl scquenc~r~ of J1(*rli (Ilila et ml.. 1!,90). ‘t’hcrc~ i: tttt(‘(‘t’taintp c~otrc~erninp the pre(ise position of’ t ranslat iotr initintion (Dila rt al.. 1990: Ross (If f/l.. l!Nn J. t,ittte is known of’ tttts tttolt~(~ular ttat ur(’ 111’ ~lcrH(:-tleprritlc~nt restric~tioti. I’tiysioloyi(~;tl ;ttrtl invest @ated the tAta of genet,ic experiments hm(‘-T1 IIS. itrsidta (YII~ antl RglK-restricted sll~@sted that ‘I’-eveti I)NA L\-ilS (.ltsavtYt t)y Ryl tZ (I stnall numtjer of times (I)hartnalinganr K- (:cJltltJerg. 197Ba). The small nutnher of c*leavapcLs su~~ehted that cleavage might he s;r’citJt’tt(.“-sfJt,(,ifi(.. tjttt f his int,erpretatioti was contestrti, haset on pti\~sioloyic~al c~onsidrratiotts (Kriiger & If: lane a) inhibit the enz)-me. but cleavage can be detected at 10 n>I (lane h). Apparent’ly maxima1 activity was obtained between 062 and 1.25 mM (compare lanes d and e). Accordingly. other nucleot,ides were tested at, 1 m&f. Eight. guanine rtucleotides acre tasted (Table 1, rows 1 to 8). Neither GDP nor (:MI’ supported cleava’ge of the substrate (Table 1. rows 4 and 3), nor did three non-hydrolyzable a,natops of GTP tested (rows 6 to 8). The guanosine deox>--. dideoxyand ribonucteotide triphosphates all support,ed the reaction (rows 1 to 3). The non-hydrolyzable GTP analogs inhibited t,he (;TT’-supported react)ion (Fig. S(h): rows 9 and 10 of

+ + + -

ITT I’ (1) d(;TP (2) ddC:TI’ 0) IGDl’ (4) rGhlP CR) GTP-y-S (6) (7) GMP--PNP (8) GJIP- PC‘P r r(“l’l’ (W r(;TP i I()) rGT P 111) rGTP (13 (13) r(:TP d>Tl’ (11) r(:TP (IS)

i L +

+ +

? r(:TP. guanosinr triphosphak: dGTP. drtrx~guanosinc triphosphatr: dd(:TP, dideoxyguanosine triphosphate: GDP. guanosim rGMP. guanosine monophosphatr: diphosphntr; (l’rP-.r+, guanosinr 5’-O-(3-thiotriphosphate). GMP-PNP, 5’ (:MF PCP. (8. y)-meth?;lew Kuarrylvl-imidodiphosphate; guanosine 5’ triphosphate; rATP. sdenosine triphosphatr; rADP. r4M P. adenosine monophosphate; adrnosine diphosphate: ATP-y-8. adenosinr 5’-0-(3-thiotriphosphate): .-\TP-PNP. 5’ adrngl~l-imidodiphosphate. $ +. Digestion pattern was similar to that for 1 mu-(iTI’ alot~~~ -, no digestion was oburrvfd: +, digestion pat,t,ern was similar to pattrrn’; observed a.t lower cwwrntr~tions of (:TP 0~’ rnz?nrc~. or at shorter timrs.

Table 1). They were not equally efficient. however. The addition of GMlilI’-PSP to the assay n-as at high concsentration (5 mAI. or IO-fold more t)han the 0.5 m&r-(>TP in the reaction). and partial inhibit,ion U’BS seen (Fig. 2(h)). GTP-y-S was much rnortb rffe~ tive. with comp1et.e inhibition ohservrd with as littlta as 10 nM of the analog. To our surprise. ATT’ also inhibited the reaction. hut non-hydrolyzable analogs of ATP did not (Table I, rows 1-C and 15). Preliminary characterization of the effects of ATP is also sholvn in Figur(h 2. I,anes i to p show that ATT’ began to inhibit SlcrKC activity when equimolar concentrations of ATP and (:TP were present (0.5 rnM in Fig. 2: taompare lanes I and n with lanes d and e). Full inhibitSion required fivefold more ATP than (:TP in this experiment (2.5 rn~: lane j). GTP alone at this cboncsentjrat,ion supported the reaction normally (lane I)). Finally. lanes q to x show that ;\MP-PXl’. a rlon-hvdrol?;7.at)le analog of ATP. did not inhibit >lcrKe. Other rxprrimrnts (data not shown) havck shown parCal inhibition of McrBC by high conceIltrations of AhIP-PSP (near 10 mill). Similarly. ATP-y-S did not) inhibit the enzyme (Table I). ((8)

iWrrH('

lens pfrrrrd

Clra,uayP sitrs

f+?gure 2 shows a site preference in the McrH(’ cleavage reaction. This is most clearly seen in lanes i

Purification of XcrtN provrci difficult, tlt3pit.t elucidation of its cofactor rcquirtbment ,111&orts to purify the active complex from cells expressing both McrB and Jlcr( were unsuccessful. tbvt’ll using pI)I)47. a very high-c*opF plasmid in whic.h Incl anti ov~‘rf”“(l~ic.i~iori w~dat~e ~~?(JWWS1011 should might 1~ esp~ctJed. HourbvtAr. a c.ruclc tkxtjrac.l f’rom culls c~oultl c~ornpleinrlit in t.rudfh mrll rr/cr(‘+ tolls: Mcrl~( a(+,-it>. was tlxtract from ntrrH+nlrr( rf~coristit.ut.ed t)y mixing t tie tsxt ra,c.ts (tln1.a not shown). This result rnahlrtcl us LO IIW c*oml)lernentat)ion assays to follow (Mach proLeirr st~lbarat~ely through u~lnmr~ chromatopi.af)h~. Sucdtr an invcstlgat,ion sho~ved that. PVW when isolated from culls expressing both proteins. ,11wl3 alld M(.rC’ (lo riot copurify (data not shown). Purifi~~ation \vas attrmptrtl with t\vo tliffi~rrnt sets of overproducers. In ow (t.hc pbNH1, 4: sta‘t‘ Slalrrials and Met,hods). nali\-cl t ranslat~it~rii~l start sites u-tare probahl>. used. Howt~ver. t hr. most fjurified frat~tiorls t)lJt;iiljeti using t,htLso ovc~rlJrt)tlllc.rrs contained a complex mixture of proteins, very little total protein. and too lit’t.lr McrK or ZVT\ilcr(: for unarrbiguous identificat,ion of the relevant prot,ein. N-t,ermina,l sequence determination was not possible. These partially puritied fractions (McrR fraction ii and Mcr(’ fraction iii) were nevertheless devoid of (~xoriucleast~ alltl rioii-spfvitit. t~ntlorlrlc~l~~~ts~~ in Mat fbrials arid activity (tested ah tlrst*rihed Metjhotls). and \vt’re usrd in some of’ t,hrk cxp~~rimt~llts t~oncerliing the site of’ t*lcavap (wts I~vltJw. scv.1ioll (g)). The sword (‘1‘7) set of OvW~JrOdllWrS \vt‘rf’ cyjnstruc~tetl such that translation init iatioli was fortwi to tj(‘cai1r at spwitit~ positions t~tiose~l iwt)i Ijot,rnli;rl siifts. t’rYjn1 t hv &nlong t)raril? HomogrntLous (or rlrarly I-rorno~Lenet)rrs) prottGn was obtaintd anti shon.n t.o C)r acd iw. All of thr t~harac~terizat with

fractioils

ion

dfw-rikd puritif~tl

aljovf~

was

(‘1’7) or partially

twritd

out Ijuritirtl

435

JlcrRC Endonucleas~ Action

[ATPI CrnM)

kb 2.3 1.9 1.4 1.3

obcdefqh

qrstuvwx

ijklmnop (al

o*NG-yoo li,AG66066

[GTP-y-S](mM) [GMP-PNP](mM) LGTPl(md

cD0D-j

Ol+g$!~~

I

ro.5

i

obcdefgh

j

k

I

mnop

(b) Figure 2. Effwts on McrUC’ activit’y of various nucleotides and analogs. McrU fraction IV and Jlcr(’ frac.tion \.a. ww used. sue% that rac*h reaction contained X pmol McrR. 8 pmol YIrr(‘ (rotnhined activity on thr same day of ,5 unit?;) and Oo’i ~~nol ~~BICCLlluT in the presence of decreasing conwntrations of nucleotidrs. Othrr wntl~tions \I’WC’ as in Materials and MClrthods. Mixtures were made up without McrK and without the indicakd nucleotide. nuc~leotitlr a-as addrd t’o thr 1st tube in the series. the samples were serially dilutrd to the conwntrations indica,tc4. and reactions \ver’r hegut~ by adtlitiotl of McrR and transfer to 37°C”. (a) Thr 3 titration series shown here demonstrate the rf&cts of (iTI’ alonr (laws a to h) and of ATP (lanes i to p) or AiNPPI’SP (lanes q to s) in the presence of 0.5 m.w-( iTI’. (hi Titration stlries for (:&II’- I’SP (lanes a to h) and GTP-7-S (lanes i to p) in ththioninr residue of the frame is sir amino acids upstream. The McrC’ start site used was nucleotidr 1367: this is the first methioniw residue acid residurs of its frame. and is ten amino upstream from t,he start s&e proposed h\, Ross rt (11. (19896). The position of translation initiation WLS verifirtf 1)~ sequencing the K terminus of’ thr pur-

McrB

a bed

Yis i

McrC

efghi

Figure 3. I’urifimtion profile of’ MwIS ant1 ~cr(‘. Protein (l-.5 pg) from each fraction (Tahk 1) was load4 as indicated. run on a IO to 20”,, polyacrvlamide SIW gradient gel and stained with (‘oomassie Brilliant t~luo a:, described in Matrrials and ~lethods. Thr JZcrB protein (arrow at left: lanes a to d) migrated with an apparent molrcular wright of -51,000, whereas the Mcr(’ protein (arrow at right, lanr~s e to i) migrat’ed with an apparrnt~ molrcula~~ M;eight of - 39,000. The - 27.000 31, wntarninant in Mcr(’ frac%ion I\’ is ribosomal protein Sd. as drduwd from K-terminal sequence of 19 amino avid wsidurs.

fied proteins (see Materials and Methods). The possible impact of the position of t,ranscription and translational start, site is discussed below (1)iscussion). (‘rude extracts made aft,er induction of ~11s cwrying t,hese construct,s were assayed thr their enzymatic activity and analyzed by SISF’AGE.

(‘olumn

t’rotrin

(rng)

t For JlcrH, 1 unit ia the amount of enzyme required in the presence of 1 ~1 of MwC’ fractiotl I\’ (about 100 fmol) to cleave site B of 02 pg (about 70 fmol) pBR322.AluI (see Fig. I) to completion (see Materials and Methods). For McrC‘, 1 unit is the amount of enzyme required in the presenw of I 1~101’ crude extract from BLdl(DE3)/pER263 (about 2 pmol) to cleave site C‘. 1 For fraction \‘a, only a portion of fraction IV was further purified this way: total protein and total units are adjusted to compensate for this. yj NA, not applicable; this fraction was titered at a different time. and thus units annot be vomparrtl.

JlrrBC

pBR322

Endonucleanv

T4gf

puc19

abcdefq

hijk

Action

I m

(a)

n

T6gt

abcdefghi

j (b)

Figure 4. Srnsitivit>- to McrB(’ digestion of differently modified substrates. SlcrH(’ nas used w a mixturc~ of JlerR f’rnction ITT. and .\‘l(~r(’ fraction Va (see Table 2). such that’ approximately 2 /ce (40 pmol) of McrB and 0~3.5pp (8 pmol) of M(~r(’ wrr~ present in each reaction. nith a most-recent tit.er of 5 unit,s: ot,her vortditionx as in Materials and Mrthods. (a) I)igrstion products of variously methylated pBR322 (lanes a to a) and ply(‘19 (lanes h to n). all tttc~tiifird irl rit,o. Mrthylases that c.onfer sensitivity to mcrB(’ in ritw are M.AluT (.i(i’YTT). M.HarIII (GGm(‘(‘), M.HhrrT ((:“(Yi(‘), Jl.,llspl (“(‘(YK:) antI M.Sssl (“CX). whereas unmethylated DNA and DSA modified by M.HpaI (CYYK:) art’ not sensitive to restriction irr ri~o. Digestions are not complete. but they do produce bands of defined sizr: the pat%icaular partial digest I)rodu& are obtained reproduribty in enzyme titrations at different stagrs of purification and from diff+rrnt sour~s. (b) l’r’odu~ts from .Vc*rtSC‘ digestion of T4yt and TGgt DKA. The DNA itt lanes tt to d and g to j were first digrstrd \vith tr:coRI or A’spI to ensure that all of the large genotne ( - 170 kb) entered the gel. These phapr I>NAs caontain hytlrox,vmethyl-

analyzed 1)~ S I )S- f’A(: F:. no JIcrI& was detected (data not shown). Since we could ha,ve detected as little as l.?Otrg itt this experiment. if any was l)resent it must have been at a concentration < 1 O. of’ the JlcrR, c,otlc,f,ntration. The characteristics of cleavage as deh(~ribecl above (sections (1)) and (c)) and helow (section (f)) wflre qualitatively consistent for all enzyme preparations from all constructs (data not showy). From these data. we conclude that McrH, is the only product of t)he mcrH gene required for ir/ vitro clea\,age act,ivity. In short. Mcrf%‘-specific activity requires McrK,. Jl(nr(‘. Jfp* ’ and GTT’. These component’s act to cleavr c.?tosint~-modified 1)SA in a sequence-specific manner. The prr(*ise specificity of YIcrfH’. i.e. the f )&A seclurnc*rs recognized and the position of cleavage. was t IW nest cbtaracteristic of the enzyme analyzcYl.

Each of the McrBCI-sensitive sequence-specific tnethylases yielded a different banding pattern (Fig. l(a)) when samples of the same D?U’A were modified and then digested with McrBC purified as described ahove. Again. the modified I>XAs that were restricted in viw were substrates in vitro: pBR322 (lanes h t,o g) or pc’C (lanes h to n) modified by M.Al/tl (lattes 1) and i). SI.HaeIII (lanes c and j). M.ffhnf (laws (1 ant1 k). M.M.spT (lanes f and m) or

M.SssT (modificat.ion specific$>-. S”CY:) itrfl clegra,ded it/ ~ifro. whereas the unmodlhed plasmid (lanes a and II) and plasmid modified by the 1\l.f[~~iIT methylasr (lanes e and 1) were not degraded. Ext.rnsive modification resulted in extensive calravage: ~I.SxsT-modified I)SA yielded a bright smudge of unresolved prodncts well helow the 0.7 kh size marker (Fig. 2(a), lanes g and n). IAM. molecular weight products were expe:cted. since thtx ( ‘I)(: recogmore nitinn site for NI.XssI occurs so rnuc~h frecluent’ly than do the sites for ot.her met hylases. The patterns in Figure 4(a) resulted from partial cleavage of the substrate. Tn most cases. the sum of fragment sizes resulting from McrH(‘ clf~avqy wits greater than the size of the substrate 1)X-A. and not all bands were represent.ed equall>-. For faxample. for pHTi322.dluT (Fig. 4(a). lane b) the largest bn~Jd (2.3 kb) is present in a lesser amount than the next largest (20 kb). M’e have mapped some of the sites of cleavage relative to ot.her restriction sites (dat.a not shown). Thp positions of the A//r1 sites on pHK322 a,nd the approximate positions of the McrRC cleavage sites on pHR322.,il/rr I are shown in Figure I. Kot.e that t,here are Ii An1 sites but onI>four principal McrEK’ sites. One clea\-aye resulted in the appearance of 2.3 and 2.0 kh fragments: another cleavage occurretl in t,he 2.3 kh fragment. resulting in the appearance of the 1% and 0.7 kh fragments seen in Figure l(a). (Jornplete cleavage of the four sites mapped on this 1.3 kh substratta wor~ld result in

f’typet~t,s

of 24). O.!). 0~75. 03% anti 045 kb. ‘T’hib digest pattern has never hrrtt acahie\-ed. ‘fhcx dificulty in obtaining a limit digest and t hc ~)it~ti(‘Ul~t’ pttvrns of protluc~ts Ivit h difi~~rettt substrates wt:rt~ observed with all ettz>pmrt sour(~t’s i1Iltl at all st*ages of purificat,ion. iZs exf)ected from in Go data. hvdro.u!-mc,t.h?-l~ c,~tositle-c,ontaiiiing DNA was also sensitive to lk~w~( c2kavage (kg. 4(h)). In vice. T2 ant/ ‘I’4 f)hagt> with unglucosylat’ed hmC in t’heir DNA HOP rc~st.rict~rd by M(LrBC* (RglB), but. 7’6 f)SA containing hm(’ is not restricted by it (Revcbl, I!+li7). piyurc> 4(b) shows dn ,vho digestion of I)SA f’rom two h”(‘-containing T-even phage. Tlqt and Tliqt. The T4qt (lanes a to e) and TByt (lanes f to j) I)N;\s tclstr,d here were first cleaved with EcoRI (lanes t). (‘. g and h) or SspI (lanes d, e, i and j) and then with .\f(arB(’ (lanes c. e. h and j). f- hI(.rIZ(‘. This result for ‘1‘4!qtwas ~~xpe(Qd. sitrcr t hr Ithag? ih scatisitivr to r&ric*tion in /%~/YJ.Possible ~.x]~IittliltiOtlh for the discVf)anc~y brtwertt ir/ r*itro and in ~ivo rtWllts with T&/t arc’ addressed later ( WP I)iscussion). c~oml~letf~

\fYe tsxamined t hr cleavage f)roducts in more d&ail. focusing on calravage sit,r A (Fig, 1). This strongly prefrrrtld McrK(’ cleavage sitr is fount1 in a cluster of five .4//11 sites within a region of 70 bft. Any or all of thr t.rn modified cytosines associated with these .4 luT sites could ],(a rcquirrd for recogttition or cleavage. LYr used the method of primrr extension on of ,11c~rIJ~‘-cleavetl suhst,rate to map tf~e position c~lravage relative to these modified .illz~T sites. Flanking primers with 3’ ends at f)p 1969 (to], stra,nd) or bp 222X of pIZR321 were used. \.\‘ithin this region we ident,ified sit)es for caontrol restricbtion c*lcavagc. /linfI cleaves at position 2031 to yield R three-base 5’ 6~xtettsion, Pr%r*Il cleaves at position 2066 t)o yield a blunt end, and Zlrdl cleaves at ftositiott 2164 t,o yield a five-f,ase 3’ ext.c,nsiott. pKR322 (met,hylatcld or unmet~t~latc~tl. as af)proftriat,e) ~42s digested by McrIX‘. or fty one of the (Gontrol enzymes. or by one of the cant rol enzytn~ attd then by 5’Ic.r lJ( ‘, End-labelled primers ( 321’) were annealed t)o the cleaved I)XA and extrtrdetl with Klenow enzyme. These rrac+ions rrsultrd in thr synthesis of labeled DNA complementary to one taut strand of pKR312 DNA, of a length determined by the position of the cleavage. The products were analyzed on tl?;, polyacryfamide-urea gets; Sanger dideoxy-sequencing reactions using t,he same fjrimers served as size standards and to locate t’he produ& in the sequence. Results from onr such experiment, are shown in Figure 5. For simplification of the Figure and clarity of the results, the sequences in Figure 5(a) and (b) have been labeled to reflect t-he sequence of the cleaved strand rather

tftan its synthesized c*otnplcment. Accordingly. 1h(x sequence reads 5’ t’o 3’ from top t.0 hot tom r;tt.ht*r tfian the rtlverse. BfcrK(’ cslcaavagr of c,losed-c~irc:ulat, and linthat. tnodifird I)NA is shown in Figure 5, lattps a ttttd tt respectively in eaczh panel; Figure’ T,(a) shows calravages made on the top strand of’ f)lis conventionally represented and Figure 5(bj shows those on the bottom strand. The most striking f’act about these results was the large number of ftroduct bands present. These bands resulted f’rom thcl intrinsic action of McrBC. not from contaminat.ittg nuclease, as discussed below. Controls designed to detect nucfease c*otttatnination of t,he McrB. McrC’ or AluI methyfa,sr f)rrf)arations, or other artifactual results, included c~lravag~~ with PPuII (T’), HinfT (H) or L)rdI (I)). I:ttmodified plasmid DNA was cleaved with one ot’ t’hesr, tftr satnplr was divided. one sample was t rc~at~t~tlwith Jlc~rB(‘. and both samples were analyzed. f3at1d patterns iti eac’h pair of lanes (c and d. t’ and f’. ilIlt I1 and i) were identical. so no ~~xottuclrasc~ contaminatrd the Sl(>rK or Mcr(’ fractions. Similarly. non specific. single or double-strand endonuc~least~ (*or>taminatiott of the Alul met,hyfase preparat.ion was ruled out by cleaving tnoditied pfasmid witfr IIinfI (Fig. 5. lane g), =\ comparison of Figure 5. IWIICS P, f and g shows no produc%s derived from tJfrcl met ftyl~ atcd DKA not found in t,ht> tmmet~hylatt4 l)SA. lsofat~ed. intensely laf)~~letl bands were ot~t~aitred f’rottr digtbsts \vith c~otivt~trtiott;~I rthst,ri(4 iott (~tixyrn(+ (IiLtlt’S (’ 10 I). 1vhic.h at’f’ litlO\4’tl t0 (‘I(‘i\\.f’ i1 sitigl(~ f)~~osf)~io(tit~sIt’,’ t)otld Ott c%c,h st rattd. ilt i1 llosit ion foutid iti a tletined rrlatiotishif) to thcl t.cWtgttitiott site. ,4c,cordingl>,. t)lici tntiltif)lt~-t)atrcI ])a1 tern obtainecl from McrK(’ dipt~stion strongly ittclic&es that M(arK(’ c~k~avagr~oc*c~ttrrt~clal tntrltiple silrss \vit h a IOOSW rc4at~ionshif) to tf>cl rtlc*ognitiott *it. f~riming sitfas. Sttcah a sit{. is (*lt~ilt~l>~ltrr%scattt itt th(b I)rdT-(*l~~a\-(~tl c*ottt rol (Fig. 5(a). Ittttt’h It atttl i). ‘I’htt f)ositiorr of’ thr> l)rdl c~lc~avagr in tftcs r+otr of’ interest is indic~ittr4 (bottottt right ot’ Fig. .;(a)). ‘I’hcs dark hand af~orr thr known c*ut site, is most likeI> due to it stJc.ondary ftriming sitta irlrnt~itic~cl ~.Ios(’ 10 thrl only othf,r ZJrdl site, on ftI oond b

G

t 49 &

2117

2136

&

d

McrBC cuts lanes o and b

A C 1

2

lllC 1

e?

McrBC cuts Iones o and b

Figure 5. (Zharaet.erizatiorl of an MerBC cleavage site. Primer tzxtension reactions were carried out as cirscrihed in Materials and Methods. The substrate, pBR3W (modified or not,). was precleaned with MrrB(’ or caontrcjl restriction enzymes so that primer extension yielded DZU’A fragments of sizes defined by the relative posit’ions of the primer and of the cleavage in the complement of the synthesized strand. Parallel Sanger sequencing reactions on non-cleaved template using the same primer yielded fragments of sizes defined by the positions of the primer and the incaorporated chaint,erminating nucteotide. Sequence lanes (to the left in each panel) are labeled to correspond t,o t,hr sequence of the complementj of the synt,hesized strand. to facilit.ate interpret,ation of (Qleavage positions. Therefore. sequence reads .i’ to 3’ from top to bott~om. rather than the reverse. (a) Reactions t.hat. tlrt.ec.t cleavages in the top strand: (b) reactions that detert, cleavages in the bottom strand. The composition of each reaction is indicated at the top of the lanes. The substrate was treated wit.h McrBC (+ ) or not (-); in some cases it was treated first with another restriction enzyme (P = I’~l1. H = HinfI and I) = D&T). The topological state of the T)KA at the time the indicated enzyme was added was c*ovalently closed (c) or linearized with CZaT(I). Modification by M.dZuT was present ( + ) or not ( - ), The positions of .IkuT sites are marked to the left in (a) and to the right in (b); numbering indicates the modified position and is acc>ording to the Genbank sequence as of December 1988. which does not rrflrct recent revision (ETatson. 1988). Th(s .4l,tI sit,e at. position 2000 is ac*t.ually beyond the top of the autoradiograph, as indicated by thr vertical arrow at, left. iI1 (a). McrIX WBS a mixture of McrB fraction iii and McrC fraction iii (see Materiats and Methods); 3 ~1 (about 3 units. defined as in Materials and Methods) of McrBC was added to each reaction. Primers were end-labeled with [7/-32P]ATP. Lanes labeled b in hot*h panels (a) and (b) are faint, hecause less template was used than in the other lanes.

The s~ond potrntial artifact can result itt the appearancr of pairs or triplets of bands n-here therr should be only one. The cause of t’his “stuttering” is slippa~ge of t)he Klrnow polytnerase near the clnd of 1)iY.A fragments. particularly in AT-rich regions. MClt~fting of the cuds followed by rrannealing out of rtyist.rr may allow addition of an extra base (oftr,n a ‘I’); or rxotiuc~lrolytic~ a,t,taczk on melted (apparrtitl>, rnismat~ht~d) terminal bases may remove a has?. This particular artifact was seen with Hinfl dipclst ion in some experiments. Tts possible contribution to t hr pat,tern in Figure 5. lanes a and b is difficult t)o assess. However. the experiment shown in Figure 5 was repeated four times using different DNA preparations and at least t,hree different preparat’ions each of SlcrlS and Me&. In all cases. the banding patt,ern was essentially identical.

The

multiplicity of c4eavagr products might if McrH(: bound in a different, particular aligrimrnt to the f)SA sequen(a( when thtx topolqg diflered. i.e. different t,opoisomrrs might br clrlavecl diffi~rrntjly. :4cc~ordingly. we analyzed the produc*ts of reaction wit’h linear DNA. which should be topologically uniform. Thr band pat,tern is not detectably altered (compare lanes a and h). alt,hough in this experiment the bands in the linear sample art’ lighter due to a lower sta,rting I>KA concentration. This particular model for generation of different c+avagSr posilions was t)hereforr eliminated. resnlt

X description of the McrKC recognition site(s) was attempted based on t.he results shown in Figure 5 experiments. The eneytnt* similar and ot hrr appeared to cleave at positions between two methyfated sites (Fig. 5). The positions of met,hylf)ct,ween thtb atvd .31ul sites. and the spacing tnrthyfated bases. are indicated to the left of Figure ,5(a) and to the right of Figure 5(b). The cleaved sit,es appeared between methylat,etl bases 49 bp apart on the top strand (bases indicated at 2068 and 21 I7 in Fig. 5(a)) and between the same two sites on thr b&tom strand (bases at 2069 atld 21 18: Fig. S(b)). These cleavages correspond to site A in Figure 1. In Figure 5(b). it is seen that clravagc also occurred between bases 57 k)p apart (2001 and 2058): these correspond to site I> in Figure 1. as will f)r discussed further below. However, no cleavage products migrated between t)he rnethylated bases I I bp apart (20.57 and 206X). or I9 bp apart (2117 and 2136) in the top strand (Fig. 5(a)); or in thth caorresponding posit,ion on the hot t om strand (Fig. 5(b)). To get, a more general view of cleavage spvcificit.~~, a preferred cleavage site on pKK322,Z~haI was also examined by primer rxtension. Results from t’hese

On the basis of tttrhe c~sperimrtits atrcl t tics f)roposed “(:Y”. rrcogriition site (ftalrigh & \I’ilsott. 1986). w(’ dcvelopc~d the fi)llowitig modr4 of’ it (*l6sii~ able sitca. (‘fravagc o(‘(aurs bfbt wren tnet h~ylatr~tl I)its(xh in an appropriatr squeti(‘tl c,orttcLxt (( i’“( ‘) \vit tr spacing between methyfatt4 bastes itr t tits ratig(’ IJ~’50 to 60 bp. This suggest,iotr foc~nsrvl oitr’ alletitioti oti aft nnexplaincd fcatitrra of’ Figur(b 5: t ht. c~l(~avagc~ on t.hr bottom strand brtwrott 1tositiotts r’O()tj atrtf 367 rt t~atltl without c9rresf)onding top c~ltw~;r#‘s hrtwt,eri positions 2OOf atitf 20.58. ‘1’0 vxl)laitr t tiih ~3% note two fh(+s. First ttrct clitstc*r of c~l~a~iqys t)(‘tWCYn trrrttiyfatetl b;tWs -C!)I)[) :lt)i\l’t (T)llltl ilihll IN* said to O(‘(‘111’ twtwrrt1 lMSW fi0 t,J, ;l]J;ll’t (]wl \LlY’ll positions 2057 ant] 21 17 rat tier 1 trati ])flt mt’r’tl 2OfiS atld 21 17). It tnight tW. iti fac.1. t trat lU’0 C'll'klVil&?f' sittas ilK' suprritnposed tiercs. Svc~)ric]. this ‘tLottItI(~ site”

(site,

;\)

wit.1)

49

atltl

ti0

]t]t

S])it(4itlg

i5

(./os(.t.

to

the primcbr in t’he ~xperimrnts itt ICgttrrx ,\(a) thatt ih “missing” *ii-base hitigl(L sit I). yic~ltlitig shorl l)rotltrc*lh itt thus iti lli(s esperitncttt itt Figurch .5(t)) Itlit Iotrg ]m)cllt(dh .?(a]. llrtt ltoAltl>.. all in Figuw c~xpcritrit~nt rnolec~ulr~s ~vould f)es c*l(~\\-c~tl at silts A (t ttt- cloltl~l~~ sit(b). vielditig lotig pro(luc~ls as hwfi itr f$,rrtrc~ S(t)) f)ut t,r’uttcbat ing th(l long prodttc.ts itt the (~\f)t~ritn~~nt~ in Figurt~ r)(a) to short ottf’s. ‘I%(~ tol, qt rati< long produc+s uould thus f~ missitlg t’rotrr ttrv gt.1. A4 partial test of the tloublr~3itr trioclr~l 4VilS c3rrircl out by disrupting ttita .4//rf sitrb a1 ltosit ioti 2OAX. This was ac~c~omplishrd by inserting an X-t)]) .Ill~l linker at the /‘c,uTT sitrl (this c~ontaitls 1h(h Altrf xii(t). LVhen met h>rlatr(f 1)~. A1..-flrr I. ottI> ;I .+iti,qlfJ’. M(arfX sit{> at sitrl A should ttr c~rc~atrtl. with (X4 1))) hrtwerti tiir~tti\~latjrtl c.J-tosittrl tGclu(~s it Itos(~ at 2 I 17 + X iltld irt 2057). :\ ])5ittrI’tl Ot’ f)ositions multipk ]twrltls wah O])tiLilld similar t(~. ljllt trot idetitic*aI with. that sho\vti in IGgurtl 5 (data ttot shown). This c~~nfirtiird that ttic, mot ttyla1 iott hitP at t;tr chivtt$y. 2Of% was not tltYws;tt’y f)ositiotr (‘leavage of’ a substrates c~arryitig t tira sites ;I1 positiott IOtiX hnt ftot 20.57 (ser sw3iott (,j )) vrt~itirtl 1 Ilat thts (+orivC’rst’ was also trite. It i h stiobvti twlo44 (~~1 iott (j)) that 1Lvo methylatrtl positiotis ill't' rc~clitir~~d. These rrsltlts art‘ c*otlsistent 1%ith thus itl(s;t t ttat tht~ rnaltped sitp A caotisisth of two c~lravabk~ (aotlfigur+ 1ions of stqut~ricc and tllay it1 f):rr1 c~sftlaitt the> apparent preft~rrttc~c~ of M(arfZ( fi)r this sit of McrB(‘. we desired an in vitro assa>- that reflected the 6n ~i~,o characteristics of the restriction system. Our initial criteria were: substrates known to br restricted in GZW should be sensitive it/ rlitro to fract*ions isolated from McrBC’ cells but not from Mcr BC cells: and substrates kno\j,ri to be insensitive to r&riction ire. ,Gw should also be resist,ant it2 vitro. Finally, in most (bllt not all) rrstric~tion. the characterized (bases of in ciw molecular basis of thr phenomenon is doublrstranded DNA cleavage, so we lookrd first for DXA cleavage a,ctivity. That is what we fount1 (Figs 2. 1 and 6). The enzyme cleaves only appropriatei> modified sites (Fig. 4) in cslosr prosimity to the inferred recognition site (Figs 4, 5 and 6). genrrat)ing well-defined bands wit,h grossly mappable ends (Fig. 1). The diverse banding patterns obsrrvrd with the sarnc subst,ra,tr differently modified (Fig. 1) are

Irot intiiiJit (‘IC’iIVU$$’ srlggf~sts t 11211, tlyfit~ol~~st,~ 01 :I’f‘f’ may tJe requirefl for inhilJitiott. tJut (lo trot I.IIIC~0111 sitnfJle vom[Jrtiti\-r inhitJitiott lJ\. :\‘I’I’. It1 ytt(.tt . ttot l)intl wf,ll 14) 1trfx (~T~‘~tJinding xit(l ant1 t lrr1s tlot fYml],f~4f~ f;lt, t hf. f~of’af~tot~

Ilot~c~ detailed f~satnittat ion sflo~v~~tl t hc>fJosit ion of’ calf2ivage to IW limited to a small region. IIII~ ttot itnic~urly fixed (Fig. 5). .\lrtltifJlr~ (‘If’iiViIyt’ posit if)nh :\ rf~ilsollak)lf~ intev itt il lOc*al rrgion wf‘r’f‘ ohsrrvrfl. fJrr,tat ion might be that (.lea\ages are ~~~rtnall~ tlist rihnted about a cvntral [Joint. This suggestion is :iclv;tllcWl with caaution. sitwfh fJOtentii~l iil’tif’;lfatS f’wti not I)fs ruled out, A similarity hf~tkvf~f~trthis sugyfw tion and a model for li:coK c~lravapt~ (Studier & I~;lntl~f)f)atlh~i~~. I !NX) is discwssrd l)f~If)\V. ‘I’hf~ hfyuenw K”(‘(S,, 80)Kjm(’ is fwoposrfl as a f~onsrtisus st~fprtif~c’. \\‘r wrrr ablf~ to design it syttthetic substratv of 8% bp and 0lJtaitt f*lfavqy (7’ahlt~ :< antI Fig. 6). JIatlifJulation of this ~fy~tf~ttt*f~ all~~etl its to determine the required c~onfiguratiort of modifirvl sites ai~tl to test some of the requirements for I ttts atljacent sequence. Two modifietl sites are rrclttircd. ancl at fsafah r~lodififd sitf,. met hylation of’ sl rtti~fl (tit her strand) is rfquirefl. Od> OIl(’ I)ou~Jl~~-strantl t*leavagc of ttrmimt~th~lated I)NA is c.onsistent \vit h restric*tion results iJc viva (Tatrle 4) attd iS ur~usual for restriction enzvnlt’s. The permissihle sfJacing between the 5°C’ tAdurs is at least 10 10 X0 hp. based on mapping data (Figs 4 and 5 and clnta ttot showtt). Thcb a(atnal t’aItg(’ of’ f)rrtnissitJIr sfJacittg may be greater. Howevt~r. sites with sfJaciny of I I. I!) or %I bp wvre not cleavf~l (Fig. 5 anfl tlitta trot

sho\\n).

Both McrB, and AMcrC are required for cleavage (data not shown). These correspond to two of the three principal prot’eins synthesized from rr,rrH and rued’. The t,hird requirement for cleavage activity is (:TP. I)?iA cleavage by MClcrK(’ absolutely required a guanosine triphosphnte with a hydrolyzable j--s phosphate linkage; the sugar moiety did not seem to be of critical importance (Table 3). Since non-hydrolyzable analogs. GTP-y-S in particular. actually inhibited the GTP-supported cleavage reaction. we infer that they were able to bind to t’hr enzyme. This suggests that lack of hydrolysis. not, failure to bind to the enzyme, may be responsible for t’he inability of t,he analogs t’o support clearagr. By cont,rast. ATP actually inhibited GTPdeFJendent cleavage (Fig. 2). when present at concentrations comparable to the GTP concentration. The fact that non-hydrolyzable analogs of ATT did

sitfb

on

thfJ

f’itx\.mf’.

~‘ot~Sif~f~t~irt~l\~

mot’f-

tletail(~cl c,tizvirtolo~ic.aI h111~ly \\ill 1~. ttr~f~- (.J. Krllvht~r. I’. f,~ prfJtidr l~f~gall ilS pt~etlic~t~tl t)y Ross rf f/l. ( 19X9). It \\.tlilt start hitrs WI’f USfdfl i/r /~il’cj. is tlOt f’lf’ill’

?‘hv Jlc~fJ(’ systfxm is not IiktB any other, rwtriv tion rnz>‘nic so far descvilJrt1. It differs in two priitcipal ways. First the c,leavage vharavterist i(as a rv different McrK(’ c4eavc~ quite c~lose to t,he rrcaogntion sit.f,. since 8% bp model substratfls ilt’f’ wttsit ivfs to Clt?tl\-i&g? (Fig, ti artfl nhk 3): arid it dof3 ho without slJeci;tl treatmeirt. sinve a varirt)y of itatttral substrat.es generate specific banding patterns (Fig. 4). It t’hus is unlike the type I enzymes. which c-leave f’ar from the recognition site and do not generate IJanding patt#erns (Bicklr, 198%; Endlic+ 8 f,inn. 1985: Murray f4 01.. 1973) except in special circumstancrs (Studier 8 Bandyopadhyay; 198X) discussed below. However. on the evidence of Figure 5, the relationship between the recognition site and the cleavage site is not defined as exac%ly as it is for type 11 enzymes. which cleave at a unique position (Modrich & Roberts. 1982) or for type IT1 enzymes. which cleave within a kJasr or two (Bickle. 1982). The scvond differrnce between iMcrK(‘ and ot,hei restricticJn enymes is that the c.ofa,ctor require merits are different. Most nucleotide-dependeiit nurleases. including type 1 and type III restriction enzymes. require ATP, not GTP as does JlcrK(: (set‘ further discussion below). The onlv nuc~l~asr \~f’ know of that will use GTP. Exe 1’. prefers ATP (Goldmark & Linn, 1972). The enzyme is nevertheless more sitnilar t,o t,he nucleotide~dependent. multisubunit type I and type 111 enzymes than it is to the single-subunit. nucleotide-independent t,ype IT enzymes. I,ike type TTT enzymes. McrBC produces a banding pattern. and like type IT1 enzymes it is difficult’ t,o achieve a limit) digest. The apparent requirement for t,riphosphat’e

McrBC

Endonuclease

hydrolysis to achieve cleavage is more similar to type I enzymes, however; ATP-y-S will support cleavage by the type III enzymes but GTP-y-S will not support cleavage by McrBC (Table 1). A further speculative connection may be made between the mode of cleavage of type I enzymes and that of McrBC. based on the model for EcoK cleavage proposed by Studier & Bandyopadhyay (1988). Briefly. Studier & Bandyopadhyay (1988) were able to observe a banding pattern, with an ordered appearance of bands, when cleavage of T7 DXA by prebound EcoK was initiated by addition of ATP. Based on this and other considerations, it was suggested cleavage is triggered when two translocating complexes collide (the “collision” model). The non-appearance of bands in ordinary digestions with type I enzymes would then result from nonsynchronicity of binding, since the position of collision presumably would be distributed to a variety of locations between binding sites. This idea is supported by genetic evidence that cleavage in vivo occurs between K sites (Brammar et aZ.. 1974). as well as by evidence that non-synchronized cleavage in c&o yields heterogeneous but non-random products (Murray et al., 1973). There is no evidence for a DNA translocation mechanism for McrBC, nor does the order of addition of enzyme and nucleotide affect, t’he pattern observed. However, cleavages are distributed between modified sites that themselves may be spaced with some flexibility. This situation could reflect a collision mechanism. Cleavage might br triggered by a conformational change induced when two bound complexes touch each other in a particular way. an event that might be less constrained to a specific position than is the case for type II enzymes. Models for events mediated by I>h’A-binding proteins in which touching triggers conformational change are common (see, for example, Ptashne, 1988; Ptashne & Gann, 1990, and references therein). Such a mechanism might also explain in part the apparent’ hierarchy of site preferences exhibited by the enzyme (Fig. 2). The results described here. taken together, argue against. but do not disprove, the possibility suggested by Raleigh $ Wilson (1986). that ,McrBC restriction operates via a glycosylascx-XI’ endonuc~lrase combination, its does the urtg-mediated restriction described by Duncan (1985). First. the action of the system is indeed sequence-specific (Figs 4 and 6. Table 3). a,nd all DNA glycosylases so far described are base-specific. not sequence-specific. This is a weak argument. Second. a hemimethylated substrate is cleaved in both strands in. vitro (Table 3 and Fig. 6). which should not occur if strand cleavage requires base removal on the same strand. Third. a hrmimrthylated substrate is restricted in /*irk (Table 4). unlike uracil-containing phage made in a very similar way (Kunkel et ~1.. 1987). The latter two arguments reflect, the same underlying sit,uat,ion. that known AP-endonucleases cleave oni)the baseless st’rand (Linn, 1982). However. it is possible to imagine a novel endonucleasr that would cleave both strands when a second protein (t,hr

345

Action

glycosylase) is bound there. It must not do so in response to a naked baseless site. or the uracilcontaining Ml3 would be restricted in cells cont)aining McrBC. More detailed investigation will be required to rule out, this more complicated model.

(d) Implications

for regulation of the enzymatic nctiuity of NcrR(’

Since both McrB, and the non-essent,ial McrK, carry a protein sequence motif similar to that in GTP-binding proteins with highly regulated activities. such as EF-Tu. Ras and the signal transdurtion G proteins (Dila et al., 1990), it is intriguing to speculate that GTP hydrolysis has a regulatory role. The binding of GTP, hydrolysis, and release of GDP can all be regulated steps (Hourne et nl.. 1991). The specific ac%ivit,y of the enzyme appears to be poor. for example. it was present in excess over sites in the experiments in Figure 6 but, did not cleave to completion. This could reflect a requirement for GTP turnover and possibly the int,ervention of a (missing) third protein to facilitate turnovrar. if the srquencae motif is a useful guide. The role of t,he small product of tncrN. McrH,. is not de&mined by this work. It is clearly not required for cleavage activity (Figs 3. 4 and 6). McrH, does not appear to alter the reaction qualitativcly. since the characterist,ics of cleavage did not differ when examined in various fractions during partial purification from constructs expressing it (purification no. 1 of McrK: see Materials and Characteristics examined Methods). included GTP-dependence, response to inhibitors. and IocBation of and preference for cleavage sites (data not shown). McrB, might modulate the level of activit’y in some manner. which could (but need not) involve its GTP-binding site. The inhibitory effect of ATP could also play a role in the level of restriction in ,r%*o. possibly in combination with McrBs. Such a role for XTP is suggestled by the observation t’hat the activity of the enzyme in wuo should alreadv be partially inhibited: the .iTP concentration “in the cell is approximatelv 3 mM. whereas the CT1 concentration is threefold lower (Bochner & Ames. 1982). The act,ivity should be responsive to the relative levels of the two nucleotides in this ra,nge (see. for example, Fig. 2, lane k). Yet another observation sugg&inp that, the activity of >lcrKC might be regulat,ed is t’he sensit,ivity of T6gt DSA to restriction in. &o (Fig. 4) but not in rain (R,aleigh et al., 1989b; Revel. 1983). In view of the weak sequence-specificity of the enzyme proposed above. the question is why t,his phage is not rest’ricted in vi7!o, rather than why it’ is cleaved in t:itro. A possible resolution might be found in the anti-rest’rict’ion protein (Am) known t.o be elaborated by T4 (Ijharmalingam & Goldberg, 1976b; Uharmalingam et aZ., 1982). The ;2rn protein can rescue superinfecting phage from in r% r&riction by R#glK (McrKC). Presumably. it does not do so

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upon single infec%ion because it is syntjhesised too late or is ineffective in small amounts. The failure to restricat TByt in r,i~o might) bc explained if the phag~ were to elaborate an Arn func?tion that actas faster or more effectively than t.hat of T4. That caould allow 1he infecting genornc t,o br rffic+nt.ly resc+uetl from the, restriction that would ot,herwisc ensur. The mechanism by which Arn acts is notj understood at present. In summary, the McrBC enzyme is most similar t.o the multisubunit nucleotide-dependent type I rextrict,ion enzymes. but appears to represent a new class of enzyme. GTP is a required cofactor for ,McrB(‘: its hydrolysis may be part of the mecharlism of action of the enzyme. The presence of ATT’ is inhibitory by an unknown mechanism. Numerous potential mechanisms for regulation of the activit) of t,he enz,ymr than be identified from thtl picture drawn here. 12’~ thank. especially, Tra Schildkraut and Bill .Jack for csontinuing encouragement and essential critical discussion. We also thank nil1 Jack for t’he gift of pAIIJ 7; Stan Hattman and Sam Schlagman for the gift of T4 and T6 I)NA: Deborah Dila for construction of st)rains and plastnids used: Ian Molineux for drawing our attention to thrx Bochner and Ames reference; and Lydia Dorner. Emma .Jean Hess and Becky Kucera for technical advice. ~111 JJurification and in vitro work with the enzyme was cnarrietl out by E.S.: in ~ivo work by E.A.R.: and W-terminal protein sequence determinat’ion by L.C. This work was suppot%ed entirely by I’Cew England Kiolabs.

References Rickle, T. A. (1982). The ATP-dependent restriction endo(Linn. S. M. 8; R,oberts. R. J.. nucleases. Tn Xuclcases Ads). pp. 85-108. Cold Spring Harbor Laboratory Press. (‘old Spring Harbor, KY. IGkle. T. A. (1987). DNA restriction and modification systems. In Escherichia coli and Salmonella t,yphimurium Cellular and Molecular Biology (h’eidhardt, F. (‘. et al.. eds). pp. 692-696. American Society f