Transcriptional activation of initiation of replication from the E. coli chromosomal origin: An RNA-DNA hybrid near oriC

Cell, Vol. 55, 113-123,

October 7, 1988, Copyright

0 1988 by Cell Press

Transcriptional Activation of Initiation of Replication from the E. coli Chromosomal O rigin: An RNA-DNA Hybrid Near OK Tania A. Baker and Arthur Kornberg Department of Biochemistry Stanford University Medical School Stanford, California 94305

Summary Transcription by RNA polymerase preceding the initiation of replication from the E. coli chromosomal origin (oriC) in vitro enables dnaA protein to open the DNA duplex under conditions when its action alone is insufficient. The RNA polymerases of phages T7 and T3 are as effective as the E. coli enzyme in activating initiation. The persistent RNA transcript hybridized to the template creates an R-loop that is responsible for activation. The activating RNA need not cross oriC, but must be less then 500 bp away. Transcripts lacking a 3’O H group are effective, proving that priming of DNA synthesis is not involved in the activation. Thus, transcription activates the origin of an otherwise inert plasmid by altering the local DNA structup, facilitating its opening by dnaA protein during the assembly of replication forks. introduction Involvement of RNA polymerase (RNAP) in replication of the Escherichia coli chromosome was inferred from the sensitivity of initiation to the RNAP inhibitor rifampicin, independent from the requirement for protein synthesis (Lark, 1972). Two classes of mutants also suggest an RNAP action in initiation from OX. Mutations in rpoB, the gene encoding the p subunit of RNAP, suppress the temperature sensitivity of dnaAtS alleles, indicating that altered RNAPs can assist defective dnaA proteins in initiation (Bagdasarian et al., 1977; Atlung, 1984). Furthermore, certain mutations in the genes for the /3 and p’ subunits of RNAP elevate the copy number of 0% plasmids and the chromosome, implicating RNAP in regulation of initiation (Tanaka et al., 1983; Rasmussen et al., 1983). Despite the strongly suggested involvement of RNAP, and RNA synthetic event required for initiation has remained unclarified. Several promoters have been identified in or near the 245 bp minimal oriC (Figure 1A) (Lother and Messer, 1981; Morelli et al., 1981; Rokeach and Zyskind, 1986; Schauzu et al., 1987), but their importance remains uncertain. Transcription from the promoter for the mioC gene (for modulation of initiation at oriC; mioC encodes a 16 kd protein of unknown function and is immediately adjacent to oriC) is regulated by dnaA protein, has positive effects on oriC plasmid stability and copy number (Stuitje and Meijer, 1983; Stuitje et al., 1986; LflbnerQlesen et al., 1987), and appears to help the origin compete for dnaA protein (Hansen et al., 1987; deWind et al., 1987). Transcription of mioC reads toward oriC and terminates at numerous locations within the origin as well as proceeding all the way through (Junker et al., 1986;

Rokeach and Zyskind, 1986; Schauzu et al., 1987). Some of these termination sites coincide with the locations of RNA-DNA junctions (Hirose et al., 1983; Kohara et al., 1985), suggesting that they may be used as primers for DNA synthesis. However, the mioC promoter can be deleted from both plasmids and chromosome without destroying oriC-dependent replication. Thus, Ihere is no firm relationship between mioC transcription and the essential RNA inferred from the rifampicin sensitivity of initiation. The crude in vitro system that specifically replicates oriC plasmids is sensitive to rifampicin, suggesting a role for RNAP (Fuller et al., 1981). However, when replication was reconstituted with purified proteins, primase supplied the priming function and the requirement for RNAP depended on the reaction conditions (Ogawa et al., 1985; van der Ende et al., 1985). High levels of the histone-like protein HU or topoisomerase I (topo I), or a several-degree decrease in temperature each provoked a requirement for RNAP The effect of RNAP on the reaction temperature was observed before the priming stage, suggesting that RNAP was involved in activating the origin rather than in priming; primase was clearly responsible for much or all of the priming when both enzymes wepe present. Replication in the absence of RNAP (Figure IB) starts with 20 to 40 monomers of dnaA protein Ibinding cooperatively to the four 9 bp recognition sequences (dnaA boxes) within oriC to form an initial complex (Fuller et al., 1984). DnaA protein, with ATP tightly bound, then opens the DNA duplex at the left boundary of oriC (in the 13 bp repeats) to form an open complex (rendering the template sensitive to linearization by the single-strand-specific nuclease Pl within the 13-mers) (Sekimizu et al., 1987; Bramhill and Kornberg, 1988). This duplex opening provides an entry site for the dnaB helicase. Introduction of dnaB helicase to form the prepriming complex is effected by dnaC protein from a dnaB-dnaC complex (Baker et al., 1986; Funnell et al., 1987). Thus established at oriC, the dnaB helicases migrate from the complex, unwinding the DNA duplex in both directions, thereby cre,ating an initiation bubble. Within this single-stranded bubble, primers laid down by primase are elongated by DNA polymerase III holoenzyme. With gyrase to relieve the topological strain associated with fork movement, replication continues around the template (Baker et al., 1986, 1987). To determine where in this replication pathway RNAP makes the crucial contribution observed in cells and crude enzyme fractions, we have examined how the factors that impart RNAP dependence affect the individual stages of replication and how RNAP func:tions to overcome their inhibitory actions. Results Factors That Cause Replication to Depend on RNAP Inhibit Duplex Opening by DnaA Protein The replication of oriC plasmids in vitro is tdependent on RNAP under certain reaction conditions: high levelsof the histone-like protein HU, lowered reaction ,temperatures,

Cell 114

Figure 1. Initiation of Replication

(A) ” gid -

,

3 mers _A..

“A-_A (I”ii.4

13 mr-ers

CruA”

HU PlTP * dnaA

Q3 0 .cJ I

)

38 dnaB dM.T ATP

INITIAL COMPLEX

SUPERCOILED TEMPLATE

PRIMING AND REPLICATION

from OK

(A) The oriC region including the proposed promoters. The open box represents the 245 bp minimal oriC sequence. The four 9-mers or “dnaA boxes” are denoted bv hatched boxes near the right boundary of or& (the fourth repeat near the 18mers), the three 13-mers as narrower hatched boxes at the left edge. The thin black line represents DNA surrounding OK, and the promoters are shown as arrows denoting the direction of transcription. Within the mioC promoter region there is a dnaA box (hatched box) and a sequence similar to the 13mer repeat. (6) Individual stages of initiation of replication. See text for details.

COMPLEX

PREPRIMING COMPLEX

Transcription by RNAP can substitute for or modulate the effects of HU. In the absence of HU, addition of RNAP stimulated replication severalfold (Figure 26). At the optimal HU level, RNAP had little effect. At high, inhibitory HU levels, transcription restored the reaction to about 50% of that seen in the presence of low HU levels. Topo I inhibits initiation of replication, probably by relaxing the template below the critical superhelical density required for formation of the prepriming complex (Ogawa et al., 1985; van der Ende et al., 1985; Funnell et al., 1986). The superhelical density needed for formation of the open and prepriming complexes was less (more negative) than -0.04 (Figure 3A). Although dnaA protein prefers negatively supercoiled DNA, it can bind to (form an initial complex with) relaxed or linear DNA (Fuller and Kornberg, 1983; Fuller et al., 1984). Thus, the defect in forming a prepriming complex on overly relaxed DNA is likely due to the inability of dnaA protein to open the DNA strands at oriC in the absence of sufficient superhelical tension. RNAP antagonizes the inhibitory effect of topo I, suggesting that it can activate partially relaxed DNA. To test this possibility directly requires that gyrase be absent, but

and high levels of topo I (Ogawa et al., 1985). The effect of these conditions on the separated replication stages was assayed to determine when the reaction was blocked in the absence of RNAP Replication and prepriming complex formation were measured by incorporation of [32P]dTTP into acid-insoluble product; formation of the dnaA protein open complex was measured by linearization of the template with Pl nuclease (Bramhill and Kornberg, 1988). HU protein stimulated replication nearly lo-fold when present at low levels (5-20 ng); higher levels (>lOO ng) completely inhibited synthesis (Figure 2A). Low levels of HU stimulated open complex formation (E. Lee and A. K., unpublished results), whereas high levels inhibited, paralleling the effects on replication (Figure 2A). HU had only minor effects on the earlier replication stage, formation of the initial dnaA protein-oriC complex, although when more HU was present higher levels of dnaA protein were required (data not shown). Thus, very low levels of HU stimulate replication by enabling DNA-bound dnaA protein to form the open complex while higher levels inhibit dnaA protein action.

Figure 2. Effects of HU and RNAP on Replication

(A) -50

1000 -

-40

-

30

% g 8 5 E x B

(A) HU in the indicated amounts (ng per 25 ui reaction with 200 ng of template) was added to standard replication and open complex reactions in the absence of RNAP. The template for replication was pTB101, and that for open complex formation was pCM959. (8) T7 RNAP (which can replace E. coli RNAP; see below) was added in the indicated amounts to standard replication reactions on pTB101. The reactions contained 0.55 ng of RNAase H to maintain dependence of replication on dnaA protein.

L

5 P -20

B

s !k2 2 5

0

50

100

150

HU PROTEIN (ng)

200

0

2

4 RNAP (units)

6

Transcriptiona! 115

Activation

of oriC Replication

Figure 3. Influences RNAP on Replication

200

(4 160

% E lZO P : 60

E iI

40

0 -0.02

-0.04

-0.06

-0.08

0

4

8 TIME (min)

AVERAGE SUPERHELIX DENSITY

in the absence of gyrase prepriming complexes are stable only at a level of Mg2+ too low to support transcription. However, the observed longer lag in initiating replication on relaxed than on supercoiled DNA (Funnell et al., 1986) was shortened by RNAP (Figure 3B), indicating that transcription counteracts the topo I inhibition by activating the relaxed DNA. HU protein wraps DNA around itself upon binding, as histones do in nucleosomes, “constraining” negative supercoils (Broyles and Pettijohn, 1986). Unconstrained supercoils partition freely between superhelical turns and regions of single-strandedness, whereas constrained supercoils cannot. Binding by HU thus effectively “relaxes” the DNA. HU titrations of free superhelicity (Broyles and Pettijohn, 1986) and the effects of relaxation on replication (see above) allowed calculation of a theoretical HU inhibition curve, which agreed very well with the observed data

60 s s Lo

60

SUPERCOILS

El E 5 m

12

of

Superhelicity

and

(A) pTB101 and pCM959 were relaxed to various extents with calf thymus topo I in the presence of various amounts of ethidium bromide, then purified and their concentrations measured by microfluimetry. Agarose gel electrophoresis showed that each sample contained about seven bands corresponding to different topoisomers. The middle of the band distribution was determined and used to calculate the average superhelical density in each sample. The template for replication was pTB101; for open complex formation it was pCM959. The replication reactions were staged to form prepriming complexes at 30%; reactions included 8.4 ng of HIJ per 200 ng of template. (B) Partially relaxed and native pTBlO1 templates were assayed for replication in the presence and absence of 2 U of T7 RNAP. Reactions included 8.4 ng of HU and 0.16 ng of RNAase H.

(data not shown). Furthermore, when HU was titrated into reactions on templates possessing, on average, 3 or 6 fewer negative supercoils than the native DNA (which had 23), proportionately less HU was required to inhibit prepriming complex formation (Figure 4). Thus, HU inhibition of replication can be attributed to the titration of negative superhelical density, making it unavailable for duplex opening by dnaA protein. DNA synthesis and prepriming complex formation require temperatures in excess of 27% (van der Ende et al., 1985; Baker et al., 1986) for opening the duplex. Open complex formation by dnaA protein has a similar sigmoidal temperature curve (shifted 2%-3% lower because less DNA is melted in the open complex than in the prepriming complex; (Bramhill and Kornberg, 1988). Transcription lowers the transition temperature, such that initiation of replication can occur at temperatures as low as 20% (van der Ende et al., 1985; data not shown). Thus, four conditions require RNAP for initiation of oriC replication in vitro: high levels of HU protein, inadequately supercoiled DNA, temperatures of less than 27X, and lack of stimulatory (i.e., low) levels of HU. Under these conditions dnaA protein fails to open the DNA duplex and introduce the dnaB helicase into the oriC complex. Transcription overcomes these inhibitory states, suggesting that it helps dnaA protein open the duplex.

40

: 20

0 0

20

40

60

80

100

HU PROTEIN (ng)

Figure 4. HU Inhibition of Replication

on Partially Relaxed Templates

HU was titrated into prepriming complex formation reactions on partially relaxed samples of pTBlO1 at 30%. Replication activity was determined by the standard prepriming assay described in Experimental Procedures.

RNAP Activates the Template by Laying Down an RNA-DNA Hybrid Rifampicin was used to separate transcription from replication. Addition of rifampicin after a 10 min transcription period did not inhibit activation when compared to a reaction lacking rifampicin; rifampicin present throughout the transcription phase completely blocked activation (Kaguni and Kornberg, 1984; data not shown). Yhus, the RNAP function can be expressed before that of any of the replication proteins, permitting the requirements for transcription

Cell 116

(6) pCM959

r

Figure 5. Phage T7 and T3 RNAPs Substitute for E. coli RNAP vation

in Acti-

(A) pTBlO1 contains a promoter for T7 RNAP located 277 bp to the right Of OriC, and a T3 promoter 272 bp to the left. Transcription is directed toward oriC in both cases. T7, T3, and E. coli RNAP were titrated into the standard replication reaction containing 200 ng of HU and 0.16 ng of RNAase H. Polymerase units indicated for T3 and T7 are those stated by the supplier; the greater activity of the T3 polymerase relative to T7 reflects a difference in efficiency of transcription rather than better activation. (B) Reactions were as described in (A), using pCM959 as the template.

to be determined independently from those for replication. All four rNTPs were essential, as was incubation at an elevated temperature (>l!YC) for at least 30 set (90 set or longer was optimal at 30%). RNAP core, lacking the sigma factor, was inactive (data not shown). E. coli RNAP could be replaced by phage T3 or phage T7 RNAP provided that the OK’ sequence was cloned into a plasmid (pTB101) containing promoters for these phage RNAPs (Figure 5A). Transcription from the phage promoters started about 250 bp outside, on either side, and proceeded through oriC. The T7 RNAP was inert on pCM959, an oriC plasmid lacking the phage promoters (Figure 5B). Activation by these structurally unrelated phage RNAPs argues against a protein-protein interaction between RNAP and a replication protein as essential to the mechanism of activation. Because the T7 and T3 RNAPs start specifically at the cloned promoters and will not recognize the E. coli RNAP promoters or terminators in and near OK, activation does not require recognition of these DNA structures. Furthermore, since the two phage promoters transcribe off opposite strands (making no RNA sequences in common) and are equally effective at activating replication, the consequence of transcription, rather than E. coli RNAP or a specific RNA, must activate 0riC. The RNA produced during transcription worked only in cis; free RNA made from transcription of the same template separately and then added to a reaction failed to activate (data not shown). Exposure of an activated template to a large amount of RNAase A, which degrades free, single-stranded RNA, caused the loss of less than 50% of the activated state. By contrast, the action of RNAase H, which degrades the RNA of an RNA-DNA hybrid, destroyed the activated state completely within 2 min (Figure 6A). (RNAase H added without RNAase A also destroyed

-0

4

8

12

TIME AFTER RNas? ADDITION

Figure 6. RNAase sensitivity

(mint

of the Activated Templates

(A) Three transcription reactions (60 ul) were set up as described in Figure 4 except that the template was pTB102, the transcription stage included dnaA protein and 10 U of T7 RNAP, and the scale was increased S-fold. After transcription for 5 min at 30°C 250 ng of RNAase A or 250 ng of RNAase A and 2.75 ng of RNAase H were added. Samples (12.5 ul) were removed at the times shown and assayed for replication by mixing with a 12.5 ul replication reaction lacking template and containing 200 ng of HU. Incubation continued for 30 min at 30%. (6) Transcription reactions for RNA synthesis were identical to those in (A) but also included [a3*P]CTP to label the RNA. After digestion for 2 min with RNAase A or RNAase A and RNAase H, reactions were stopped by addition of EDTA to 20 m M and SDS to 0.1%. Samples were electrophoresed on a 0.7% neutral agarose gel. An autoradiogram of the dried gel was developed to visualize the radioactivity labeled RNA. The position of hybrids was determined by ethidium bromide staining of the template band.

the activated state, as shown below). While almost all of the RNA was digested into short pieces by RNAase A, a small amount of RNA remained bound to the template (Figure 6B). RNAase H had no effect on the short RNA but removed the hybridized RNA, suggesting that an RNA hybridized to the template is essential for transcriptional activation. To determine whether RNA hybridized to the template was sufficient for activation, the transcribed template was separated from RNAP and other reactants by gel filtration. Template-containing fractions, assayed for replication in the presence of inhibitory levels of HU, were 4- to IO-fold more active than DNA that had not been transcribed (Table 1). Replication of the activated template was independent of rNTPs and was destroyed by either heating to 90% for 3 min or by a pretreatment with RNAase H, two conditions that destroy RNA-DNA hybrids. Thus, such hybridized transcripts confer activity on oriC plasmids under otherwise inhibitory conditions. RNA-DNA Hybrids in oriGIndependent Replication In the presence of RNAR specificity factors are needed to ensure that replication is dependent on 01% and dnaA protein, RNAase H is such a specificity factor in replication both in vivo and in vitro (Ogawa et al., 1984; Lindahl and Lindahl, 1984; Torren et al., 1984). When RNAP is omitted, the specificity factors are not required (Ogawa et

Transcriptional 117

Activation

of oriC Replication

Table 1. Activity of Isolated RNA-DNA

Table 2. Activation

Hybrids

Template

DNA Synthesis

Untreated Hybrid Heateda RNAase Hb No dnaA protein

49 214 10 7 18

200 150

E 5 z

100

2 50

0 0

0.25

0.5

0.75

1.0

1.25

1.5

RNAseH (ng)

Figure 7. Effect of RNAase H on the Specific and Nonspecific tions

Locations

Reac-

RNAase H was titrated into the standard replication reaction on pTB101, containing or lacking dnaA protein. The reactions included 140 ng of HU and 5 U of T7 RNAP

(pmol)

Hybrids Formed with 3’dATPC

Promoter Locationa

al., ?985). RNA-DNA hybrids are presumed to be responsible for the nonspecific replication because replication is effectively inhibited by RNAase H. Thus, whenever RNAP is present in a replication reaction, low levels of RNAase H are needed to maintain specificity. How then does transcriptional activation of oriC-specific replication, dependent on an RNA-DNA hybrid, occur in the presence of FiNAase H? Upon titration of RNAase H into reactions that contained or lacked dnaA protein, the sensitivities of the o&-specific and dnaA-independent reactions were strikingly different. The nonspecific reaction was very sensitive to RNAase H, while the dnaA protein-dependent reaction was resistant at levels lo- to 50fold higher (Figure 7; additonal data not shown). In fact, RNAase H must be added before the initiation proteins for a decisive demonstration of the sensitivity of the specific reaction (as was done in experiments shown in Figure 6 and Table 1). This apparent insensitivity of the hybrids required for activation may be explained by their shorter length or more rapid use than those utilized in the nonspecific reaction, or they may be protected from digestion by the initiation proteins.

;

in Different

DNA Synthesis

Transcription was on pTBlO1 with T7 RNAP for 10 set, and the reaction was quenched with EDTA. The transcribed template was then purified away from the RNAP and rNTPs by passage over a small BioGel A15m column. DNA-containing fractions were assayed for replication in the presence of 140 ng of HU. a Template heated to 90°C for 3 min before assay. b Template digested with 0.55 ng of RNAase H for 10 min at 30°C before assay.

Fz 5

by Transcripts

(pmol)

Plasmid

Distance

Side

+ RNAPb

1.5 pm

3.0 pm

pTBIOld pTB102 pTB103 pTB104 pTB105 pTB106 pTB107

272 257 92 257 470 623 2676

L, R, L, R, L, R, L,

403 310 619 450 248 182 176

347 202 354 75 7 7 110

222 295 91 106 10 7 36

in in out out out out in

a Location (see also Figure 9) is summarized as base pairs from the start site of transcription to the nearest edge of OK. IL and R, promoter is to left or right of oriC (left edge of oriC contains the 13-mers; see Figure 1); in and out, transcription is directed toward or away from OK. b Templates were assayed under standard reaction conditions in the presence of 140 ng of HU. In the absence of RNAP, replication was between 9 and 44 pmol. c Templates were transcribed in the presence of 1.5 or 3.0 PM 3’dATP and isolated as described in Experimental Procedulres. Assays were in the presence of 140 ng of HU. Mock-transcribed (no rNTPs) pTl3103 gave 7 pmol of DNA synthesis. Replication activity of each template, determined in the presence of 8.4 ng of HU, was between 191 and 864 pmol. d Transcription from the T3 promoter; transcription of the others was from the T7 promoter.

The Transcript Must Be near oriC to ,Activate Plasmids were constructed with the phage ‘T7 promoter at different locations with respect to oriC (lkbk 2). Replication of these plasmids depended on dnaA protein and was inhibited by high levels of HU protein; ad&on of T7 RNAP relieved the HU inhibition. Only minor differences in the level of activation, regardless of the promoter location, were observed in the standard replication reaction with T7 RNAP present throughout the 30 min incubation (Table 2). Under these conditions, transcription pra’ceeded many times around the plasmid, such that no location-dependent activation could have been expected; addition of the T7 transcription terminator led to termination of only about 70% of the RNA and allowed an intolerable amount of readthrough (data not shown). To determine the template locations for transcriptional activation of initiation, 3’dATP (cordycepin triphosphate) was used to limit the transcript lengths. This analog, lacking a 3’O H group, terminates chain growth when incorporated into RNA in place of ATP; an increase in the ratio of 3’dATP to ATP decreases the average RNA chain length. Various amounts of 3’dATP were added to transcription reactions on pTB102 and pTBW7, plasmids that differ only in the distance between the promoter and oriC: 257 bp on pTB102 and 2676 bp on pTB107. The transcribed templates were isolated by gel filtration and assayed for replication in the presence of a high level of HU. Increasing the ratio of 3’dATP to ATP inhibited replication, showing that a minimal transcript length is needed to activate (Figure 8). At low HU levels, where transcriptional activation is not required, replication was insensitive to S’dATP present in the transcription reactions (data not shown).

Cell 118

a Q B 300 2 F *f 200 2

100 0 INACTIVE

0

I

I 3.0

I 1.5

I

I 0.6

0.3 [3’ dATP]

.

Figure 9. Activation

I I 0

(PM)

Figure 8. Effect of RNA Length on Activation 3’dATP was titrated into transcription reactions on pTB102 and pTBlO7, and transcribed templates were isolated as described in Experimental Procedures. pTB102 and pTBlO7 are identical except for the T7 promoter position, which is 257 bp away from OK on pTB102 and 2676 bp away on pTB107. Purified templates were assayed for replication in the presence of 140 ng of HU (and also in the presence of 8.4 ng to check for recovery in the purification; data not shown). The average length of RNA was calculated by dividing the sum of the nucleotide concentrations (ATP, CTP, GTP, and UTP at 125 uM each; the 3’dATP concentration varied between 0.3 uM and 30 FM) by the concentration of 3’dATP This calculation assumes a random nucleotide sequence and no bias between incorporation on 3’dATP and ATP The calculated lengths were in good agreement with those measured by gel electrophoresis of the RNAs at various 3dATP concentrations. The average measured lengths of RNA at 3’dATP concentrations that gave 50% inhibition of replication were 140 nucleotides for pTB102 and 880 nucleotides for pTBlO7, compared with calculated values of 100 and 1100 nucleotides, respectively.

The plasmid pTB107, with its promoter remote from oriC, was 8-fold more sensitive to 3’dATP than was pTBlO2. The approximate average RNA length at different 3’dATP:ATP ratios was calculated (Figure 8 and legend) and measured by gel electrophoresis of transcription reactions (data not shown). Based on these estimates, an RNA of 200 nucleotides was optimal for activating pTB102, whereas RNAs longer than 1000 nucleotides were required for pTB107. Thus, the required length for an activating RNA varied depending on the distance between the promoter and oriC; proximity of the RNA to oriC is required for activation. Activation of plasmids with the T7 promoter at various distances from OK, in both orientations, was assayed in the presence of 3’dATP concentrations sufficient to block transcription from proceeding entirely around the template (Table 2 and Figure 9). Transcription that was initiated less than 300 bp away from OK, and directed toward it, activated initiation; transcripts initiated 92 bp away on the left or 257 bp away on the right, and directed away from oriC, activated to a similar extent. However, transcription initiated about 500 bp away from either side of oriC and directed away from it failed to activate. Effective activation by limited transcription from promoters directed away from oriC clearly demonstrates that RNA does not need to cross oriC to activate. Promoters less than 300 bp

of oriC Depends

on Location of the Transcripts

The location and orientation of transcripts are shown as arrows. Numbers refer to the last number in the plasmid name (see Table 2 for plasmid names and replication activity). Transcripts are shown approximately 200 nucleotides long. Dark, thickened arrows represent RNAs that activate when they are this length; the thin arrows represent inactive RNAs.

away from oriC activated, while those 500 bp away did not. Thus, activation requires transcription to be close to oriC. Transcripts That Cannot Prime Can Still Activate Whether transcriptional activation can be attributed to priming of replication was answered decisively by the use of 3’dATP Transcripts terminated with 3’dATP lack a 3’O H group and thus cannot be used as primers. Templates produced in transcription reactions with the analog at several levels were isolated and assayed for activation in the presence or absence of primase (Figure 10). RNAase H, usually included as a specificity factor, was omitted to avoid recreating 3’ O H ends by cleavage of the hybrid. The dependence on dnaA protein confirmed that nonspecific (oriGindependent) replication was not a significant problem. Transcripts made in the presence of 3 PM 3’dATP (average RNA length of about 140 nucleotides) activated replication nearly as well as those made in the absence of the analog; still higher levels of 3’dATP inhibit by limiting the length of the RNA (Figure 8). Furthermore, replication on all the activated templates was almost completely dependent on primase. Discussion RNAP Transcription Helps DnaA Protein Open the DNA Duplex Initiation of replication from 0riC in vitro requires transcription by RNAP when the histone-like protein HU is present at a high level, the template superhelicity is reduced, or the temperature is lowered a few degrees. Each of these conditions stabilizes the DNA duplex and inhibits the opening of about 40 bp of oriC by dnaA protein, which allows the entry of dnaB protein. Inasmuch as duplex opening has been identified as the stage inhibited by these conditions, the relief by RNAP implies that transcription facilitates dnaA protein opening of the DNA strands at the origin. Dependence of replication on transcription was also ob-

Transcriptional 119

i-

Activation

of OK

Replication

(A) pTBlO2

[3’ dATP]

Figure 10. Replication

(yP.4)

on Activated Templates Requires Primase

Activated templates were made on pTB101 (A) and pTB103 (B) in various concentrations of 3’dATP as described in Figure 8. Templates were assayed for activation in the presence of 140 ng of HU in complete reactions or in reactions lacking primase or dnaA protein.

served in the absence of the low levels of HU that stimulate initiation. These low levels of HU, which coat only about 4% (180 bp) of the template, in some manner, stimulate by facilitating opening of the duplex by dnaA protein. Thus, HU has two distinct and antagonistic effects on initiation. The inhibitory effect by high HU levels is likely due to constraint of several superhelical turns. Modulation of free template superhelicity by low HU levels would relax, at most, only one or two superhelical turns (Broyles and Pettijohn, 1986); DNA templates relaxed by this amount are still stimulated by HU (data not shown). These low levels of HU may act by stabilizing the dnaA protein-o% complex and become dispensable when transcription assists duplex opening. An RNA-DNA Hybrid Activates Opening of the Duplex A region of RNA-DNA hybrid near oriC is necessary and sufficient for activation of initiation. E. coli RNAP can be replaced by phage T7 and T3 RNAPs even though the host and phage enzymes are structurally unrelated. Furthermore, transcribed templates, purified away from RNAP, remain activated. Thus, the protein-protein interaction between RNAP and dnaA protein often inferred from the suppression of temperature-sensitive dnaA alleles by mutations in the gene for the 6 subunit of RNAP (Atlung, 1984; Bagdasarian et al., 1977) seems unlikely. While our data cannot rule out the existence of such a complex, clearly RNAP can help dnaA protein function during initiation without a direct interaction. The similarities between transcriptional activation in phage h replication (see below), which does not depend on dnaA protein for initiation, and oriC also argue against a dnaA protein-RNAP complex being involved. Although the size of the hybridized RNA required for activation was not analyzed in detail, the extent of hybrid formation observed during transcriptional activation is surprising In transcription complexes, the newly synthesized RNA forms a hybrid for only about 12 bp before being dispiaced from the template (Yager and Von Hippel, 1987). In

our work, hybrids were detected by the following: association of labeled RNA with the template, with (or without OriC, on sucrose gradients and agarose gels; cross-linking with a psoralin (AMT); and RNA resistant to RNAase A and sensitive to RNAase H (data not shown). Stopping the transcription reaction by denaturing the RNAP reported to cause hybrid formation (Richardson, 19775),was not responsible for hybrid formation in our work because the hybrids were detected in the absence of protein denaturants. One possible basis for extensive hybrid formation is our use of negatively supercoiled templates, which readily take up linear single-stranded DNA (Beattie et al., 1977) and can stabilize an RNA-DNA hybrid. In the replication of plasmid ColEl, an RNA-DNA hybrid of about 40 bp is formed on supercoiled DNA in vitro (Itoh and Tomizoma, 1980). Furthermore, the requirement for RNAase H as a specificity factor for replication in vivo and in vitro implies that RNA-DNA hybrids form often during transcription and must be removed so that they are not used as nonspecific origins (Ogawa et al., 1984). Activation Does Not Depend on Priming RNA terminated with 3’dATP (cordycepin triphosphate), and thus lacking a 3’ O H group, activated initiation. The RNA was effective 200 bp away from oriC and could have the polarity of either strand, features unlikely for a primer. Finally, replication on activated templates was almost completely dependent on primase. Although RNAP can prime DNA synthesis feebly in vitro and may do so to some extent in vivo, its principal effect on replication from oriC, at least in vitro, is not priming. Transcriptional Activation of ColEi and OriC Differ Replication of ColEl requires an RNA-DNA hybrid that is normally cleaved by RNAase H to serve as a primer for DNA synthesis (ltoh and Tomizawa, 1980). However, in the absence of RNAase H, a 40 bp hybrid is still required to activate the template (Masukata et al., 1987; Dasgupta et al., 1987). The model proposed for activation is that a DNA helicase (e.g., dnaB protein) binds to the DNA strand displaced in the R-loop and extends the single-stranded region to create a loading site for the replication complex. Activation bypasses the need for either the primosome assembly sites or the dnaA box, which are normally required for assembling the replication complex l(Zipursky and Marians, 1980; Seufert and Messer, 1987). The mechanism proposed for ColEl is consistent with some of the observations about activation of oriC: an RNA-DNA hybrid is the activating structure and the step affected is helicase loading. However the strand displaced by the hybrid in oriC replication appears not to serve directly as a dnaB protein loading site, and the oriC sequence, including the 13-mer repeats, is essential for replication on the activated templates (Bramhill and Kornberg, 1988). DnaA protein in the ATP form is also required (Sekimizu et al., 1987); SSB protein, which inhibits the binding of dnaB protein to single-stranded DNA, does not inhibit initiation on the activated templates (data not shown). Inasmuch as a dnaB protein loading site should work anywhere on the template, this mechianism fails to

Cell 120

explain why the hybrid needs to be near oriC. Clearly, transcriptional activation of oriC involves synergy between the hybrid and the prepriming events occurring at the origin. The model proposed for transcriptional activation of ColEl may relate to the events that occur during initiation of replication at RNAase H-sensitive sites that are not dependent on oriC and dnaA protein, but this remains to be tested experimentally. Transcriptional Activation of h and oriC Are Similar Replication of h in vivo requires transcription of the template in cis even when the essential replication proteins are provided in tram (Furth and Wickner, 1983). Mutations that allow the phage to replicate when the normal activating transcription is repressed create new promoters (Furth et al., 1982). These new promoters, as far away as 95 bp from the origin and directing transcription away from it, suggest that RNA synthesis activates the origin rather than supplying a primer. The orih sequence is still required, as are the phage-encoded replication proteins 0 and P Thus, h activation has many features in common with oriC. In the initiation of replication at the h origin, the 0 protein (the dnaA protein analog) binds to reiterated sequences within the origin, creating a large complex (Dodson et al., 1985), and opens the DNA duplex (Schnos et al., 1988). The P protein (the dnaC protein analog) complexes with dnaB protein and brings it to the 0 complex at the origin (Dodson et al., 1985, 1986; Zylicz et al., 1984). As with 06, the crude in vitro system for replication is sensitive to rifampicin, implying the involvement of RNAP (Wold et al., 1982; Toshiki and Matsubara, 1982) while replication with purified proteins does not require RNAP. Purification of the component of the crude system responsible for the RNAP dependence yielded HU protein (McMacken et al., 1988). Thus, both or& and oriC appear to undergo transcriptional activation by similar mechanisms. Transcriptional Activation of oriC In Vivo Analogies of transcriptional activation of oriC to that of phage h in vitro argue that the strong case for such activation of h in vivo also applies to oriC. In addition, several observations of oriC replication in vivo establish an interplay between dnaA protein function, template topology, and transcription similar to that found in vitro: First, dnaAtS mutants are hypersensitive to gyrase inhibitors; rpoB alleles suppress both the temperature-sensitive phenotype and this hypersensitivity (Fitulowicz, 1980; Fitulowicz and Jonczyk, 1981, 1983). Second, gyrase mutants specifically defective in initiation are suppressed by the same rpoB alleles that suppress dnaA mutations (Fitulowicz and Jonczyk, 1983). Third, plasmid DNA isolated from these rpo6 mutant strains is more negatively supercoiled than that isolated from wild-type cells. Fourth, some oriC plasmids transform gyrase mutant strains poorly; the transcriptional organization of the plasmids may be important in determining this sensitivity (Leonard et al., 1985). Fifth, mutations in topo I, which increase

negative superhelicity, suppress dnaAtS alleles (Louarn et al., 1984). In vitro, a decrease in negative superhelicity evokes the need for transcriptional activation. Remarkably, the free superhelicity value of -0.025 in E. coli (Bliska and Cozzarelli, 1987; Lilley, 1986) is significantly more relaxed than the threshold required for initiation in vitro (<-0.04). While the inhibitory levels of HU that elicit the need for transcriptional activation in vitro are higher than those believed to coat DNA in vivo (Dixon and Kornberg, 1984; Rouviere-Yaniv and Gros, 1975), other proteins and polyamines may combine to achieve the same effect in the cell (Drlica, 1987). Transcription units located in and around oriCcould activate initiation in vivo. Transcription from the mioC gene promoter initiates about 490 bp to the right and reads toward oriC. Transcripts terminate at the edge and within or/C as well as proceeding through it (Rokeach and Zyskind, 1986; Junker et al., 1986). Transcription from this promoter increases or/C plasmid copy number and stability as well as the ability of oriC to compete for limiting amounts of dnaA protein (Stuitje and Meijer, 1983; Stuitje et al., 1986; LQlbner-Olesen et al., 1987; Hansen et al., 1987; deWind et al., 1987). Our results also suggest that transcription from the gid promoter, at the other edge of oriC and reading away from it, may also be able to activate initiation of replication, as could transcription from the promoters within oriC. The Mechanism of Transcriptional Activation Two general mechanisms can be considered to account for how an RNA-DNA hybrid near oriC enables dnaA protein to load dnaB protein on the template when dnaA protein by itself is unable to open the DNA duplex. The hybrid may alter the DNA structure near oriC in a manner that facilitates melting by dnaA protein (“melting model”). Alternatively, dnaB protein first joins the orjC-dnaA protein complex and is then transferred to the opened DNA strands at the hybrid instead of to the 19mer region (“loop model”). The melting model is more attractive because it explains why the hybrid needs to be near oriC and why the 13-mers within oriC remain essential on activated templates. Also, transfer of dnaE? protein from the origin to the hybrid region in the loop model would be expected to be effective over longer distances and not require the 13-mer region. In the melting model, the hybrid, rather than altering the template topology, nucleates and facilitates melting of neighboring DNAover modest distances. Inasmuch as the plasmid is only one topological domain, a topological change anywhere on the template should be equivalent. Yet the hybrid must be near oriC to activate and thus operates through a localized change in DNA structure. Interestingly, the hybrid activates over a distance of about 250 bp, the amount of DNA that could be unwound if all of the negative superhelicity present in the plasmid was taken up as a single-stranded bubble. Recently, the effect of !ranscription on DNA topology has been recognized (Liu and Wang, 1987; Wu et al., 1988). Transcription can

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generate positive superhelicity ahead of the polymerase and negative superhelicity behind it. By this mechanism, transcription some distance removed from oriC could facilitate opening of the strands. However, this appears not to be the mechanism of activation, at least in vitro. Neither a single transcription unit on a circular plasmid nor an isolated R-loop should cause underwinding of the template, yet they both activate oriC. Effects of DNA sequence on the melting properties of neighboring sequences have been observed by others. Extrusion of the ColEI cruciform occurs by the melting of an extended region of duplex to create a single-stranded bubble before folding of the DNA into hairpins. This pathway is facilitated by neighboring AT-rich regions that can act at a distance of 100-200 bp (Sullivan and Lilley, 1986, 1988; Lilley, 1988). An AT-rich region can lower the melting temperature of linked GC-rich stretches at a distance, a phenomenon termed DNA telestability (Burd et al., 4975a, 1975b). Transcription as a General Mechanism of Activating DNA Replication and Recombination Altering DNA structure by transcription to activate DNA may occur in systems other than prokaryotic replication origins. C/s-acting transcription elements are inseparable from eukaryotic origins of replication (DePamphillis, 1988). Furthermore, in mammalian cells transcriptionally active genes replicate earlier in S phase than do inactive genes (Hatton et al., 1988). In addition to stimulating replication, transcriptional activity appears to stimulate recombination in yeast (Klar et al., 1981; Voelkel-Meiman et al., 1987) and has been proposed to activate immunoglobulin rearrangement during B cell differentiation (Blackwell et al., 1986; Y’ancopoulos et al., 1986). This interplay between replication or recombination and transcription may provide a way for these processes to respond to the physiologic state of the cell, to which the transcriptional machinery is exquisitely sensitive. Experimental

Procedures

Reagents Sources were as follows: Tricine (N-tris[hydroxymethyl]methyl glycine), ribonucleoside triphosphates, deoxynucleoside triphosphates, and 3’dATP were from Sigma; [c@P]CTP was from Amersham; bovine serum albumin (BSA) (RNAasefree) was from Bethesda Research Laboratories; BioGel A15-m was from BioRad; restriction site linkers were from New England Biolabs.

to +448 bp) fragment from the oriC region and 16 bp from the polylinker, into the EcoRl and Pstl sites of the Bluesci’ipt vector (M13+) (Strategene). The oriC fragment used in constructing the remaining plasmids came from cutting pTBlO1 with Smal, yielding a 538 bp fragment (-44 to +489 bp) and including 3 bp from the polylinker. This fragment was blunt-end ligated into the Pvull site of pGem-3 (Promega Biotec) to make pTB103 and pTB104, and into the BamHl site of PAR2529 (A. H. Rosenberg, J. J. Dunn, and F. W. Studier, Brookhaven National Laboratory) to make pTBlO2. The blunt ends were converted into Ndel ends by using synthetic linkers, and this fragment was cloned into the Ndel site of PAR2529 to make pTB107. pTBlO5 and pTBlO6 were made by first cutting out the T7 promoter fragment from PAR2529 with Bglll and reinserting it in the opposite orientation (pAR2529flip), and then inserting the Smal fragment into the EcoRl site using EcoRl linkers. Reconstituted DNA Replication The standard reaction (25 ~1) contained Tricine-KOH (pH 7.6), 30 mM; magnesium acetate, 8 mM; ATP, 2 mM; GTP, CTP, and UTP, 500 I.LM each; dATP, dGTP, and dCTP, 100 NM each; [c@P]dTTP, 100 NM (~200 cpm/pmol); template DNA, 200 ng (600 pmol of nucleotide); gyrase A subunit, 300 ng; gyrase B subunit, 150 ng; dnaA protein, 120 ng; SSB protein, 400 ng; dnaB protein, 60 ng; dnaC protein, 20 ng; primase, 10 ng; DNA polymerase Ill*, 70 ng; and p subunit of DNA polymerase Ill, 75 ng. RNAase H, HU, and RNAP were added as described in figure legends. Replication mixtures were assembled at O°C and incubated for 30 min at 30°C. Total nucleotide (pmol) incorporation was measured by liquid scintillation counting after precipitation with trichloroacetic acid and filtration onto Whatman GF/C g?ass-fiber filters. Prepriming complex formation was assayed by dividing the replication reaction into two stages. The first stage contained Tricine, template DNA, dnaA, dnaB, and dnaC proteins as described above and also included the following: glycerol, 20%; EDTA, 0.38 mM; Brij 58, 0.01%; BSA, 0.3 mg/ml; potassium glutamate, 60 mM; and ATP, 5 mM; magnesium acetate was omitted. Incubation was at 30°C for 20 min. Reactions were then shifted to 16OC, the remaining replication components were added (including magnesium acetate to 10 mM), and incubation was continued for 15 min before precipitation with trichloracetic acid. Detection of Open Complexes by Linearizatiofl with Pi Nuclease The standard reaction (50 )II) contained 40 m M HEPES-KOti (pH 7.6), 8 m M magnesium acetate, 30% glycerol, 320 pglmi BSA, 150 fmol of supercoiled plasmid DNA, 240 ng of dnaA protein, and 5 m M ATP. Following incubation at 30°C or 38°C for 2-5 min, 1.2 U of Pi nuclease was added in 3 ~1 of 30 m M potassium acetate (pH 4.8). The reaction was stopped after 5 set by the addition of 40 ~1 of 25 m M EDTA, 1% SDS, and the samples were electrophoresed through a 0.7% agarose gel in 89 m M Tris-borate (pH 8.3), 1 m M EDTA (TBE), at 6 V/cm. The gel was stained with ethidium bromide and photographed using Polaroid film. Densitometric scanning of the negative was used to determine the proportion of linear molecules.

Enzymes Highly purified replication proteins were as previously described (Kaguni and Kornberg, 1984); dnaA protein and DNA polymerase Ill* were purified by modified procedures. Restriction endonucleases and T7 RNAP were from New England Biolabs. T3 RNAP and calf thymus topo I were from Bethesda Research Laboratories. PI nuclease was from United States Biochemical Corp. RNAase A was a gift of the Baldwin group in this department.

Formation and Isolation of Activated Templates The transcription phase (50 pl) included the following: Ricine (pH 7.6), 30 mM; magnesium acetate, 10 mM; 2 pg of template DNA; and 20-40 U of T7 or T3 RNAP. Reactions were incubated at 30°C for 2 min, and transcription was initiated by the addition of the four rNTPs (including B’dATP, as indicated) to 125 PM each. Incubation was continued for IO-20 set, and transcription was stopped by addition of EDTA to 20 m M and transferring the reaction to ice. Reactions were gel filtered through a 1 ml BioGel A15m column (in 30 m M Tricine [pH 7.61); SDS was added to 0.1% just before loading. Template-containing fractions were collected and pooled (-120 ~1). These activated templates were stable on ice for several days. Samples (10 1.11)of the pooled fractions were assayed by the standard replication assay except that CTP, UTP, and GTP were omitted.

Plasmid DNAs pCM959, a gift from M. Meijer (University of Amsterdam) (Meijer et al., 1979), is a minichromosome (4012 bp) consisting solely of E. coli DNA from the origin region (-677 to +3555 bp). pTBlO1 was constructed by ligating the EcoRI-Pstl fragment from M13oriCRE85, a gift of D. Smith (University of California, San Diego), containing the Hincll-Pstl (-189

Topoisomers Topoisomers were made by relaxing the DNA with topo I. Relaxation reactions (100 ~1) contained Tricine (pH 7.6), 20 mM; KCI, 20 mM; magnesium acetate, 5 mM; dithiothreitol, 2 mM; BSA, 12.5 pglml; glycerol, 5%; template, 10 pg; ethidium bromide, O-12 PM; and topo I, 20 U. Incubation was at 37OC for 35 min. Reactions were stopped by the addi-

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tion of EDTA to 20 m M and SDS to 0.1%. The DNA was extracted with phenol-CHCIs twice and CHC& once, ethanol precipitated, and redissolved in 25 ~1 of 10 m M Tris-HCI, 1 m M EDTA (pH 8). Concentrations were measured in a minifluorimeter (TKO 100, Hoefer). Superhelical densities were determined by electrophoresis in 1% agarose gels (TBE) containing various concentrations of chloroquine and subsequent band counting. Acknowledgments We thank Mark Dodson, Boyana Konforti, Celeste Wilcox, LeRoy Bertsch, and members of the laboratory for careful reading of the manuscript. This workwas supported bygrantsfrom the National Institutes of Health and the National Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “‘advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received July 11, 1988. References Atlung, T. (1984). Allele-specific rpofl mutations in Escherichia

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Bagdasarian, M. M., Izakowska, M., and Bagdasarian, M. (1977). Suppession of the DnaA phenotype by mutation in the rpo6 cistron of ribonucleic acid polymerase in Salmonella fyphimurium and Escherichia co/i. J. Bacterial. 730, 577-582. Baker, T. A., Sekimizu, K., Funnell, B. E., and Kornberg, A. (1986). Extensive unwinding of the plasmid template during staged enzymatic initiation of DNA replication from the origin of the E. coli chromosome. Cell 45, 53-64. Baker, T. A., Funnell, B. E., and Kornberg, A. (1987). Helicase action of dnaB protein during replication from the Escherichia co/i chromosomal origin in vitro. J. Biol. Chem. 262, 6877-6885. Beattie, K. L., Wiegand, R. C., and Radding, C. M. (1977). Uptake of homologous single-stranded fragments by superhelical DNA. J. Mol. Biol. 776, 783-803. Blackwell, T. K., Moore, M. W., Yancopoulos, G. D., Suh, H., Lutzker, S., Selsing, E., and Alt, F. W. (1986). Recombination between immunoglobulin variable region gene segments is enhanced by transciption. Nature 324, 585-589. Bliska, J. B., and Cozzarelli, N. R. (1987). Use of site-specific recombination as a probe of DNA structure and metabolism. J. Mol. Biol. 794, 205-218. Bramhill, D., and Kornberg, A. (1988). Duplex opening bydnaA protein at novel sequences in initiation of replication at the origin of the E. coli chromosome. Cell 52, 743-755. Broyles, S. S., and Pettijohn, D. E. (1986). Interaction of the Escherichia co/i HU protein with DNA: evidence for formation of nucleosome-like structures with altered DNA helical pitch. J. Mol. Biol. 787, 47-60. Burd, J. F., Wartell, R. M., Dodgson, J. B., and Wells, R. D. (1975a). Transmission of stability (telestability) in deoxyribonucleic acid. J. Biol. Chem. 250, 5209-5113. Burd, J. F., Larson, J. E., and Wells, R. D. (1975b). Further studies on telestability in DNA. J. Biol. Chem. 250, 6002-6007. Dasgupta, S., Masukata, H., and Tomizawa, J. (1987). Multiple mechanisms for initiation of ColEI DNA replication: DNA synthesis in the presence and absence of ribonuclease H. Cell 57, 1113-1122. DePamphillis, M. L. (1988). Transcriptional elements as components eukaryotic origins of DNA replication. Cell 52, 635-638.

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