Inceptor and origin of DNA replication in lambdoid coliphages II. The λ DNA maximal replication system

Gene, 6 (1979) 173--197 173 @ Elsevier/North-Holland Biomedical Press, Amsterdam -- Printed in The Netherlands

INCEPTOR AND ORIGIN OF DNA REPLICATION IN LAMBDOID COLIPHAGE8 IL THE X DNA MAXIbfAL REPLICATION SYSTEM (Recombinant DNA; E. coil phage ),; ),dv; pBR313; or/; ice; transcriptional activation; replicational repression; initiator proteins; compartmentalization) MONIKA LUSKY and GERD HOBOM

lnstitut fiir Biologie III, Univemit~ft Freiburg, Sch~'nzlestrasse 1, D-7800 Freiburg i.Br.

(F.R.O.) (Received October 19th, 1978) (Revision received and accepted March 4th, 1979)

8UMMARY

In pBR313-Xdv hybrid plasmids a second system for initiation of DNA replication has been detected in iambdoid replicator DNAs (in the absence of the P0 promoter). This "maximal" (or "magi") initiation system depends on the origin of replication (or/) sequ-ehce, in c0njunction with the "inceptor" (ice) element located in the lambdoid cII genes. Only leftward, but not bidirectional, primer RNA synthesis seems to be initiated at or/in its newly defined boundaries, and it appears to be catalysed by dnaG-coded primase. Only if transcriptionally activated, will or/effectively initiate ), specific, O and P-dependent "maximal" hybrid.plasmid replication. In addition, it will repress a complete ), "minimal" initiation system in e/s, i.e., if present on the same plasmid molecule. This newly discovered repressive activity of the or/ system depends on only three factors: an intact left section of or/, the O product, and transcriptional activation of or/(rightward or leftward). A repressed minimal initiation system will regain its activity as soon as a segment carrying either part of the O gene or a promoter for transcriptional activation is deleted from such a plasmid which was combining both the "mini" and "maxi" systems of ~ replication. INTRODUCTION

In the accompanying first paper of this series (Lusky and Hobom, 1979; hereafter called part I) we have described a hybrid plasmid system for studying ~ b d o i d DNA replication. This system consists of ~(~dv) DNA fragments cloned into pBR313 vector DNA (Bolivar et al., 1977), and studied in the

174 polAts strain £scher/chia coU SC294 (a derivative of TS214, Kingsbury and Helinski, 1973) at both permissive and non-permissive temperatures. Dissection of the lambdoid replicator DNAs by restriction enzyme cleavage reactions, and analysis of cooperative effects observed in a serial cloning of small replicator fragments has led us to the detection of a DNA replication system present in DNA, which is able to initiate DNA synthesis in the absence of the origin of replication (or/). This~ "mmunal" replication system (or mini system) ~ been shown to consist of the leftward p o (or substitute) promoter site ~and a newly discovered "inceptor" (/ce) element, which is located with the vii gene. The sequence and potential secondary structure of/ce resembles ~ r i p t i o n a l terminators, and it appears to control both termination of leftward primer RNA and inception of daughter strand DNA synthesis. The autonomous ~ (or 21) minimal initiation system has been observed to be independent of the transcomplementing O function, but to require P-complementation. Fragments containing the ), origin of replication are unable to initiate DNA replication in hybrid ColE1-), plasmids as long as fragments containing/ce are missing (I, Fig. 1). The observation that ~ e ), minimal initiation system consists of the specific inceptor signal in conjunction with a leftward transcriptional promoter led us however to explore whether or/might not function as an equivalent to a transcriptional promoter, at least if analysed in conjunction with ice. This prediction was indeed fulfilled, and a further analysis of this ), "maximal" initiation system (or maxi system), which is described in this part of our ), replicator analysis, shows that replication primer synthesis is initiated within the ori boundaries and only in leftward direction. This is in agreement with the presence of a putative primase recognition site, also oriented leftward in the center of several lambdoid origin DNA sequences, which has been noted in structural comparisons with the phage G4 "minuHtrand" ori sequence (Fiddes et al., 1978; Sims et aL, 1979; Grosschedl and Hobom, 1979). The same inceptor signal as in the ~ minimal initiation system is also used for inception of DNA synthesis i n the maxi system, and is therefore recognized by both RNA polymerase and primase, MATERIALS AND METHODS

Strain E. coil K-12SC294 (polAts) and others used for transformation have been described in part I, including a number of minimal replication piasmid strains which are explained in I, Table I~ and in figures throughout RESULTS. ~he lambdoid maximal replication plasmids constructed in this part of the work are listed in Table I. The methods used are those described in Part I, including a range of a~plications of the s ~ p ~ e cloning procedure d e ~ r i ~ in RESULTS, stepwise cloning has involved up to five individual restriction fragments, see construction of hybrid plssmid pilL73.

175 TABLE I HYBRID PLASMIDS CONSTRUCTED BY IN VITRO INSERTION OF ~,, 434 AND 21 DNA FRAGMENTS INTO pBR313 Plasmid

Parental plasmid

Fig.

pilL19 pilL26 pilL32 pilL47 b pilL48 b pHIA9 b pilLS0 pilL51 pilL52 pilL53 pilL54 pilL55 pilL58 pilL61 pilL62 pilL63 pilL64 pilL65 pilL66

pilL18 4 pilL22 3 ~dvh93 2 pilL34 2 pilL47 3 pHIA7 3 pilL49 3 (footnote) pilL32 1,2,3 pilL51 " 1,2 pilL32 1 pilL34 3 pilL34 3 pilL22 1,4 pilL22 1 pilL32 1 pilL32 1 pilL71 3 pilL58 4 pilL65 4

pilL67

pilL58

4

pilL68 pilL69 b pilL70 b pilL71 pilL72 pilL73

pilL53 pHIA8 pHIA8 pilL26 pilL26 pilL71

4 4 4 3 3 3

pilL81 pilL82 pilL84 pilL85 pilL89

(434) pilL81 pilL81 pilL81 pilL85

5 5 5 5 5

Nucleotide positions of integrated DNA fragments a (-1050)- 1150

(-1050)-86 ~ 105-457 ~ 457-105 ~ 793-1150 454 - 646 646 - 454 793 - 1558 ~ 646 - 454 1558 - 7931646 - 454 1150 - 793 ~ 646 -454 454 - 646 ~ 793 - 1558 1150- 793 ~ 646- 454 ~ pBR313d :379---2 ~ 1146--1558 454 - 646 ~ 1558 - 793 793- 1558 ~ 646 - 454 1558- 793 I646- 454 (-1050) - 85 ~454- 543 ~ 738- 1150

(-1050) - 85 ~(21): 3 7 8 - 6 4 6 ~ 793- 1150 4 5 4 - 646 ~ (21): 798 - 1188 4 5 4 - 646 ~ (9.1): 1673 - 1736 ~ (21): 988- 1188 1793-2824/0-85 ~ 4 5 4 - 646 ~ 457-105 ~ 793-1150 (-1050)-85 ~ 454-543~ 738-796 ~793-1097~ 1110-1150 c (-1050)-85 ~ 454-543 ~ 738-796 ]~85-0/2324-1793~ 798-1097 ~ 1110-1150 c (-1050)-85 ~ 454-548 ~ 738-796 ~85-012324-1793~ 793-1150 454-646 ~ 85-012324-1798 ~ 1558-793 793- 1558 ~ 85- 0/2824- 1793 ~ 646- 454 793-1558~ 1798-2824/0-85 ~ 646-454 4 5 4 - 646].457- 105~ 793- 1150 (-1050)-85 ~ 105-4571 454-6461457-105 • 798-1150 454-646 ~457-I05 ~ 793-1150 ~(21):1146-1183~ 82-563 (434): (¢I- Q) (434): (-380) - 736 (.Q.) (434): (-380) - 732]I (21): 1151 - (3600) (484): (-380) - 1586 (434): (-380) - 786 ~ 793- 1586

Nucleotide positions for the lambdoid DNA inserts refer to sequence data provided by Schwarz et al., 1978; Scherer, 1978; Grosschedl and Schwarz, 1979; and E. Schwarz, unpublished result& They have been normalized to a reference position 1146 for the first nucleotide position (G) of the common A/lambdoid E¢oRI cleavage site at 81%x. Symbols (21): and (434): refer to DNA of phages 21 and 434, respectively. (See footnote to Table I in Part L) bSubstitution of a 50 bp E¢oRI-BamHI segment of ~80 DNA for the 375 bp E¢oRI-BamHI segment of pBR31& eThe B&III-EcoRI fragment 793-1097 ~(110-1150 contains the xori- deletion of 12 bp (1098-1109) U present in Xr99, and was prepared from pKDT99 (Denniston-Thompson et al., 1977). dNucleotide positions of pBR313 correspond to those of pBR322 (Sutcliffe, 1978; and personal communication). a

:rye .l+

" " Sa

-'-'"'-

,',r L

•

-O" - ~'~

D ..,

i

Y

.....

T

" [

pHL~ pHI.S2 __

! pHL6~ k

pHI.IS3 pilL66__ ~

n

~

pI.L61

restriction analysis

A

6 C .6.22;1.95; pHL53 IHndIll:9.95 Lelllest, is t :e.97;o.u

helper plamld assay (pHL18~ pHL4) nmber of colonies grovlng at 32°I: 42°C ratto

7.9 x 104 0

0

pHL51 Itind111:9.95 L~,al-.5.47;!~.95; E.~RI :9.02;0.93 Psti "I.78;0,75

9.2x10 4 4.3x10 3 5

pHL52 Ht._qdlll:9.95 Hi._ndlll-:6.12;l_..9.~; [aoRI :9.02;0.93 Ps..ttl 1.13;0.75

6.6x104 1.1x102 1.5x10 .3

pHL62 Htndlll:9.58 H(n--dlll-:8.55;1.03 [£~oRI :8.65;0.93

4.2x104 2.6x103 6

pHL63 Hi.__ndill:9.46 Ht---ndllJ-:8.55;0.91 E_..coRI :8.68;0.78

3.2x104 1.5x102 4.3x10 -3

sazJ

Sa l

x10 ~

x10- 2

pHL58 Sail

:9.6

0.6; .0

L.dllJ-.O. ;0.74 "O.30

1.6x104 1.5x102 , 9.3x10 .3

~HL61 Sail

:9.76 6_~Jl- :9.15;0.61

H_.~ndlll:8.6;1.16

2.4x104 2.6x102 1 x10- 2

Fig. 1. Schematic maps of ori and ice minimimtion hybrid plumidL The inserted ~, (~,dv) frafpnents (heavy lines) and surrounding parts Of the circular hybrid plasmids are shown in linearized form. Points of fusion of different DNAs are indicated by a fusion symbol (~). Capital letters refer to ~,dvh93:Sau3A fragments (for their mapping order compare part I, ¥ig. 4) and orientations of inserted fragments are marked by fragment-internel arrows, which refer to their orientation (left to right) in ~DN&. Arrows below every hybrid plasmid map in addition indicate the functional orientations of transeripti0n and replication signal structures, more fully explained for pHI,68 and pilL68 (see ice, ovi, to, PE and Ptet symbOls) indicated byarrows for other hybrid p l ~ d ~ , ~D ~ i b d lines between I)i4L62 and pilL63 correlate corresponding origin segments, The£eoRl.truncated, funetionaliy ineom-

177 RESULTS

(1) The origin o f replication (ori) as an alternate start site for replicational primer synthesis Exploration of the role of the ori site as a possible promoter for synthesis of a replicational primer would be facilitated by the absence of a leftward promoter, such as Po- Otherwise, the complete (Po.ice) mini-replication system would interfere with the analysis of the replication potential in ~;he maxi or/-/ce system. Most 0fthe initial "maximal" (i.e., ori-dependent) replication plasmidstherefore carry only inceptor and origin fragments, 8au3A-D and 8au3A-A (A1+A2) or derivatives thereof. (For a genetic and physical map of ~,dvh93 and its 8au3A fragments see Part I, Fig. 4.) In addition, a rightward promoter has often been included to provide the transcriptional activation of or/. Rightward transcription of the inceptor fragment will by itself neither initiate DNA replication, nor will it interfere with DNA replication being initiated by leftward transcription into that fragment (compare pilL32 and pilL30 in Part I~ Fig. 4). The first of such maxi initiation systems of ~, DNA replication has been constructed by inserting the Sau3A-A fragment in a second BamHI serial cloning step into pilL32 (Part I, Fig. 4) at its insert Sau3A-D right-hand junction. The resulting pilL51 plasmid (see Fig. 1) is able to sustain replication at 42°C ff complemented in trans with the O and P gene products. This experiment shows that the ori fragment substitutes for Po or other leftward promoters in initiation of plasmid ~, DNA replication. The or/fragment, however, does not have the properties of a divergent, bidirectional promoter in DNA replication. Upon inversion of the origin fragment in an otherwise identical set of DNA signal elements (pilL53) no replication is ob~rved under O ÷ P* conditions. The ori.ice constellation as in pilL51, which is equivalent to their positions and orientations in ~ DNA, therefore, appears to act as a (leftward) unidirectional system for initiation of replica-

plete origin of replication is marked by a specific symbol (.-I). A key to the restriction site symbols is specified in Part I, Fig. 1 (b). pilL52 has been obtained from pilL51 by inversion of the hybrid segment between its two E¢oRI cleavage sites. The Xdvh93:Sau3A-D' fragment in pilL§8 refers to the 90 bp 8au3A-TaqI fragment (x:454-543) also used in pilL59 and pilL60 (Part I, Fig. 9), and the D x analogous Ximm21:Sau3A-D 2' (21:378-646) fragment has been used in pilL61. Tabulation of restriction endonuclease cleavage analyses in the lower left part follows the principle outlined in Part I, Table H. Only one representative digestion has each been included for linearisution (column A), junctional (or substitute) (B), and orientation (C) cleavages. Several examples of hybrid plasmid digestions listed in,this figure are shown in Fig. 2. Helper plasmid complementation results are tabulated in the lower right part. po|Ats strains carrying plasmids pilL53 to pilL63 have been transformed by pHLI~8 DNA; pilL58 and pilL61 strains by pilL4 DNA (see Part I, Fig. 1). Three to eight individual hybrid plasmid strains isolated in parallel have been analysed separately and averaged for tabulation.

178

tion, in agreement with a structurally asymmetric organization of both individual signal sequences (see Fig. 6, and Grosschedl and Hobom, 1979). In pilL51 and pilL53 the rightward promoter Ptet has been included to substitute for the X PR promoter in providing transcriptional activation to the X maximal replication system (see below).

(2) Delimitation of the ~(21) origin of replication structure For the initial study of the ~, maxi system, tile large (765 bp) fragment ~dvh93:Sau3A-A (~,:793-1558) was chosen, which contains most of the coding sequence for gene O (codons 44-298, see Scherer, 1978) and the 63 bp core segment of the ori structure (defined by ~,or/- mutants; Denniston-Thompson et al., 1977; Scherer, 1978), which is located in the middle of that fragment (X:1068-1130). Using the initial data (pilL51) as a control, we then reduced the size of the origin of replication fragment in two consecutive steps, and examined the resulting activity. The first of these minimization steps made use of an additional 8~u3A cleavage site present in phage 21 or ~imm21 DNA (both of which are identical in this region, Grosschedl and Hobom, 1979) but not in ~, DNA, at 21:1180, i.e., 34 bp to the right of the EcoRI site at ~/21:1146. Because the DNA sequences for both phages differ in this region by only a few nucleotide exchanges, results obtained with Ximm21 DNA should also apply to ~, DNA. The relevant fragment (Xdvimm21:Sau3A-A') was cloned into the junctional cleavage position of pilL32, and the resulting hybrid plasmid (pilL62) did show full replicational activity in the presence of O, P
179

Fig. 2. Restriction endonuclease analysis of pilL51, pilL52 and pilL69 DNA. Digestions of hybrid plasmid DNAs, and conditions of agarose gel (0.8%) electrophoresis are described in Part I, METHODS. Slots d and k display standard molecular weight markers: mixtures of partially EcoRI digested ~.dv021 octamer DNA, together with H/ndII digested ~.dvl DNA (d) or with HaeHI digested ;~dvl DNA (k). (a)--(c) double digestions BamHI-PstI of pilL51 (a), pilL53 (b), and pilLS2 DNA (control) (c); (e)--(g) EeoRI digestions of pilL51 (e), pilL53 (f), and pilL32 DNA (control) (g); (h), (i) E¢aI (Hobom et al., 1979b) digestions of pHIA7 DNA (control) (h), and pilL69 (DNA (i). segment to the origin of replication function (Grosschedl and Hobom, 1979), which would leave rather small tail sequences, 30 bp and 14 bp, without specific functions (other than coding for the O product) inside the present limits of minimization. Defining the outer limits of a DNA signal structure through cloning of progressively smaller fragments can be cross-checked by experiments, which deliberately cut into the (potentially) active parts of that structure. Out of three structurally defined subsections of the origin of replication sequence (Grosschedl and Hobom, 1979), both A and B had already been shown by DNA sequence analysis of three deletion mutants and one point mutation (Denniston-Thompson et al., 1977; Scherer, 1978) to be indispensable for their full activity. The rightmost origin section C structure (~ :1136-1168) consists of two asymmetrically overlapping inverted repeat DNA sequences, which extend to b e y o n d the ~:1146 E c o R I restriction site and are identical in ), and ~80 DNAs, unlike the DNA sequences on either side. Since this is in contradiction to an earlier report, which concluded the ?,:1146 EcoRI site to be to the

180

right of the )` origin of replication structure (Moore et al., 1977), the ori DNA t~mcated by the E¢oRI cut has been t e ~ inthe hybrid ColE1-)` system. To accomplish this in the most conservative way, without adding or deleting even a single nucleotide as compared to the immediate control plasmid (pilL51), construction of pilL52 has been carried out through EcoRI cleavage of pilL51 DNA, and inversion of the fragment between the two E¢oRI sites (see Fig. 1). As a consec~uence of this remmmgement the whole group of relevant replicator signal elements: Ptet, ice, and ori left section (Sau3A-Al), though inverted with respect to the external parts of the vector DNA, are kept in the same relative positions and orientations to each other. Only the origin right section (Sau3A-A2), though actually staying in its place uncbanged, becomes disconnected from the origin left section. The resultinghybrid plasmid (pilL52) yields only 3% of the activity determined for pilL51 in the helper plasmid colony growth assay. Data from a number of similar, though mostly EcoRI deletion rather than EcoRI inversion experiments also always yield 3% to 5% activity as compared to their origin complete counterparts (see below). From these results we conclude that the or~ sequence extends to the right of the ),:1146 EeoRI site, and that the or/section C sequence ()`:1136-1168) is part of its active structure. As can be concluded from the residual activity of 3--5%, however, this outer part C of the ori sequence may have only an assisting function in the composite or/activity (see Grosschedl and Hobom, 1979). This is indirectly s~Jppo~ed also by some of the experiments to be presented below, which for technical reasons at least initially have been performed on the level of an £¢oRI truncated, i.e. incomplete or/fragment. In all cases in which the origin has been either restored by adding the missing rightmost part or in which the respective experiment has be~.n repeated using a complete origin fragment instead, only quantitative rather than qualitative changes have been observed.

(3) An identical inceptor signal is used in both )` replication systems The requirement in the maximal initiation system for ice in addition to or/, has been concluded from the deletion experiments described in Part I, Fig. 1. Therefore, the (larger) ice fragment )`dvh93:Sau3A-D has been included in our construction of maximal system hybrid plasmids. However, U~e ice signal employed in the ), maximal replication system might not necessarily be identical to that observed in the ), minimal initiation system. For introducing the smaller 90 bp inceptor fragment ()`dvh93:.Sau3A-454/ TaqI-543) into the maximal system plasmids in vitro ligation of ~ fragment to the )`:738-1150 TaqI.EcoRI fragment via their common TaqI ends has been used (compare construction of pilL59 and pilL60 in part I). The resulting Sau3A-£coRI fusion fragment has been inserted into pilL22 (Part I, Fig. 1) DNA, after BgHI-EcoEI cleavage deletion of the intervening segment. This procedure exchanges an EcoEI-trtmcated or/fragment without conjunctional ice (pilL22), into a similarly £coEI-terminated or/ fragment, which now includes a properly oriented ice fragment of the 90 bp Sau3A.TaqI size (pilL58,

181

Fig. 1). The 90 bp inceptor/origin plasmid has full level replication potential (if the incomplete, EcoRI-tnmcated ori is taken into account) and should be compared toits immediate positive (pilL52, Fig. 1) and negative (pilL22; zero; Part I, Table II) controls. The/ee 2~ signal has also been assayed in the maximal replication system through insertion of the Xdvimm21 derived Sau3A-D 2~ fragment (21:378-21/ ),:646) into the BglII site of pilL22. For the resulting incomplete.ori.ice ~ plasmid pilL61, a replication activity similar to pilL52 or pilL58 has been observed (Fig. 1). Analogous regions in both the )`, 434 and 21 DNA, therefore, yield the same/ee activities in either of the two replication systems (see pilL74 in Part I, Fig. 9). For additional data see Fig. 6 and DISCUSSION. (4) The 0 and P requirements in the )` maximal replication system Independence from the O gene function in the minimal replication system indirectly suggests that this protein interacts with ori; a similar conclusion has also been reached when the specifity of interaction of various lambdoid O proteins with their corresponding origins of replication was determined (Furth et al., 1978; Furth and Yates, 1978). Indeed O is required in ), maxi replication. This can be concluded f~om the inability of pilL58 (see above) to be complemented by P-donating minimal system plasmid pilL84 (Part I, Fig. 6), in contrast to a standard level of colony growth if complemented for both O and P by pilL4 (Fig. 1). The second initiating factor, P, is also needed for ~ maxi replication. This has been determined for 434 hybrid plasmids, because P is not required in the 434 mini system (see Part I). A hybrid 434 P-deletion plasmid pilL85 has been constructed from pilL81 DNA by HpaI cleavage (included in Fig. 5). The HpaI site in ), or 434 DNA at 1587, which has been used for fusion onto the vector Hpa I site, is located at a short distance to the right of the O gene terminator codon at 1563 (E. 8chwarz, unpublished). The 434 on~O÷P- plasmid is able to grow at 42°C only if complemented with P. This result points out a second function for the P protein, this time in the 434 (and therefore l~robably also in the )`) maximal initiation. (5) Mode o f replicational primer synthesis in the )` DNA maximal initiation system With the origin of replication postulated to act as an alternate start site for the replicational primer synthesis (pilL51 and controls, Fig. 1), we also wanted to study the process that actually extends the leftward primer synthesis into the/ce-carrying fragment. Similar to the corresponding analysis in the ), minimal initiation system (Part I, Fig. 4), we inserted a tR1 terminator carrying 8au3A-C fragment in between the origin and inceptor fragments in two similar but slightly different experiments. A two-step BglII-Sau3A cloning procedure, first adding 8au3A-C fragment(s), into the BglII site of pilL22 (formation of pilL26), and then inserting a Sau3A-D fragment into the reconstituted BglII site of pilL26 resulted in

189.

pilL72 (Fig. 3). pilL72 contains two Sau3A-C frsgments, due to an initial insertion of two such fragments head to head in pilL26, which then became separated by Sau3A-D in the second step, In addition, a second plasmid has been constructed, starting with pilL26 DNA. After deleting in a BglII.BclI double digestion all the fragments between~the~vector Bell site located below t h e a m p i , region (seePart I, Fig. 7), and the/~/II~ site at the Smz3A-C headto-head junction, a Sm~3A-D fragment hasbeen ligated t 0 ~ e isolated BcffBgHl fragment (pilL71, Fig. 3). Thereafter, a Sau3A-B fragment has been ins e ~ into the resulting BclI junctional cleavage site (pilL64, Fig. 3). In both plasmids, p H i l 4 and pilL72, transcriptional activation of the £¢oRl-terminated o ~ ' i s achieved through rightward p a transcriptions (in pilL72 only because of a leak-through of that transcription across tax , which is equivalent to the plasmid ~,dv situation in general). The pa-promoted /ranscriptions will not initiate replication at the inversely oriented replicational incepton and will not be terminated at the inversely oriented tax terminators, as observed in pilL32 and pHIA6 as controls (Part I, Fig. 4). In these maxi systems, initiation of replication is dependent upon the off-initiatedp~mer synthesis. Primer RNA synthesis should be ~terminated at the leftward oriented tax signsk inserted between origin and incept0r, if it were ca~lysed by RNA polymerase. In contrast to the related experiment in the A minimal system (pilL42 in Part I, Fig. 4), primer synthesis here is not blocked by the inserted transcziption terminator frsgment since plasmid replication is observed in the presence of a helper, as seen in Fig. 3 (pilL§2 and pHIA9 are other relevant controls besides pHIA2, pilL32, and pHIA6). These e~pedments indicate that RNA polymerase is not catalysing the maximal system primer synthesis, which therefore must be performed by another enzyme, probably dr~G coded primase. This conclusion is in sgreement with earlier observations that d ~ G is involved in RNA synthesis related to initiation of ). replication (Hayes and Szybalski, 1973, 1975; S. Hayes, pen. commun.). It is also supported by a structural comparison between the ~, ¢80 Fig. 3. Analysis of the transcriptional ori activation in hybrid plasmids. Fragments of ~, (~,dv) DNA are inserted either to the left (pilL26, pilL72, pilL64, pHLT1) or to the right (pHI,67, pHIAS, pH!A' 9, pilL56, pilL54) of the pBl~13 vector DNA EcoltI site, pilL73 is extending aczoss this site. Functional orientations oflrelevant transcription a~d replication signal structures are.~'~dicated by arrows and symbols for p ~ 5 1 and p I~72;:onl¥ the respective arrows are ~t]rawn for other p|Mmids. ~ e n t (B) in bracket8 ~i~e~s~toa fragment which is equival~nt to ~,dvh9$:Sau3A-B, but bes been derived from ~dvl (or pilL22, which has been used for constructing IpHL26, see Part I, Fig. I), For other... notations and symbols see Fig. I, and Part I, Figs. I and 4. For tabulation Of restriction cleavage data (lower left) and helper plmanid complementstion results (lower r~ht) corn-. _pare Fig. ! ; and Part-I, Table II. In each case three to seven individual h y b r i ~ ~ isolated

~80 DNA segn~.ents being removed) which cann0t becomplemented for g r o ~ ;

,~

183 oBR313.---.-~

:

pOR313 T

pkL51

l

'•,lk ....

....

pHL,W" ,,

p l t L 4 ~ t':l'

" ....

~ (ooo)

/

--Jh---

(i;80) ~ ('~Ptet)

!'" ^

,,----A?--

- ° - - '

:,

--As-!

...---A,--

--;

--"'-

..L_

l.-_!~--:_L

IJ -

T -.

A

t*

p H L ~ ~ pHLS4

T

-.

.

,.,.m,,m,._

73~

....

II

--~Ul-iktt

---II

.....

restriction analysis A pilL51 Htndlll:9.95

8

helper plaseid assay (pHL18, pHL4) number of colonies groutng at 32°C 42°C ratio

C

OLHHI- .5.47;1.95; EcoRl:9.02;0.93 -

-

8.2 x 104 4.3 x 103 5

x 10-2

pilL26 B~ll

:10.0 Hlndlll:8.4;1.6

Aval :4.4;3.8; w 1.5;o.51

2.5x 104 0

pHI.72 Sa]l

:10.2 Htndlll:8.4;1.6

Aval :4.4;3.6; -~;o.7

1.3 x 104 1.4 x 102 1

Hpal :4.42;2.18

1.6 x 104 1.5 x 102 9.3 x 10.3

pHL64 Hindlll:6.6

Hindlll"5 3"1 3

pHI.71 Htndlll:6,O w

~Hindlll"5..35;0.65 Bglll: Ps....ttl

--

2,9 x 104 0

0 0

pilL48

Sa_.ll :9°45 Hi.._.~III:

~

(coR1:9.1;0.35

5.1 x 104 0

pilL49

SaJI

--

Ec._.oRl:9.05;0.4

4.5 x 104 6

:9,45 Hi._gdlll:

0 x 10.2

~ 101 1.3 x 10-3

pHL55 Iflndll 1:9,95 ~Hindl l l 11°:8"45;1"55 EcoRl:9.17;0.T8

8.2 x 104 2.5 x 102 4

x 10.-3

pilL54

Hindlll"O• •45"1 HJLndlll:9.95 ~']1 , . 55 EcoRl:9.22;0.73

5.8 x 104 1.2 x 101 2

x 10-4

pilL73

Hlndlll:5.6

3.2 x 104 1.3 x 102 4

x 10..3

Ht--p.ndlll'.8 0;0 6 F,.f~dil " " '

Hindll-3.0;2-O; m "0.91;0.65

184

and phage G4 minus strand origins of replication sequences which has led to detection of a putative, right-to, left p~mase start site in the central section of lambdoid origins (Fiddes et al., 1978; Sims et al., 1979; Grosschedl and Hobom, 1979). In spite of their apparent differences in primer RNA synthesis, maxi and mini iuitiation of DNA synthesis are both controlled by the same inceptor signal, which thus should be recognized by both RNA polymerase and primase.

(6) Rightward transcriptional activation in the hybrid plasmid system A characteristic feature of the )` replication initiation is its dependence upon a "transcriptional activation" mechanism, which usually is under control of the PR promoter but can be provided by other rightward promoters as well (Dove et al., 1969, 1971; Nijksmp et al., 1971). Since in the ), mini system the inceptor is independent of a rightward activation promoter, the most obvious candidate in the )` maxi system for a signal activated by transcription is the or/site. We wanted to confirm the dependence upon transcriptional activation for the hybrid plasmid )` maximal initiation system, and therefore used ori-/ce hybrid plasmids that did not contain a rightward (or leftward) promoter. Except for plasmid pilL71 already described above, these were constructed starting from pilL47 DNA, which has the pBR313 EeoRI-BamHI (Ptet) fragment exchanged for a small, promoter-free ~80 DNA fragment, and which does contain the )`dvh93:Sau3A-D inceptor fragment in a ),-inverse orierttation (Part I, Fig. 4). The )`dvh93:Sau3A-A origin fragment was inserted into its junctional BamHI cleavage site in both possible orientations (pilL48, pilL49; Fig. 3). In the O,P-donating helper plasmid assay, only plasmid pilL49 with the correct ori.iee orientation was able to sustain replication, but did so at highly reduced levels (2--3% of the transcriptionally activated pilLS1 control plasmid), when the transcriptional or/activation was absent. Plasmid pilL48 with an inversely oriented ori fragment did not initiate )`-replication. This proves unidirectionality of the ori activity, even in the absence of transcriptional activation. The replication potential of E¢oRI-truncated ori plasmids was not detectable in the absence of transcriptional activation, even for the correct ori-ice orientation. This was observed for inserts in two different vector locations, for pilL71 (Fig. 3) and for pilL50 (obtained from pilL49 DNA by EcoRI deletion, not shown). (7) A leftward transcriptional activation experiment Recently we have proposed a model for the mechanism of transcriptional activation exerted upon the ),(lambdoid) ori structure (Hobom et al., 1979a). This model employs RNA polymerase as a strand separation agent, and predicts DNA single-strand hairpin loops which may be transiently formed in the non-coding strand to represent "activated" initiator protein binding sites. Durability of such "secondary structures" should be influenced by adjacent DNA sequences; the extremely asymmetric (oligo-A:oligo-T)strand composition in the central ori section is expected to differently affect the stability

185

of the/-strand or the r-strand structures depending on which strand is used as the non~oding strand in transcriptionalactivation.W e therefore wanted to try whether leftward transcriptionalactivation of ori is possible, and it does result in a significantlydifferentdegree of activation to be observed in the plasmid colony growth assay. The construction of the respective plasmid (pilL73, Fig. 3) had to take into consideration that leftward transcription across ori might also lead to initiation of replication in the 7~ mini system. Therefore, results obtained in any simple dual leftward system such as pilL55 (which has been constructed in a inverted orientation, see Fig. 3) are not readily interpretable,but useful as a control. The parallelpilL54 experiment tellsus that transcription across the origin (inverted with respect to the inceptor) indeed leads to initiationas expected for the ),mini system. The reduced yield observed in colony growth complementation (5% of the pilL55 or other fulllevelresults,see Fig. 3) may be attributed either to the increased distance between Ptet and ice,or to ori acting as a transcriptionalbarrierin its part-time activated state caused by the "right-to-left"activation (pilL53, pilL48, pilL69, and pilL70 are other relevant cont~rolsto this experiment). The replicationpotential of pilL54 in the presence of only the P protein has not yet been analysed. Both plasmids, pilL54 and pilL55, have been constructed through junctional insertion of a Sau3A-A fragment into pilL34 D N A (Part I, Fig. 4). In order to avoid m1~~ confusion due to transcription across ori in a leftward "wrong-way" activation experiment, a tR1-carryingfragment inserted (in leftward orientation) between the origin and inceptor signalshad to be used, similar to pilL72. According to the data obtained for pilL72 (Fig. 3) and for pilL42 (Part I, Fig. 4), the tR1 terminator signalshould discriminate between the origin started,dr~aG catalysed primer synthesis, and the PR promoted R N A polymerase transcription.In pilL73 only the progressing primase reaction should be able to bypass the terminator signal,and to extend up to the inceptor sitein a double leftward situation (Fig. 3). For construction of the pilL73 plasmid, pilL71 D N A (Fig. 3) was cleaved at itsEcoRI and HindIII sites,and the small 31 bp pBR313 fragment was replaced by an EcoRI-HindIII fusion fragment produced by in vitro ligation. It consisted of the )~dvimm21 right terminal origin fragment (21.EcoRI:1147/ 21-Sau3A:1183) and a ),dvl-PR-Prm~arrying fragment ()~.HindIII:-563/)~BgIII:+85) ligated via their c o m m o n G A T C single-strandedends. Due to orientational cloning, tlijs£coRI-HindIII-fusion fragment was inserted in such a way as to complete E~c origin of replication sequence, and have the ;xpR promoter located next to it,in right to leftorientation (see Fig. 3). The pilL73 plasmid turned out to be tetracycline resistant,which has to be attributed to the action of the )"Prm promoter, oriented towards the tetracycline region, and unrepressed because of absence of the cro protein (the pilL73 strain

is sensitive for ~vir or ),* infection). In helper plasmid complementation pilL73 shows moderate capability of plasmid replication at 42°C, which in the lack of structurally very close plasmid

186

controls is difficult to be rated quantitatively (it appears t o be 10% of the rightward transcriptionally activated pilLS1 figure, and about three "times higher then the pHLA9 result, without transcriptional activation, see Fig. 3), The results at least qualitatively are in agreement with what has been predicted from our model and may indicate that the leftward transcriptional activation of ori is 5--10 times less effective than the rightward one. (See below for or/ leftward transcription activation in repression of the minimal replication system.)

(8) Repression of the X minimal replication system With the potential for an alternative mode of replication initiation built into the replicator system of bacteriophage Z, c/s-dominant ~or/'-mutants should n o t exist, and also ), DNA replication should n o t necessarily depend upon the 0 protein, which is dispensable in plasmid mini replication. Since both of these conclusions are in apparent contradiction to many expe~ments dealing with phage ), DNA repfication, we suspected that the maxi system might repress a complete and otherwise functional minima~ initiation system in ¢/8, i.e. if present on the same molecule. In order to test this assumption we constructed several hybrid plasmids, which in contrast to the ~ maxi replication plasmids described so far, had a P o or substitute leftward promoter fragment inserted to the right of their ice signals. Repression exerted by the origin of replication upon such a complete po.iCe replication system is measured in the helper plasmid colony growth assay. A first series of these plasmids has been constructed with the origin fragment inserted in its inverse, inactive orientation (see pilL§3 in Fig. 1). In this way, only the origin repression effect becomes measurable, and initiations due to the maxi system will n o t confuse the results. The construction has been r ,

Fig. 4. Schematic maps of hybrid plmmids used for determining tran~riptionsl activation and P-dependence in ori-exerted replication repression. The f'ust group contains its ~, DNA fragments inserted into the BamHI cleavage site of pBR313, while the eeeond group of hybrid plasmids has been derived from pilL22 (Part I, Fig. 1); pilL19 is parental to pilL22. The xdvh93:Sau3A-D' fragment in pilL65 to pilL67 refers to the 90 bp minimized inceptor fragment (~,:454-543) also used in pilL60 (Part i, Fig. 9). In pilL65 and pilL66 the wildtype BgHI-EcoRI fragment As (compare Part I, Fig. 4a), which was present in the parental pilL22 plasmid, has been removed and replaced by the corresponding A~* fragment which carries the or/- mutation of xr99 and was isolated from pKDT99 DNA (Deqniston-Thompson et al., 1977). For other notations and symbols see Fig. 1, and Part I, Figs. I and 4. Tabulation of hybrid plasmid restriction analysis (bottom left) followed the scheme explained in Fig. I, and Part I, Table II. Helper plasmid eomplementation results (bottom right) have been included for both O,P-donsting transformation (pHLI8 DNA was used for the pilL68, pilL70, and pilL69 transfomations; pilL4 DNA was u~d " for the pilL65 through pilL19 transformations) and for P-donating transformations with pilL84 DNA (see Part I, Fig. 6). (I) Plasmids p H I l 9 and pilL70 are derivatives of pHIA7 (Part I, Fig. 4), constructed by Xdvh93:Sau3A-B insertion into pHIA8 DNA (Fig. 3)and carry the ~80 DNA derived substitution fragment, which replaeasptet. (2) Only the size of the ~, origin containing Avall-EcoRl ~ e n t is indicated, as o ~ e d in parallel to a xdvh93 control, a difference in size was clearly detectable for pilL65 versus pilL58 DNA. ~.r99 contains a deletion of 12 bp (~,:1098-1109), see Denniston-Thompson et al., 1977).

187

achieved through insertion of a ~dvh93:Sau3A-B fragment (Part I, Fig. 4a) into the junctional BamHI cleavage site between fragments 8au3A-D and Bau3A-A in plasmid pilL53 DNA. The resulting 91asmid (pilL68, Fig. 4) has its PR promoter oriented leftward, towards the inceptor signal, and its Ptet promoter oriented,rightward, in a position to support (a leftward) transcriptional activation of the inverted origin. Plasmid pilL68 is not able to replicate under helper assay conditions in spite of its containing a complete ), mini initiation system; it should be compared to nonrepressed minimal replication plasmids such as pilL34 or pilL82 in Part I (Figs. 4 and 6). ,.=-llmlmo

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188

The dependence of the pilL68 repression upon transcriptional activation has been studied in two closely related plasmids. Both of these (pilL69 and pHLT0, Fig. 4) have been constructed by inserting the ~,dvh93:Sau3A-B fragment into the junctional BamHI cleavage site of pilL48 (Fig. 3), which is equivalent to pilL53, but d e l e t ~ for transcriptional activation (order of elements inverted). Oneof the two ~sulting plasmids (pilL70) contains ~'PR in inceptor-conjugated orientation, similar to pilL68. With or/not activated by transcription (~"Prm should be blocked by pilL18 cro complementation), the maxi system in pilL70 isnot able to repress the mini system, and pilL70 fully replicates under helper assay conditions (see Fig. 4). The related control plasmid (pilL69) has the Sau3A-B fragment inserted in the inverse orientation. Therefore, ~Plt would not promote/¢e-dependent initiation, but only the transcriptional or/activation, and the pilL69 plasmid is unable to replicate. This proves that the pilL70 replication is in fact due to the Pa-promoted mini system, which remains active in the presence of or/ in ¢is configuration as long as the transcriptional activation of ori is absent, even if complemented by the 0 and P gene products. In a second set of repression experiments we tried to explore the influence exerted upon the )~ mini system by the origin in standard orientation. To accomplish these experiments without any maxi.system interference the hr99 or/- allele had to be used, and for reasons that will become apparent below we chose an £¢oRI.tnmcated or/. The ~r99 or~ mutant in form of a Bglll-£¢oRI fragment isolated from plasmid pKDT99 (Denniston.Thompson et al., 1977), was inserted into plasmid pilL58 DNA after deletion of its equivalent on" fragment. The ;tr99 hybrid plasmid (pilL65) is not able to replicate under helper assay conditions, while the control plasmid pilL58 can do so (Fig. 4). Thus, the r99 allele has the same phenotype in hybrid plasmid replication as in phage replication. For the repression experiments, a ~dvh:Sau3A-B fragment has been inserted into their single BgllI cleavage sites, and the plasmid strains with inceptorconjugated Pa orientations have been picked among the transformants. While the transcriptionally activated ),r99 plasmid (pilL66) does not replicate under O*+P* conditions, the corresponding control plasmid (pilL67) replicates better than pilL58 (Fig. 4). This set of four plasmids has also been used to determine whether P is also involved in the repression effect exerted by the activated origin. In additional helper experiments, instead of the standard pilL4 (O+,P*) DNA (or pilL81 DNA, not shown) also pilL84 DNA has been used for complementation, which contains the P but not the O gene (see Part I, Fig. 6). Under these conditions both plasmids, pilL66 and pilL67 will grow, irrespective of the ori alleles present. Their growth ratios are above the ratios observed for ~, maxi systems with incomplete origins (see Fig. 1) andin the range character- " istic for the ~, mini (or complete ~ maxi) replication systems. The necessary controls are provided by their c o u n t e r p ~ pilL65 and pilL58, which under these conditions do not grow at 42°C (Fig. 4). Their

189

inability to grow has to be attributed to their missing the leftward PR promoter fragment which is required for minimal initiation (pilL58 also cannot initiate according to the maximal system because O is missing). Results similar to pilL67 have also been obtained for pilL19, which instead of a leftward pa promoter carries a leftward Po promoter in a standard kdv (or A) Configuration, including also to (Fig. 4). From this set of data we conclude that a transcriptionally activated origin is not able to exert repression if complemented only with P. Another series o f repression experiments asked whether O alone would be

enough to yield that reaction. We used a group of plasmids carrying 434 DNA inserts, because the P gene can be deleted from 434 plasmids without losing the potential for minimal replication (Part I, Fig. 6). Deletion of the P gene

(pilL85, Fig. 5) results in inability to replicate at 42°C. Thus, the O function, together with a transcriptionally activated origin structure is enough to yield repression of the minimal initiation system. It is not enough, however, to

initiate replication in the maximal system, for which also P is required. The next deleticn step in this series of 434 hybrid piasmids was achieved through A/434:BgIII-Gdeletion of 60 bp from the NH2-terminus of the O gene ~R313 '

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190

in pilL85 DNA, and resulted in an O- on" derivative, pilL89 (Fig. 5; compare pilL5 in Part I, Fig. 1). Plasmid pilL89 has regained the ability to replicate at 42°C, indicating that repression is lifted in t h e absence of both O and P, in spite of the presence of a complete, transcriptionally activated origin. Add'~ional deletion of t h e o ~ and remaining parts of the O gene (pilL82, con~pm also pilL83 in Part i, Fig. 6 ) d i d not change the phenotype, which therefore must be attributed to the minimal replication system, also in pilL89. It has not been tested whether central or C-terminal mutations in the O gene would have a similar or different effect, due t o the different functions of the NH2-terminal and C-terminal domains of that protein. Repression of complete mini systems by O complemented, transcriptionally activated origins, is exerted only in ¢is, i.e. if present on the same plasmid molecules. As has been exemplified in part I in essentially all of the positive complementation expe~ments involving any of the minimal replication systems plus pilL18 as a helper, plasmids replicating according to both mechanisms are able to coexist in the same cell under coselection pressure. (pilL18 because of its inherent cLs-repression effect should initiate its own replication according to the maximal system). In a number of experiments, maxi and mini replication plasmids have also been shown to be present in about equal copy numbers (see Part I, METHODS). From the inability of the origin exerted repression effect to operate also in trans, we would like to conclude that both an activated and Oocomplexed origin, and an "activated" i.e. primer-containing ice structure appear to be bound to two different replication sites during initiation. The affinity for or/ binding as compared to ice binding appears to be considerably higher, even if no effective initiation of replication can be achieved due to or/" mutations, inversion of the origin, or absence of P. DISCUSSION

The analysis of ), DNA replication in a ColE1 hybrid plasmid system had resulted in the detection of an ori-independent minimal initiation system, which consists of a leftward promoter (Po) and a new element located in the eli gene, the replicational "inceptor" (ice; see Part I). The ice site is also required for replication of hybrid plasmids containing or/, since they are unable to replicate in poIAts cells at 42°C as long as fragments containing ice are missing. The experiments described in this paper provide evidence that the ~ or/site can substitute for a leftward Po promoter in the activation of the inceptor. The maxi system of ~, replication therefore, basically consists of ori (in gene O) and ice (in gene cII). For potentiation of its activity ori needs to be attached to a (rightward) transcriptional promoter, and ori in its structure is far superior in priming initiation of replication in [th ice. Our data are consistent with the ideathat a primer RNA is initiated within the (newly defined) boundaries of the ~ or/sequence in leftward direction,

191

which then is extended until it reaches the ice signal for primer termination and inception of DNA synthesis. Insensitivity of the ), maxi initiation system to insertion of terminators (e.g. tR1) between or/and ice strongly suggests that probably dnaG-coded primase is catalysing the maxi system replicational primer synthesis. The well known overall rifampicin sensitivity of )k (or ;kdv) initiation (Klein and Powling, 1972; Hayes and Szybalski, 1973; Hobom and Hobom, 1973) then would have to be attributed to the preceding transcriptional activation reaction. This interpretation of the ~ maximal initiation mechanism is supported by the observation that the DNA sequence in the center (section B) of the )~ and ~80 origins very much resembles the DNA sequence located around the 5' end of t h e G 4 minus strand primer RNA (Fiddes et al., 1978; Sims et al., 1979; Grosschedl and Hobom, 1979), while the ), and 434 inceptor DNA sequences similarly resemble the DNA sequence corresponding to the 3' end of that primer RNA (see Fig. 6, below). G4 primer RNA (Bouch~ et al., 1978) is known to be synthesized by primase on single stranded G4 DNA (Rowen and Kornberg, 1978), and the template's signal structures controlling its initiation and its termination must be recognized by this enzyme. What is clearly different in the lambdoid DNA templates is the distance between both vital replicator elements, which is nearly 600 bp (varied between 320 and 800 bp in different ice-dependent hybrid plasmids). Most importantly, however, DNA replication in ), DNA appears to be initiated on a double-stranded template, while in G4 it is single-stranded. The G4 DNA priming reaction therefore may be more readily compared with initiation of an Okazaki fragment on a single-stranded lagging strand template during replicational elongation than with initiation of replication itself on a double-stranded template. Here, more elaborate origin structures may be necessary to allow in a preceding step its conversion into an "activated" structure exposing the single strand prirnase recognition sequence. Based upon structural considerations we have proposed a model mechanism for such an origin activation reaction, which includes initiator protein binding, in particular O protein binding to the origin (Grosschedl and Hobom, 1979). Since the ), minimal replication system is repressed (in c/s) by the ), maximal replication system, ori appears to be the only site of primer initiation in a complete (or/-po-iC,e) replicator structure which resembles the )~ wild type situation. According to this interpretation both of the very first steps leading to replication are being initiated at or/: DNA strand separation, and primasecatalysed start of a replicational primer. Therefore, this site, legitimately in our opinion, deserves the name of the "origin of replication", even if DNA synthesis is started elsewhere. Because it is repressed, the )k mini (Po or oop dependent) :replication system may be regarded as a back-up mechanism rather than providing wild type )k with an alternative initiation pathway. It remains to be seen whether ), replicator mutants exist or can be constructed that will use the ), mini instead of the maxi system for phage zeplication. In Part I we showed that mini

192

initiation depends solely on the ~ (lambdoid) replicator DNA, and is independent of the vector-plasmid system (see pilL88; Part I, Fig. 7). The repression exerted by a (rightward or leftward) transcriptionally activated, O~omplemented origin upon the mini system explains why this back-up pathway has not previously been observed in ~. The series of consecutive deletion steps as summsrJzed in Fig. 5 exemplifies a controlled, stepwise reduction from ), maxi to mini replication. Plasmid pilL81 is equivalent to a complete ),dv or ~dvgal (Berg et al., 1974) and its replication is not expected to be different from the (early) mode of ~ replication, while pilL82 represents a mini replication plasmid (Fig. 5) and may be further reduced to pilL88 without significant changes (Part I; Fig. 7). A similar argument is provided by the ori99 plasmid pilL66 (Fig. 4) which is unable to grow in the presence of O and P, but will regain this capability ff only P is present. With RNA polymerase catalysing primer RNA synthesis in the mini system and (probably) primase synthesizing the replicational primer in the maxi system it is surprising that both should recognize the same inceptor signal. The/ce function, however, has been minimized to within the same 90 bp fragment in either system (pilL60 and pilL58, respectively), and comparison with the analogous phage 21 sequence as well as a dissection of the remaining 90 bp sequence (see DISCUSSIONin part I) supports our interpretation of a 21 bp ice structure. Also ~e" mutants have been observed to be defective in either system (unpublished results). The sequence of ice resembles both the structure of a majority of transcriptional terminators suggesting its recognition by RNA polymerase (see Part I, Fig. 10) the DNA-template complementary to the 3' end of the G4 primer RNA sequence (Bouchb et al., 1978; Godson et al., 1978). Instead of a G.rich taft sequence [six out of six (k) and a five out of six (434)] in the lambdoid sites, the G4 primer RNA 3' end contains four out of six G's in the taft, and displays the same asymmetric location relative to the overlapping inverted repeat sequence (see Fig. 6). The close similarity between the G4 DNA signal and the lambdoid DNA structures (in particular for the P-independent 434 sites) suggests that they can be recognized by primase, as a singl,~-stranded template. It remains to be seen whether the ~ primer RNA-DNA transition point will also be located two nucleotides beyond the hexa-G taft sequence as suggested by analogy to the G4 structure. The observation of a back-up start site for the replicational primer synthesis poses the question whether there might not also be back-up termination sites for replicational primer synthesis, that is back-up inceptor signals. All of the deletion studies shown in Fig. I of Part I (in particular the ori÷ice- plasmids pilL6 and pilL22, 11.4 kb and 9.5 kb in size), argue that a substitute inceptor signal is not available either onthe ~ DNA within its kdv boundaries, or on the pBR313 vector DNA. All other negative helper assay experiments, e.g., employing an inverted inceptor fragment, argue the same way. Our results indicate the indispensability Of the only k inceptor signal avail-

193

template, strand:

A GAA C G C A CA G G=C C=G A-T iC A-T G=C C=G A-T G.TIGI C=G !IT G=C i =~ A-T ~ C-~'-CTCGGTA ~T1GGGGITA-5'~'-AGCTCGG rAAG-s, ~,-CGTCATC ACATAG-5,

).-incept0r

434-inceptor

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Fig. 6. Structural comparison between the template strand DNA sequences predicted to constitute the ~, and 434 inceptor signals, and the G4 DNA sequence known to be used as template in the synthesis of the minus strand primer RNA 3' end. Direction of primase catalysed primer synthesis is left to right, inverted relative to the ~. map. G-rich hexanucleotide groups in close asymmetric relation to the center of the inverted repeat (hairpin) sequences are indicated by boxes. The arrowhead below the G4 DNA sequence indicates the position of the 3' end for the majority of in vitro synthesized primer RNA molecules (Bouch6 et al., 1978), two nuc!eotides beyond the hexa-G "tailsignal". For a structural interpretation of the lambdoid ice signal in the RNA polymerase-dependent minimal initiation system see Part I, Fig. 10. ~,, 434 and G4 DNA sequences have been taken from Schwarz et al. (1978); Grosschedl and Schwarz (1979); and Godson et al. (1978).

able within the ),dv bou~daries. However, topological constraints may be involved for a DNA sequence to become active as an inceptor signal, i.e. a potential inceptor sequence may have to be located at a specific distance relative to the primer start site and to be correctly oriented. Since not every segment of the ),de DNA has as yet been fu~ed to ori in both possible orientations (through deletion or otherwise), we cannot rigorously exclude the presence of some dormant substitute inceptor signals, though there is no positive evidence. Despite all the negative results obtained in the ), DNA-ColE1 hybrid plasmid system, substitute inceptor signals have to be postulated to be present in several ~spi virulent phages (Oppenheim and Kappeler, 1976; Smith et al., 1976), in which deletions extend beyond the ),cIIam41 marker and, therefore, their ice site is replaced by a segment of E. coil DNA. All of the ~spi vir ,~,~"" -~ isolated so far grow rather poorly, and are not repressed in cI-immune cells. Since the bacterial DNA segment of the ~spi vir phages cannot be e x p e c t ~ to include the K coli chromosomal replicator, there appear to be some other ice-like signals present elsewhere in E. coli DNA. We suspect that these E. coil DNA ice-like signals may have been recruited from among some of the Okazaki restart signals. These signals which may or may not resemble a G4 minus-strand origin sequence must also have the built-in property of a primer start and a primer termination/transition signal, even if dealing only with much shorter RNA molecules synthesized on single-strand templates by primase (Ogawa et al., 1977). Some of these signals if transferred into close conjunction with or;, (i.e. into a ),-cH position) may be capable of substituting for a deleted ~ ice*

194 inceptor site. It remains to be seen, whether this is indeed the case. Our results on the indispensability of an inceptor signal located in the ~¢II gene, and in particular the inability of pilL22 containing cells to grow at 42°C in the helper plasmid complementation assay (Part I, Table II) is in apparent contradiction with a similar experiment assaying an almost identical ~ DNA insert in a different vector system, which allows plaque formation if replica/ion is initiated in the insert. Under these conditions, however, ~ DNA~lerived inserts internally deleted for the BgffI-E and G fragments (~:82-795) yield a ¢onsiderabi2 reduced burst size, though clearly above background levels (Moore et al., 1979). This result suggests the presence of a substitute inceptor signal not observed in the similar pilL22 experiment. The _segment of DNA transferred into a ~eII equivalent location in the phage vector expe~ment does however not only consist of ~ DNA, but also includes part of a 1400 bp insertion of foreign DNA (KH100) into the X¢I gene which has been called IS5 (Blattner et al., 1974; Szybalski, 1977), while the corresponding segment in the pilL22 experiment only consists of X DNA extending to beyond the left end of the ¢I gene. Based upon the observed inability of the XeI DNA (and also the pBR313 vector DNA) to provide the pilL22 system with a substitute incel~tor signal, we would suspect the foreign KH100 DNA segment to perhaps cont..in such an element, resembling therefore, the £. ¢oli DNA inserts in the ~spi oil" phages. From dissecting the ~, replicator signal elements in this hybrid plasmid analysis, several conclusions can also be drawn for the functional activities of the ~, initiator proteins O and P. In addition to the two activities discussed for P in Part I, ;t maxi initiation is also dependent upon P. Since also the 434 hybrid plasmids require P for initiation in the maxi system (pilL85 in Fig. 5) this activity clearly is different from the mini system function. It is not known whether P would act in inceptor or origin recognition in this system. O/P interaction (Tomizawa, 1971) would argue for an or/function, because O has been proposed to be bound to the ori section A cloverleaf conformation (Grosschedl and Hobom, 1979). P is dispensable in the repression effect exerted by an O~omplemented, transcriptionally activated origin upon the minimal initiation system (pilL85), and only the left part (section A) of the origin may be needed in such a reaction. This conclusion is based on the pilL66 experiment (Fig. 4) which results in complete repression though half of both sections B and C of the or/sequence has each been deleted (B: 12/3"/bp; C: 17/32 bp; see Table I). Either of these deletions alone has been shown to result in a substantial reduction (EcoRI deletion: pilL52, Fig. 1) of the potential for initiation of plasmid replication (or may result in its loss; ori99 in a complete origin, not yet done). Thus, the pilL66 experiment also provided an independent argument for interaction of O with the left section of or/and it explains the ori- phenotype. We cannot yet explain hhe O" phenotype of the standard O amber mutants in relation to the pilL89 experiment (Fig. 5). No amber mutants have, however, been •

r

J

195

isolated for the DNA binding NH2-terminal domain of the O protein (amino acids 1-106; Scherer, 1978) and the phenotype of mutations located in this region, such as the fragment G deletion, may be different. Apparently there is no difference in repression by ori if activated by right~ ward or leftward transcription (inverted ori experiments, see Fig. 4), which contrasts with an approximately tenfold difference in initiation of replication activity for ori .in rightward as compared to leftward activation (Fig. 3). The left section of ori which probably binds O has a (highly) symmetrical structure, while the total or/structure as required for initiation is altogether asymmetrically organized (see Grosschedl and Hobom, 1979). From several series of repression experiments we have concluded that replication site compartmentalization should discriminate between plasmid molecules initiated according to the maxi and mini replication systems (see RESULTS). The active component guiding a preinitiated molecule into a cellular compartment reserved for the ~ maxi replication mechanism appears to be the gene O protein upon interacting with the left section of the transcriptionally activated ori (see above). One may imagine that the replication site on the membrane binds to the C-terminus of the O protein (or to another protein of a larger complex attached to it) while the NH2-terminus is bound to the origin cloverleaf conformation (Hobom et al., 1979a). This kind of an interaction may be a necessary prerequisite for ~, maxi initiation, and appears to be kept unchanged throughout all of an individual replication cycle (Takahashi, 1975; Klinkert and Klein, 1978) though it is not part of the elongation mechanism as such. This latter conclusion is drawn from the ~, or 21 mini initiation system which is only P-dependent, and from the 434 mini system which requires neither O nor P. There is no reason to suspect that elongation which is catalysed by a group of cooperating host proteins should be different for either of the three initiation systems. We therefore conclude that the P protein has several roles in initiation, while the O protein is involved in initiation and in compartmentalization of the replicating molecule. ACKNOWLEDGEMENTS

We would like to thank G. Schlingmann for her expert technical assistance, R. Grosschedl, H. KSssel, G. Scherer, and E. Schwarz for valuable discussions, and K. Denniston-Thompson for her sending us the pKDT99 plasmid strain. This work has been supported by the Deutsche Forschungsgemeinschaft. REFERENCES Berg, D.E., Jackson, D.A. and Mertz, J.E., Isolation of a ~,dv plasmid carrying the bacterial gal operon, J. Virol., 14 (1974) 1063--1069. Blattner, F.R., Fiandt, M., Hass, K.K., Twose, P.A. and Szybalski, W., Deletions and insertions in the immunity region of eoliphage lambda: revised measurement of the promoter-startpoint distance, Virology, 62 (1974) 458--471.

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Bolivar, F., Rodriguez, R,L,, Betlach, M,C. and Boyer, H.W., Construction and characterization of new cloning vehicles, L Ampicillin.resistant derivatives of the plssmid pMB9, Gene, 2 (1977) 75--93. • Bouch6, £P., Rowen, L. and Kornberg, A., The RNA primer synthesized by primase to initiate phage G4 DNA replication, J. Biol. Chem., 253 (1978) 765--769. Denniston-Thompso~ K,, Moore, D.D., Kruger, K,E., ~ ME, and Blattner, F.R., Physical structure of the replication origin of bacteriophage lambda, Science, 198(1977) 1051--1056. Dove, W.F., Hargrove, E., Oheshi, M., Haugli, F. and Guha, A., Replicative activation in lambda, Japan. J. Genet., 44 (1969)SuppL 1, 11--22. Dove, W.F., Inokuchi, 14. and Stevens, W.F., Replication control in phage lambda, in Hershey, A.D. (E&), The Bacteriophage Lambda, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1971, pp. 747--771. Fiddes, J.C., Barrel, B.G. and Godson, G.N., Nucleotide sequences of the separate origins of synthesis of bacteriophage (]4 viral and complementary DNA strands, Proc. Natl. Acad. Sei. USA, 75 (1968) 1081--1085. Furth, M.E., McLecster, C. and Dove, W.F., Specificity determinants for bacteriophage ~. DNA replication. L A chain of interactions that controls the initiation of replication, J. Mol. BicL, 126 (1978) 195--225. Furth, M.E. and "fates, J.L., Specificity determinants for bacteriophage ~. DNA replication. IL Structure o f O proteins of ~-080 and ~.-082 hybrid phages and of a ~. mutant defective in the origin of replication, J. Mol. Biol., 126 (1978) 227--240. Godson, G.N., Barrel, B.G,, Staden, R. and Fiddea, J.C., Nucleotide sequence of bacteriophage G4 DNA, Nature, 276 (1978) 236--247. Grosschedl, R. and Hobom, G., DNA sequences and structural homologies of the replication origins of lambdoid bacteriophages, Nature, 277 (1979) 621--627. Grossehedl, R. and Schwarz, E., Nueleotide sequence of the ero-cH.oop region of bacteriophage 434 DNA, Nucl. Acids Res., 6 (1969) 867--881. Hayes, S. and Szybalski, W., Synthesis of RNA primer for lambda DNA replication is controlled by phage and host, in Hamkalo, B.A. and Papaconstantinou, J. (Eds.), l~olecular Cytogenetics, Plenum, New York, 1973, pp. 277--288. Hayes, S. and Szybalski, W., Role of oop RNA primer in initiation of coliphage lambda DNA replication, in Goulian, M., Hanawalt, P.C. and Fox, C.F. (Eds.), DNA Synthesis and Its Regulation, Benjamin, Menlo Park, CA, 1975, pp. 486--512. Hobo~-1, B. and Hobom, G., Rifampicin insensitive replication of dimer ~.dv DNA, Nature New Biol., 244 (1973) 265-261. Hobom, G., Grosschedl, R., Lusky, M., Scherer, G., Schwarz, E. and K6ssel, H., Functional analysis of the replicator structure of lambdoid bacteriophage DNAs, Cold Spring Harbor Symp. Quant. Biol., 48 (1979a) in press. Hobom, G., Schwarz, E., Melzer, M. and Mayer, H., Isoschizomeric restriction endonucleases from Enterobacter cloacae DSM30056 and from Bacillus stearothermophilus ET, Nucl. Acids Res., 6 (1979b) submitted. Kingsbury, D.T. and Helinski, D.R., Temperature sensitive mutants for the replication of plasmids in £scherichia toll: requirement for desoxyribonucleic acid and polymerase I in the replication of the plasmid ColE1, J. Bacteriol., 114 (1973) 1116--1124. Klein, A. and Powling, A., Initiation of ~. DNA replication in vitro, Nature New Biol., 239 (1972) 71--73. Klinkert, J. and Klein, A., Roles of bacteriophage ?. gene products O and P during early and late phases of the infection cycle, J. Vkol., 25 (1978) 730--737. Lusky, M. and Hobom, G., Inceptor a~.d origin of DNA replication in lambdoid coliphages. I. The ~DNA minimal replication system, Gene, 6 (1979) 187--172.

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