The EMBO Journal vol.13 no.24 pp.6152-6161, 1994

Chromosome end formation in phage X, catalyzedby terminase, is controlled by two DNA elements ofcos, cosN and R3, and by ATP

R.Rachel Higgins and Andrew Becker1Department of Molecular and Medical Genetics, University of Toronto,Toronto, Ontario, Canada M5S 1A8

'Corresponding author

Communicated by H.Eisen

The terminase enzyme of phage X is a site-specificendonuclease that nicks DNA concatemers toregenerate the 12 nucleotide cohesive ends of themature chromosome. The enzyme's DNA target, cos,consists of a nicking domain, cosN, and a bindingdomain, cosB. cosB, situated to the right of cosN,comprises three 16 bp repeat sequences, RI, R2 andR3. A similar sequence, R4, is present to the left ofcosN. It is shown here that terminase has an intrinsicspecificity for cosN which is independent of the R sites.The interaction with cosN is mediated by binding totarget sites that include 12 bp on the 5', and 2-7 bpon the 3' side of the nick. Of the four R sites, only R3is required for the proper formation of ends. When R3is present, an ATP-charged terminase system correctlycatalyzes the production of staggered nicks in cosN, atsites Ni and N2 on the bottom and top strands,respectively. When ATP is omitted, the bottom strandis nicked incorrectly, at the site Nx, 8 bp to the left ofNi. If R3 is removed or disabled by a point mutation,nicking in cosN becomes dependent upon ATP but,even in the presence of ATP, bottom strand nicking isdivided between sites Ni, the correct site, and Nx, theincorrect one. Thus, R3 is an important regulatoryelement and must reside in cis in respect to cosN.Furthermore, cosN substrates bearing point mutationsat Ni and N2 are nicked at sites Nx and Ny, 8 bp tothe left of Ni and N2, respectively. When R3 is presentand ATP is added, nicking is redirected to the Niand N2 positions despite the mutations present. Thus,terminase binding to R3, on one side of cosN, regulatesthe rotationally symmetric nicking reactions on thebottom and top strands within cosN.Key words: cosAambda/terminase

IntroductionThe essential catalyst in the maturation and packaging ofk DNA is the phage-coded enzyme terminase (for reviewssee Feiss and Becker, 1983; Feiss, 1986; Becker andMurialdo, 1990). Terminase holoenzyme, as purified (Goldand Becker, 1983), is a hetero-oligomer assembled fromtwo polypeptides, gpNu 1 (Mr 21 kDa) and gpA (Mr74 kDa), the products of the X genes Nul and A,respectively (Sumner-Smith et al., 1981). Terminase inter-acts with X DNA concatemers at a site called cos, which

is a complex DNA target that encompasses the sequencescosN, to be nicked by the action of terminase to generatethe sticky ends of the mature phage DNA (Figure iB),and cosB, where the enzyme must bind for its properfunctions (Figure 1A).

Genetic and biochemical studies on terminase reveal anintricate system of reactions that includes binding to cosB(Feiss et al., 1983a,b; Miwa and Matsubara, 1983), cosNcleavage (Becker and Gold, 1978), prohead binding(Becker et al., 1977; Frackman et al., 1984) and DNAstimulated hydrolysis of ATP (Gold and Becker, 1983).The known roles of ATP in terminase action include thoseof an allosteric effector that stimulates DNA binding atcos (R.Higgins and A.Becker, in preparation; also seebelow), promotes cosN cleavage (Becker and Gold, 1978;Gold and Becker, 1983) and fine-tunes the nick-sitespecificity in cosNR (Higgins et al., 1988).A number of the activities of terminase can be correlated

with domains strung along the enzyme's two polypeptidechains. For example, genetic studies with hybrid viralproteins have localized the DNA binding domain to theN-terminus of gpNul, the prohead binding domain to thecarboxyl end of gpA and subunit assembly to the C-terminus of gpNul and the N-terminus of gpA (Frackmanet al., 1984, 1985). Near the N-terminus of gpNul, ahelix-turn-helix motif is present and likely represents adomain for specific cosB DNA binding (Feiss, 1986; Kyprand Mrazek, 1986; Brennan and Matthews, 1989). TheATP binding sites, first inferred from sequence analysis,reside in gpNul and gpA (Guo et al., 1987; Becker andGold, 1988; Parris et al., 1988). Recently, Davidson andGold (1992) have described point mutants near the C-terminus of gpA that are specifically defective in cosNcleavage. One of these mutations lies within a putativeleucine zipper region (bZIP) (Vinson et al., 1989) whilethe other lies 150 amino acid residues upstream within asequence that bears homology to a motif conserved amonga number ofDNA polymerases and primases, and believedto be involved in nucleotide binding (Delarue et al., 1990).One or both of these two regions within gpA may definedomains involved in specific cosN recognition and/ornicking and in the dimerization of terminase protomers.

cosN, displayed as a one-dimensional array (Figure 1 B),shows imperfect 2-fold rotational symmetry (Weigel et al.,1973) and the nicks, NI and N2, are symmetrically placedwithin this dyad. The arrangement invites comparisonwith a type II restriction enzyme target and suggests themodel of a complementary protein dimer (say, of gpAsubunits) forming a symmetric interaction with its DNAtarget (Becker and Murialdo, 1990) along the lines of theEcoRI paradigm (McClarin et al., 1986). A number ofobservations concerning the terminase reaction, however,suggest a less direct, perhaps more complex presentationof enzyme to cleavage target, as follows. (i) The two half-

62 Oxford University Press6152

Specificity of cos cleavage by x terminase

A

B

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Fig. 1. Summary of structure of cos DNA. (A) A view of cos represents -280 bp, predominantly from the left end of X DNA. cosN is drawn as therectangle with the potential sticky ends and bisected by its axis of 2-fold rotational symmetry (vertical, dashed line). The alternating orientation ofthe four gpNul binding sequences, called R and one IHF binding sequence, called 11, are shown. Below R3 is displayed the 16 bp R3 duplex; theR-site sequence given is offset by 2 bp from that proposed in Bear et al. (1984) to reflect more accurately the region protected in DNase Ifootprinting experiments (R.Higgins and A.Becker, in preparation). The relationship of cos, a non-coding region, to the beginning of the Nul gene isshown. The distance from the center of R3 (+53/+54) to the NI site (+6/+7) is shown to be 47 bp (Higgins and Becker, 1994). (B) The sequence

of cosN is given, again bisected by its axis of 2-fold rotational symmetry. Boxes denote perfect symmetry matches. Bases to the left are calledcosNL and can be numbered negatively from right to left, beginning with -1. Within cosNL, the top strand of X DNA is nicked by terminase in thenucleotide interval -6/-7, called N2, or aberrantly at nucleotide interval - 14/-15, called Ny. Similarly, cosNR is the half-site to the right of thesymmetry axis, with bases numbered left to right, starting with + 1. Within cosNR, the bottom strand of k DNA is nicked, normally in the nucleotideinterval +6/+7, designated NI, or aberrantly at -2/-3, called Nx (Higgins et al., 1988). The r- and 1-strands correspond to bottom and top strand,respectively.

cos sites are not treated symmetrically during the nickingreactions at cosN; nicking in cosNR appears as the leadingreaction, with nicking at cosNL following closely behind(Higgins et al., 1988). (ii) Each of the two half-cosN sitesis readily nicked in the absence (by deletion) of its partner(Higgins et al., 1988). (iii) Symmetrically placed, identicalmutations in cosN can have very unequal effects on coscleavage (Xu and Feiss, 1991). The question then ariseswhether binding to cosB might somehow govern thenicking activities of terminase at cosN.

In the bipartite structure of cos, cosB comprises a regionof some 160 bp between cosN and Nul (Feiss et al.,1983a,b; Miwa and Matsubara, 1983) (Figure lA). In thiscosN-Nul interval there are three 16 bp segments calledR3, R2 and RI that bind gpNul (Bear et al., 1984; Shinderand Gold, 1988). To the left of cosN, an analogoussegment, R4, failed to bind gpNu 1, but that can beshown to bind holoenzyme (R.Higgins and A.Becker, inpreparation). In addition, cos contains consensus bindingsequences for the Escherichia coli DNA bending protein,IHF (Bear et al., 1984; Feiss, 1986), and one of these, theone that binds IHF most strongly (Xin and Feiss, 1988),is shown in Figure lA. IHF binds to cosB and promotesthe activities of terminase (Feiss et al., 1985, 1988;Granston et al., 1988; Miller and Feiss, 1988).What is the function of cosB on the nicking activities

of terminase at cosN? Earlier studies by Feiss et al.(1983b) and by Miwa and Matsubara (1983) indicatedthat the requirement for cosB in the cosN cleavage reactionwas not absolute and that the total deletion of this regionreduced cleavage to -10%. Cue and Feiss (1992a) studiedx phages that carried single base pair transition mutationsin the R sites of cosB. The mutation of any one of theR3, R2 or RI sites rendered the phage dependent on IHFfor plaque formation, but the double mutation of pairs ofR sites resulted in the failure of plaque formation in eitherIHF- or IHF+ cells.

Is the formation of a cohesive end by the action of

terminase an essential step in the initiation of DNApackaging? Xu and Feiss (1991) showed that substratesbearing mutations in cosN were poorly cleaved in vitroand led to the accumulation in vivo of proheads thatwere not expanded, suggesting that packaging, if at allattempted, was aborted in the absence of cos cleavage.Thus, the role of cosB in the initiation of DNA packagingmight be examined from two points of view: (i) thepromoting of efficient and correct nicking at cosN byterminase or (ii) the promoting of the assembly ofterminase protomers into an oligomeric apparatus requiredfor prohead capture and subsequent packaging (Beckerand Murialdo, 1990), or both. In the present study, weexamined the role of the R sites in the nicking reactionsat cosN. We also examined the consequences of certainbase changes within cosN. The results show that R3, thebase pair sequence of cosN and ATP are critical elementsfor the proper formation of the X chromosome ends.Mutations in either the sequence of cosN or R3 or theomission of ATP alter the biochemical properties of theterminase reaction at cosN and result in the formation ofX chromosomes with aberrant ends.

ResultsRole of the R sites in cosN nickingPrevious studies on the nicking of cosN by terminasedetected no difference between substrates that containedall four R sites and those that lacked R2 and/or RI (Higginset al., 1988). In both cases, cosN nicking took place inthe presence and absence of ATP. When ATP was presentnicking occurred at sites NI, position +6/+7(13/12) andN2 (-6/-7), on the bottom and top strands, respectively.In the absence of ATP, however, the bottom strand wasnicked incorrectly at site Nx, at position -2/-3(4/5), 8 bpto the left of the NI site. The same studies also suggestedthat R3 might be important. When a half-cosN substrateof structure R4+cosNL+(cosNR-R3R2RJ)' was used and

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Fig. 2. Schematic diagram of cos-containing fragments used assubstrates in cosN cleavage studies. The plasmid source of each andthe corresponding cos-containing fragments are shown. The restrictionfragments are drawn to approximate scale; the locations of R sites areindicated, as are the nicking sites N1, N2, Nx and Ny in cosN. Xderived sequences are solid lines; polylinker sequences are zigzagged.The fragment in (1) from pWPI4, contains the complete X cos site(cosN in continuity with cosB). The fragment in (2) from pRH121,lacks R2 and Rl. The fragment in (3) from pRH4N3 1, lacks cosB R3,R2 and RI sites. The fragment in (4) from pIB13-, bears a transitionmutation at position +52(58) of R3 (Cue and Feiss, 1992a). Thefragment in (5) from pRHN3 1, carries the sequence of cosN, from-22 to +31 and is deleted for all four R sites. The fragment in (6)from pRHN 14, carries the sequence of cosN from -22 to + 14. Thefragment in (7) from pRHN8, carries the sequence of cosN from -22to +8. The fragment in (8) from pSX124, is deleted for cosB andbears transversions at positions +7 (Ml) and -7 (M2) of N1 and N2,respectively. The fragment in (9) from pSX125, carries a CG to ATtransversion at position -7 of N2 (M2). The fragment in (10) frompSX126, has a GC to TA transversion at position +7 of NI (Ml). Thefragment in (11) from pCOSNSL, carries cosB in continuity withcosN, but cosN carries both transversions at positions +7 and -7 ofNI and N2 (Ml/ M2) (Xu and Feiss, 1991).

nicking of the top strand was examined, nicking at N2became strictly dependent on ATP. We asked if this changewas due to the removal of R3 and/or cosNR, by testing asubstrate that lacked R3, R2 and RI, but that had an intactcosN element. Figure 3 shows the results of a nickingexperiment where the two DNA substrates, differing fromeach other by the presence or absence of R3, werecompared [R4+cosN+R3+(R2RI)A (Figure 3A) andR4+cosN+(R3R2RJ)A (Figure 3B), respectively]. Eachsubstrate was labeled at the 3' end of either the top or thebottom strand to examine nicking at cosNL or cosNR.

The typical result for the R3+ substrate (Higgins et al.,1988) is shown in Figure 3A. Nicking of both the top andbottom strands takes place in the absence of ATP, but thepredominant bottom strand nick is at Nx (lane 5). In thepresence of ATP, the bottom strand nick is redirected toN 1, the correct site (lane 6). Figure 3B gives the resultsobtained for the (R3R2RJ)' substrate, specifically labeled

at the 3' end of either the top strand (lanes 1-3) or thebottom strand (lanes 4-6) and shows that the propertiesof the nicking reactions at cosN are altered in two waysby the removal of R3. First, in the absence of ATP, nickingof either the top (lane 2), or bottom strand (lane 5) is notobserved. Nicking activities in these strands are only seenwhen ATP is present (lanes 3 and 6). Secondly, unlike thecase of the R3+ substrate (Figure 3A), in the presence ofATP, the location of the bottom strand nick is not uniqueto NI but is divided between NI and Nx to a roughlyequivalent extent (Figure 3B, lane 6). The results indicatethat R3 serves two functions. First, it enables terminaseto nick at cosN in the absence of ATP and secondly, itenables the enzyme to discriminate between the NI andNx nick sites on the bottom strand.

A specific, unique role for R3 in nickingIn order to verify that these differences in reactionproperties were due to the specific deletion of R3 and notdue to the removal of the entire R3R2RI block, orthat R2 and/or RI, had they been present, would havesubstituted for R3 to restore these putative R3-specificproperties, we tested a DNA substrate that carries a pointmutation in R3 but has normal R2 and RI regions incontinuity. This R3 mutation, which is a G to A transitionat position +52(58), exhibits a deleterious phenotype inan IHF- host (Cue and Feiss, 1992a,b). Figure 4 comparesthe normal substrate (Figure 4A, lanes 1-5) with the R3-counterpart (Figure 4B, lanes 1-5) with respect to nickingof the bottom strand, the more diagnostic strand. Theresults for the normal substrate are typical, with nickingpredominantly at Nx in the absence of ATP, while in thepresence of ATP the reaction is stimulated (5- to 15-fold,with increasing enzyme concentration) and the principalpoint of nucleolytic attack is shifted to NI. The resultwith the R3- point mutant substrate is qualitatively thesame as with the (R3R2RJ)A DNA, namely, no detectablenicking at either N 1 or Nx in the absence of ATP andnicking at both N 1 and Nx with roughly equivalentefficiency in the presence of ATP.

These results strongly suggest that the altered nickingactivity at cosN is due to the inability of terminase tobind the mutant R3 site of cosB. Indeed, this issue hasbeen examined by DNase I footprinting. There was noevidence of binding of terminase to this mutant R3 site,neither to the bottom nor to the top strand while, bycontrast, R2 and RI were clearly protected from DNaseI attack (R.R.Higgins and D.Cue, unpublished data);furthermore, a normal R3 site is readily protected byterminase binding (R.Higgins and A.Becker, in pre-paration).

R3 functions in cisOne role of R3 might be that of a specific allosteric ligandthat altered the conformation of the enzyme upon bindingand thereby changed its nicking properties. To test this idea,we again examined the R4+cosN+(R3R2Ri)A substrate fornicking by terminase, but added R3- or R3R2RJ-containingDNAs in trans in the form of restriction fragments ofDNA, but this did not change the nicking attributes of theR3A substrate (data not shown). Furthermore, in order tomaximize the concentration of R3 present in trans, weadded a synthetic 30mer of duplex DNA containing the

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Specificity of cos cleavage by k terminase

1i 2`3 G Nel X ;4 5 6 X, '.%

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Fig. 3. The properties of the nicking reactions at cosN are altered when R3 is deleted. A comparison of cos DNA nicking by terminase on twosubstrates. (A) The EcoRI-HindIII fragment from plasmid pRH121(R2RJ)A radiolabeled at the 3' tip of the top strand at the EcoRI site (lanes 1-3)or at the 3' tip of the bottom strand at the HindIII site (lanes 4-6). (B) The EcoRI-HindIII piece from plasmid pRH4N31 (R3R2RJ)A, labeled at the3' tip of the top strand, at the EcoRI site (lanes 1-3) or at the 3' tip of the bottom strand, at the HindIlI site (lanes 4-6). DNA fragments were

reacted with terminase, but in the absence of ATP (lanes 2 and 5) or in the presence of ATP (lanes 3 and 6) and electrophoresed throughpolyacrylamide gels containing urea. For lanes marked 1 and 4, there was no enzymatic treatment and lanes marked G are the Maxam and Gilbert(1980) G-specific sequencing ladder. The positions of the terminase nicks Nx, NI and N2 are indicated, as are the positions of the nicks byrestriction enzymes XmnI (X), MnlI (M) and FnuDII (F). The terminase- and ATP-dependent nick called N I is in the interval + 10/+11 (Higginsand Becker, 1994).

16 bp R3 sequence and surrounding nucleotides to afinal concentration of _1015 molecules/ml (_10-6 M). Nodifferences were observed. In particular, as shown for thebottom strand in Figure 5, there was no relief from theATP requirement (compare lanes 2 and 5 with lanes 3 and6) and no evidence of any Nx to NI correction (comparelanes 2 and 3 with 5 and 6, respectively). The resultssuggest that, for the R3 dependent effects to take place,R3 and cosN must be in cis, that is, in DNA continuitywith one another.

Failure to find a role for R4 in cosN nickingHaving shown that R3 exerted control over certainparameters of the nicking reactions at cosN and that RIand R2 did not, we asked the related question about R4.To test if R4 could in any way modulate nicking activityat cosN in either the bottom or top strand, R4 deletionswere combined with the normal cosB and with variousdeletions that extend into cosB from the right, systematic-ally removing the R sites on the right of cosN (Higginset al., 1988). Reactions were performed in the presence or

absence of ATP. No significant differences were observedbetween the R4+ and R4- substrates (data not shown).Only the presence or absence of R3 made a difference tothe reaction parameters, in the manner described above.The results indicate that R4 does not exert any controlover nicking by terminase at cosN as studied in thisin vitro system. Binding to R4, however, might inhibit the

production of an incorrect nick on the top strand at Ny(see below and Discussion).

Binding to R3 by ATP-charged enzyme modulatesnicking events at cosNAberrant nicking of R3-negative substrates bearing basepair substitutions in cosN. Analysis of the nicking patternsof certain cosN mutants further revealed how the nickingspecificity of terminase at cosN can be modulated by theavailability of R3 for enzyme binding. First, substrateswere used which bear deletions of RI, R2 and R3, andwhich carried either the normal cosN sequence, or mutantsequences in which the base pairs adjacent to, andbracketing, the normal N1 and N2 nick positions were

transversions, +7C to A, called Ml, and -7G to T, calledM2 (Xu and Feiss, 1991, wherein these mutations are

called 13A and -1T, respectively). These transversionswere present either as individual changes, or together as

the double M1/M2 change. In addition, x DNA to theright of NI, shown to be important in cosB-independentnicking at cosNR (see below), was retained up to position+31(37).As for all substrates which lack R3, nicking of both the

bottom (Figure 6, R3AA) and top (Figure 6, R3AB) strandswas dependent upon the addition of ATP (compare lanes2 with 3, 5 with 6, 8 with 9 and 11 with 12 in Figures 6,R3AA and B). For the normal substrate and for that withthe single -7 (M2) change in cosNL, nicking at NI was

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Fig. 4. A point mutation in R3 alters the nicking properties in thesame way as does the deletion of R3. Nicking by terminase of asubstrate containing normal cos (A) and one bearing the transitionmutation in R3 at G+52 (B). In (A) the EcoRI-AvaII piece fromplasmid pWPl4 was radiolabeled at the 3' tip of the bottom strand, atthe Avall site. In (B) the same EcoRI-AvaII piece from plasmidpIBI3- was also labeled at the 3' tip of the bottom strand, at the AvaIlsite. Samples were incubated with terminase and without ATP (lanes2); or with ATP and with increasing concentrations of terminase (0.8,1.6 and 3.2 mg; lanes 3-5) and analyzed on 8% urea gel. For lanesmarked (1) there was no treatment with terminase. Abbreviations andsymbols used are the same as in Figure 3.

in -3-fold excess over nicking at Nx (Figure 6, R3AA,lanes 3 and 9). This observation indicates that the mutationin cosNL had no effect on the nicking operations in cosNR(i.e. on bottom strand nicking). The cosNR +7 (Ml)transversion, however, present alone or in combinationwith the -7 (M2) substitution resulted in a markedreduction in nicking at N 1 (compare lanes 3 and 9with lanes 6 and 12 in Figure 6, R3AA, respectively).Furthermore, nicking at Nx was enhanced by a factor oftwo (lane 6) or three (lane 12) in the case of the cosNRtransversion when compared with that of the normal cosN(lanes 3 and 9).

For the substrate with the normal cosN and that bearingthe cosNR +7 (Ml) mutation, nicking of the top strandat N2 proceeded as usual in the presence of ATP (Figure

Fig. 5. R3, provided in trans, fails to restore the normal nickingproperties. cos DNA fragments used as substrates were theHindIII-EcoRI piece from plasmid pRH4N31 radiolabeled at the 3'end of the bottom strand at the HindIII site. Lanes 2 and 3 show theproducts of reactions carried out in the absence of the R3-containingduplex deoxyoligonucleotide, while lanes 5 and 6 are reactions wherethe synthetic 30mer was added. In lane I terminase was omitted, inlanes 2 and 5 ATP was omitted and in lanes 3 and 6 ATP was added.The multiple banding pattern observed is due to non-uniform end-filling with nucleotides during radiolabeling. The nick called N-9,commonly seen, is in the interval -8/-9 (Higgins and Becker, 1994).

6, R3AB, lanes 3 and 12). For the DNA bearing the cosNL-7 (M2) transversion, however, nicking at N2 was reducedby a factor of three (compare lanes 9 with 3 in Figure 6,R3AB) or six (compare lane 9 with lane 12 in Figure 6,R3AB). The data indicate that the -7 (M2) bp substitutionin cosNL results in reduced nicking activity at N2. Strik-ingly, in the case of the substrate bearing both the -7(M2) and +7 (Ml) substitutions, nicking at N2 is abolished(Figure 6, R3AB, lane 6). Together these data imply thatthe normal interaction of a terminase protomer withthe N 1 site in cosNR can promote some productiveterminase-DNA interaction at a mutant N2 site in cosNL.However, when the enzyme-DNA interaction is alsoimpaired at the N 1 site by mutation, productive

6156

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Specificity of cos cleavage by k terminase

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Fig. 6. Binding to R3 restores nicking to the mutant NI and N2 sites. cos DNA fragments used as substrates were: in R3A, the AvaII-EcoRI pieces

from the plasmids pSX123(normal cosN, lanes 1-3); pSX124 (M1/M2, lanes 4-6); pSX125 (M2, lanes 7-9); and pSX126 (Ml, lanes 10-12). In (A)the 3' tip of the bottom strand was labeled at the Avail site. In (B) the 3' tip of the top strand was labeled at the EcoRI site. In R3+A theAvaII-EcoRI pieces from plasmids pWPI4 (normal cosN, lanes 1-3) or pCOSNSL (MI/M2, lanes 4-6) were uniquely labeled at the 3' end of thebottom strand, at the AvaIl site. In R3+B the same AvaIl-EcoRI pieces were used and were labeled at the 3' end of the top strand, at the EcoRIsite. For lanes marked 1, 4, 7 and 10 there was no enzymatic treatment, for lanes 2, 5, 8 and II incubation was in the presence of terminase but inthe absence of ATP and, for lanes 3, 6, 9 and 12, incubation was in the presence of terminase and ATP. Lanes F and U display the products ofnicking by restriction enzyme FnuDII (F) or Fnu4HI (U). The sequence of cosN (-18 to + 18) is given below the gels; the position of the terminasenicks (N1, Nx, N2 and Ny), the FnuDII (F), the Fnu4HI (U) sites and the mutations are indicated. N1 17 is a prominent terminase- and ATP-dependent nick commonly seen in the interval + 116/+117 (Higgins and Becker, 1994). The vertical dashed lines to the left of NI and N2 represent

the axes for the NI/N2 and Nx/Ny nicks, respectively. Fnu4HI nicks DNA at positions -2/-3 and - 1/-2 on top and bottom strand, respectively.Note how the Ml mutation abolishes nicking by FnuDII which restricts DNA at the interval +8/+9.

enzyme-DNA interactions at the mutant N2 site are

greatly diminished. One other feature concerning topstrand nicking in these mutant substrates is noted. For thesubstrates bearing the -7 (M2) substitution at the N2 site,as well for the case of the single +7 (Ml) Nl site lesion,a novel nick is introduced in the top strand in the-14(48495)/-15(48494) nucleotide interval. We havecalled this site Ny (Figure 6, R3AB, lanes 6, 9 and 12); itbears the same relation to Nx, the 12 bp stagger, as N2bears to N1.The results indicate that the base sequence of cosN

plays an intimate role both in the specificity and in thedual nature of the nicking reactions at cosN. Binding ofterminase to a normal cosN results in the formation of a

nicking complex that nicks at Nl and N2. If Nl and N2are mutant, as above, the nicking complex is displaced as

a unit by 8 bp to the left, to nick at sites Nx and Ny.Re-establishment of nicking specificity on mutant cosNsubstrates due to R3 binding. The nicking ambiguities,described above, imparted by the base pair substitutionsin cosN are rectified when R3 is available for enzyme

binding. In the experiment of Figure 6, R3+ for example,two R3-containing substrates, one with a normal cosNand the other bearing the double -7 (M2), +7 (Ml)substitution, were examined for nicking of the bottom(Figure 6, R3+A) and top (Figure 6, R3+B) strands.Bottom strand nicking of the substrate with the normalcosN is typical, at Nx in the absence of ATP andpredominantly at N1 in the presence of the nucleotide.Interestingly, the identical result is also seen for thesubstrate bearing the two transversions (Figure 6, R3+A).In the case of the top strand (Figure 6, R3+B), nicking ofthe normal cosN is at N2, with or without ATP. Nickingof the mutant substrate is again interesting, almostexclusively at Ny in the absence of ATP and almostexclusively at N2, the normal site, in the presence of ATP.In the case of the mutant substrate, nicking of cosNLappears inefficient relative to nicking at cosNR under anycondition. Together, these results indicate that through thebinding of terminase to R3, and in the presence of ATP,the endonucleolytic domains of the enzyme are positionedto interact correctly within the mutant cosN despite the de-

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R.R.Higgins and A.Becker

stabilizing effect of the base changes present. Furthermore,binding to the R3 site of cosB regulates both the top andbottom strand events at cosN.

These results extend those of Xu and Feiss (1991) whoobserved -10-fold reduction in cos cleavage (Nl plus N2)for this mutant substrate (cosB present, in their case), aresult that can be explained by the relative inefficiency ofnicking at cosNL (Figure 6, R3+B, compare lanes 2 and3 with 5 and 6, respectively).

DNA target for R3-independent nicking of cosNIn view of the ability of terminase to nick within cosN,at N1/N2 or Nx/N2, in the absence of cosB, we asked,for such an enzyme which is not bound to R3, about theextent to which bases on the 5' or 3' side of the nick mayor may not play a role in nicking. Deletion/substitutionDNA substrates were prepared from which all four of theR sites had been deleted and replaced by heterologouspGEM polylinker DNA, in each case from position-23(48486) leftward and from (i) position +31(37) right-ward, (ii) position + 15(21) rightward and (iii) position+9(15) to the right, as shown in Figure 2. The resultantcos-containing fragments carry 24, 8 or 2 bp of k-specificduplex DNA, plus an additional 9 bp of heterologouspolylinker duplex DNA, 5' to NI; or 31, 16 and 10 bp ofk-specific DNA, as well as the additional 9 bp of polylinkerduplex, 5' to Nx. As shown in Figure 7, for the +31substrate (lane 2), nicking at N 1 and Nx took place in thepresence of ATP with the usual approximate equivalence.For the + 15 substrate (lane 4), however, nicking at N 1failed to take place while nicking at Nx appeared to proceednormally. Upon further impingement toward cosNR (the+9 substrate), nicking at NI again did not take place,while nicking at Nx was significantly reduced (lane 6) ascompared with the + 31 and +15 cases.Our results with these substrates can be interpreted in

either of two ways. One possibility is that certain X DNAsequences in the interval +15 to +31 are required fornicking at NI and, similarly, X sequences in the interval+9 to + 14 are required for efficient nicking at Nx. Weexamined cos DNA in the +9(15) to +31(37) region forrepeats in base sequence, spaced eight nucleotides apart,as possible substrates for specific terminase binding duringnicking at Nx and N1. We noted the presence of a GCdinucleotide at positions 11 and 12, counting bases in the3' to 5' direction from either the NI or Nx nick. TheseGC dinucleotides are changed in the + 15 and +9 deletion/substitution substrates that inactivate nicking at N I and Nx,respectively. Interestingly, GC doublets are also present atequivalent positions with respect to both N2 and Ny inthe top strand. Other data (not shown) indicate that 12 bpof X-specific, duplex DNA 5' to N2, which carries thisGC doublet, is all that is required for normal nicking atthat site. We have not pursued this issue further.An altemative explanation emphasizes the extent over

which the enzyme must contact the DNA, rather thanspecificity of interaction. Thus, 8 bp of duplex k DNA 5'to the N 1 position, as in the case of the + 15 substrate, isinsufficient DNA for interaction and nicking at N I.Similarly, 10 bp of X DNA duplex 5' to Nx (the +9 case)is insufficient for proper binding and nicking at Nx,whereas 15 bp of duplex is sufficient, as in the + 15 case.According to this interpretation, terminase requires >10,

r _ _

1 I1-

I

12 3 4 5 6

N1

Nx

Fig. 7. DNA requirements for R site-independent nicking in cosNR.cosN DNA fragments used as substrates were the BamHI-EcoRIfragments from plasmids pRHN31 with 25 X-specific base pairs 5' toNI (lanes 1 and 2), pRHN14 with eight X-specific base pairs 5' to NI(lanes 3 and 4) and pRHN8 with two k-specific base pairs 5' to NI(lanes 5 and 6). To the 5' side of these k-specific sequences, thesesubstrates carried an additional 9 bp of heterologous duplex DNAderived from the pGEM polylinker. The bottom strand of eachfragment was uniquely labeled at the 3' end, at the BamHI site. Forlanes marked 1, 3 and 5 incubation was in the presence of terminasebut in the absence of ATP and for lanes marked 2, 4 and 6 incubationwas in the presence of terminase and ATP. Samples were processedand electrophoresed through denaturing polyacrylamide gels. Thepositions of the terminase nicks on the bottom strand, N1 and Nx, areindicated.

but <15 bp of X DNA 5' to its nicking position forproductive binding/nuclease activity in cosNR; in cosNL,as stated above, 12 bp of kDNA is sufficient for nickingat N2.

In a previous study (Higgins et al., 1988), we showedthat N2 and N1 are nicked efficiently in half-cosN sub-strates that carry seven and nine k-specific base pairs 3'to N2 and NI, respectively. Nicking at Nx however,appeared inefficient in substrates with only one k-specificbase pair 3' to that site. This suggests that the number ofX-specific base pairs that may be required 3' to the nickis between two and seven. Together these results implythat cosN comprises four potential binding sites for thegpA subunit of terminase, Nx overlapping NI on thebottom strand, and Ny overlapping N2 on the topstrand.

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DiscussionWe summarize this work by stating that the base pairsequence of cosN, the R3 site of cosB and ATP are criticalelements for the specificity of chromosome end formationby phage k terminase.We have shown that deletion of the RIR2R3 DNA

interval of cosB, but not of the RJR2 block, alters thebiochemical properties of the nicking reactions at cosN intwo ways: (i) nicking of both DNA strands becomesdependent on ATP and (ii) when ATP is added, terminaseis not discerning on the bottom strand, nicking at NI or

Nx, a site eight nucleotides to the left, with about equalfrequency. The result with the (RIR2R3)A DNA substratecan be duplicated by a point mutation in R3 that disablesenzyme binding to R3 (it makes no difference whetherRI, R2 and R4 are present or absent). An R3-boundenzyme, on the other hand, nicks within both top andbottom strands of cosN in the absence of ATP, butpredominantly at the incorrect Nx position in the bottomstrand. Further, in the presence of ATP, the R3-boundenzyme efficiently redirects its point of nuclease attack inthe bottom strand from Nx to Ni, the proper site. Thus,R3 is a dominant regulatory DNA element that cannot besubstituted by RI, R2 or R4. Attempts to restore this R3function to an R3A substrate by providing R3-containingDNAs in trans did not succeed. Hence, to perform its rolein nicking, R3 must be in cis configuration with respectto cosN. The precise relationship between an R-site boundenzyme and its point of nucleolytic attack is elaboratedin an accompanying paper (Higgins and Becker, 1994)The observation that an R3-bound terminase can nick

the top strand in the absence of ATP, while a terminasethat is not bound at R3 is impaired in this task, suggeststhat an R3-bound terminase can influence nicking eventson both sides of the cosN symmetry axis. We propose thatthe protomer which is to operate at cosNL is bolstered tonick at N2 even in the absence of ATP by virtue of itsspecific interaction with the R3-bound protomer operatingat cosNR; the interaction being suggested is one betweenthe two gpA subunits across the symmetry axis of cosN.When R3 is missing, terminase protomers, by virtue ofthe intrinsic specificity of their gpA subunit for cosNsequences, will interact with, and nick, both the top andbottom strands of cosN, but both reactions require thebinding of ATP to enzyme.A further compelling argument for the assembly of a

dimeric system at cosN is provided here by experimentsthat tested the nicking of substrates which bear pointmutations in cosN and the effect of R3 on these reactions.In the absence of R3, binding of terminase to a normalcosN results in the production of nicks at Nl/N2 or Nx/N2. If, however, NI and N2 are mutant and R3 is missing,the bottom and top strand nicks are displaced as a block,by 8 bp to the left, to nick at sites Nx and Ny. The siteNy bears the same spatial relation to Nx as N2 bears toNI. The top strand half-site Ny (5'CGACIAGGT) more

closely resembles the bottom strand Ni site (5'CGCGItA-GGT) than the top strand N2 site (5'TACG1GGGC).Concurrent nicking at Nx and Ny, and subsequent disen-gagement of the DNA strands upon ATP hydrolysis(Higgins et al., 1988) would generate complementary endsof 12 nucleotides, but with a sequence differing from that

of mature X DNA. Thus, the nucleotide sequence at cosNplays an intimate role in the specificity of the nickingreactions.On the other hand, when R3 is present on these mutant

substrates and ATP is added, nicking is redirected to theNI and N2 intervals. This result indicates that both of theendonucleolytic domains of the enzyme are repositionedto interact correctly within the mutant cosN, and thatbinding to the R3 site of cosB regulates the location ofboth the top and bottom strand nicks at cosN, overridingthe sequence recognition problems in cosN caused bythe mutations. Such repositioning of the binding/nickingdomains of gpAs due to the binding of gpNul to an Rsite suggests a system in which the location of the nicksis gauged from that R site, a mechanism confirmed byother studies (Higgins and Becker, 1994).A significant outcome of these studies is that terminase

retains its intrinsic nicking specificities for the top (at siteN2) and bottom (at NI) strands of cosN even when allthe R sites are deleted. Thus, terminase has a dual sequencespecificity, for the R sites of cosB through its gpNu 1subunit and for cosN through gpA. In view of the intrinsicspecificities of terminase for cosN, we asked if basesoutside of the nick sites might be required for interactionwith its gpA subunit and, if so, to what extent. Accordingly,certain deletion/substitution DNA substrates that lackedR3 were tested for their ability to be nicked by the enzymeand were found to be impaired in this capacity. Our resultssuggest that the binding sites for the gpA subunits ofterminase consist of 12 bp of duplex X DNA 5' to thenick and 2-7 bp in the 3' direction. Although it is notclear if specific DNA bases play a role here, we note thepresence of a GC dinucleotide at positions 11 and 12,counting bases in the 3' to 5' direction from either theNI or Nx nicks. These GC dinucleotides, also present atequivalent positions with respect to both N2 and Ny inthe top strand, are changed in the deletion/substitutionsubstrates that inactivate nicking at NI and Nx.

Becker and Murialdo (1990) presented a model for theinteraction of terminase with cos for cosN cutting and theinitiation of DNA packaging. One feature of this model,pertaining to cosN nicking, was based on the apparentcontinuity of terminase's interaction with the R4-cosN-R3 region as evidenced by footprinting studies (R.Higginsand A.Becker, in preparation). A protein-DNA interactioncontinuum extending from R4 to R3, across cosN, wassuggested and the observation invited symmetric modelsin which a terminase protomer bound at R3 was nickingin cosNR, while another protomer bound at R4 was nickingin cosNL. The results given in the present study do notsupport this model. The model predicts that, in the nickingreactions studied, the R4 site would be the site of bindingof the terminase protomer that nicked in cosNL. However,no effect of R4 on cosN nicking could be demonstratedby comparing substrates that were deleted for R4 (thesesubstrates carried heterologous DNA in place of R4)with ones that had intact R4, under a variety of cosBconfigurations. The results imply that R4 is not involvedin the nicking reactions in cosN, either in the top orbottom strands. Cue and Feiss reached the same conclusionand uncovered a different role for the R4 element, that inthe termination of DNA packaging (Cue and Feiss, 1993).Finally, our recent studies (Higgins and Becker, 1994)

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establish the unique directionality that is imparted to aterminase protomer upon binding to an R site. If an R3-bound protomer is directed to nick at cosN, then anyputative terminase protomer bound to R4, which is in thesame orientation as R3, would have its nucleolytic domainsdirected to the left, away from cosN (Figure IA).We propose a model where the protomer nicking at N1

is bound to R3, while the protomer nicking at N2 is notspecifically anchored to an R site via its gpNul subunit.Rather, it is held in register at cosNL by the associationof its gpA subunit with the gpA subunit that is interactingwith cosNR. The interaction between the gpAs bolsterstheir specific binding to cosN DNA. The model canincorporate the putative bZIP region in gpA (Davidsonand Gold, 1992) as the structure by which the protomersdimerize, while the basic region domain of the bZIPsystem is specific for the vicinity of NI and N2 (Vinsonet al., 1989; Ellenberger et al., 1992). In this model, thebinding of a terminase protomer to R4 would block bindingand/or nicking at the incorrect Ny site on the top strand.The DNA comprising R4 (-18 to -33) and that whichmight subtend a protomer nicking at Ny (-26 to -14)overlap, which may explain why nicking at Ny has notbeen detected in normal cosN substrates. Formally, anothermodel of terminase action (suggested to us by CarlosCatalano, University of Colorado) would mimic themechanism of the class IIS restriction ENase enzymes(Szybalski et al., 1991), in which a single protomer boundat R3 would catalyze both the N 1 and N2 nicks by virtueof a complex nucleolytic active center. We do not favorthis mechanism because we find that the top and bottomstrand nicking activities can be uncoupled, suggesting theoperation of two protomers in cosN nicking, one for eachof the top and bottom strands (Higgins and Becker, 1994).

Materials and methodsSequence designationsThe numbering convention that we have used previously (Higgins et al.,1988) gives the interval, in bases, extending away from the axis ofsymmetry of cosN to emphasize distances from the nicking domain ofcos. It uses negative numbers to the left of the symmetry axis, beginningwith -1 and proceeding leftward and positive numbers to the right ofthe axis beginning with +1 and proceeding rightward (Figure IB). Inaddition, the standard numbering system for k DNA of Daniels et al.(1983) is sometimes included in parentheses.

cos-containing plasmids and cos fragment resectionsPlasmids were constructed, from which small restriction fragmentscontaining cos DNA, or portions thereof, could be resected withrestriction enzymes for use as DNA substrates (Figure 2) and some ofthese have been described in Higgins et al. (1988) and in Higgins andBecker (1994).The plasmids constructed for this study were: pRH4N3 1, pRHN3 1,

pRHN14 and pRHN8, all derived from pRH121. Plasmid pRH4N31carries R4-cosN DNA and is deleted for cosB,(R3R2RJI)A. It was derivedfrom pRH121 by restriction with HindlIl and XmnI and subcloning intothe HindIII-SmaI sites of plasmid pGEM4Z. Plasmid pRHN31 isdeleted for all of the R sites, (R4R3R2RI)A, but carries the sequence ofcosN between positions -18 to +31. It was derived from pRH 121 byrestriction with Sau3AI and XmnI and subcloning into the BamHI andSmaI sites of plasmid pGEM7Z. Plasmid pRHN14 carries the sequenceof cosN between positions - 18 and + 14. It was derived from pRH 121by restriction with Sau3AI and Mnll and subcloning of the cos-containingfragment into the BamnHI and SmaI sites of plasmid pGEM7Z. PlasmidpRHN8 carries the sequence of cosN between positions -18 and +8and was constructed by restricting pRH 121 with Sau3AI and FnuDIIand subcloning of the cos-containing fragment into plasmid pGEM7Z,as above.

Plasmids pIB13- [with a CG to TA transition mutation at position+52(58) of R31, pSX123 (same as pRH4N31), pSX124, pSX125,pSX126 (cosBA and GC to TA transversion mutations at NI and/or N2)and pCOSNSL (cosB in continuity with CG to TA transversions mutationsat NI and N2) were the gift of Dr Feiss.

Enzymes and reagentsTerminase holoenzyme was purified as outlined in Higgins et al.(1988). Restriction enzymes were purchased from New England Biolabs,Boehringer Mannheim and Pharmacia. T4 DNA ligase, T4 polynucleotidekinase and calf intestinal alkaline phosphatase were from New EnglandBiolabs; AMV reverse transcriptase from Boehringer Mannheim andDNase I from Promega Biotechnologies. dNTPs and ATP were fromSigma Chemical Co. Vz-32PIdATP and [y-32PIATP (3000 Ci/mmol) werefrom Amersham.

Preparation of end-labeled DNA fragments32P end-labeled restriction fragments were 3' end-labeled using AMVreverse transcriptase and Ioa-32P1dATP or were 5' end-labeled using T4polynucleotide kinase and [y-32P]ATP after dephosphorylation of theends with calf intestinal alkaline phosphatase. Fragments labeled at oneend were then prepared by digestion with a second restriction enzyme(Higgins et al., 1988). The labeled fragments were isolated by electro-phoresis on 5-10% polyacrylamide gels, followed by electroelution andwere concentrated by ethanol precipitation.

Other methodsThe assays for nicking at cosN catalyzed by terminase were performedas described previously (Higgins et al., 1988). For electrophoresis onsequencing gels, samples were heated at 90°C for 5 min in a solutionof 80% formamide, 10 mM NaOH, 1 mM EDTA, 0.1% xylene cyanoland 0.1% bromophenol blue (gel-loading buffer). Autoradiography ofdried gels was done at -80°C using XAR-5 X-ray film with a Du PontLightning Plus intensifying screen. When required, band intensities weremeasured by scanning the relevant regions of the autoradiographs, usinga Bio-Rad 620 video densitometer. The results were integrated using aBio-Rad 3302A integrator.

AcknowledgementsWe thank Clarence Fuerst and Paul Sadowski for valuable discussionsand Michael Feiss for the gift of plasmids. The work was supported bya grant from the Medical Research Council of Canada.

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Received on June 27, 1994; revised on September 22, 1994

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