Cell, Vol. 63, 325-331,

October

19, 1990, Copyright

0 1990 by Cell Press

A Novel Protein Binds a Key Origin Sequence to Block Replication of an E. coli Minichromosome Deog Su Hwang and Arthur Kornberg Department of Biochemistry Stanford University School of Medicine Stanford, California 94305-5307

the entire 13-mer region with specificity, blocks initiation at a very early stage in vitro, and may prove to be an important negative effector for chromosome replication in vivo. Results

Summary A sequence of three tandem repeats of a 13-mer in the replication origin (oriC) of E. coli is the highly conserved site of opening of the duplex for initiation of DNA synthesis. A protein that binds this sequence has been discovered in E. coli and purified to homogeneity. This novel 33 kd polypeptide behaves as a dimer. Binding to the Wmers is specific and limited to this region. At a ratio of lo-20 monomers per or/C plasmid, the binding blocks initiation by preventing the opening of the 13-mer reglon by dnaA protein. Once the 13mers are opened by dnaA protein action, the 33 kd protein is without effect on the subsequent stages of replication. The specificity of binding and profound inhibitory effect suggest a regulatory role for this protein at an early stage of chromosome initiation. Introduction The autonomously replicating oriC piasmid behaves in most physiologic respects like the E. coii chromosome and may be regarded as a minichromosome (Kornberg, 1982; von Meyenberg and Hansen, 1987). The availability of a replication system, reconstituted from purified proteins (Kaguni and Kornberg, 1984) makes it possible to explore the numerous parameters that may control the firing of initiation at the origin. The level of dnaA protein (the initiator protein) (Lsbner-Oiesen et al., 1989) the state of the DNA (Russell and Zinder, 1987) nearby transcription (Baker and Kornberg, 1988; Skarstad et al., 1990) and membrane attachments (Yung and Kornberg, 1988) are among the factors that can profoundly affect initiation. it seems likely that in addition, negative as well as positive effecters act to coordinate ceil mass with initiation of repiication at 0riC. Within the minimal oriC sequence of 245 bp (Zyskind et al., 1983), certain highly conserved and biologically essential regions have been identified with the four 9-mer sequences bound by dnaA protein (Fuller et al., 1984). Other essential regions have yet to be associated with unique proteins that might affect their function. in particular, the three tandem repeats of a 13-mer, the site at which the origin is opened for initiation of DNA synthesis (for review see Bramhiii and Kornberg, 1988a), might be a reasonable target for binding by a regulatory protein. Furthermore, each of the 13-mers contains a GATC sequence, the site for methyiation that modulates chromosome attachments and initiation (Ogden et al., 1988; Russell and Zinder, 1987). We report here on the discovery of a protein that binds

A Protein Binds the DNA Fragment Containing the 13-mer Region of oriC A DNA fragment (638 bp) containing oriC and its left fianking region was purified from an oriC piasmid, pBSoriC (Bramhill and Kornberg, 1988b). Further cleavage of this fragment by Sau96i produced three fragments (Figure 1): agidfragment of 169 bp containing part of the open reading frame for the gid protein, an L-ori fragment of 198 bp from the left region of oriccontaining the three tandem 13mers and the nearby dnaA box (Qmer Rl), and an R-ori fragment of 271 bp from the right side of oriC containing the other three dnaA boxes (9-mers R2, R3, and R4) and part of the mioC open reading frame. A mixture of the three 32P-labeied fragments was incubated with a crude protein fraction prepared from E. coli and subjected to eiectrophoresis through a 5% poiyacryiamide gel foilowed by autoradiography (Figure 2). Of the three fragments, the retarded migration of the L-ori fragment was by far the most significant. Under these assay conditions (without added magnesium), purified dnaA protein failed to retard any of the three fragments (data not shown). Purification of the L-ori Binding Protein Based on the gel shift assay (Figure 2) the protein bound to the L-ori fragment was purified to near homogeneity (Table 1). Repeated precipitations of the soluble iysate (fraction I) with ammonium sulfate were needed to eliminate the nucleic acids that interfered with chromatographic separations. Neither nuciease nor topoisomerase activities, which inhibit oriC piasmid replication in vitro, were detected in the final fraction. Overall purification of the 33 kd protein from fraction ii was about 1300-fold (Table 1); activity in the crude iysate could not be reliably assayed. The purified protein migrated as a 33 kd poiypeptide upon eiectrophoresis on a 12% SDS-polyacryiamide gel (Figure 3). About 200 monomers are present per E. coii ceil based on the activity in fraction ii-l. The N-terminal sequence of 41 amino acids found no match in the Swiss Protein data bank. Upon gel filtration (Superose 12 of FPLC), 67% of the loaded protein activity was recovered in a peak that coincided with that of bovine serum aiumin (66.2 kd); no measurable activity was detected in other fractions (Figure 4). Based on this criterion, the protein behaves as a dimer of 33 kd subunits. The Protein Binds Specifically to the Three 13-mers in 0riC The purified protein bound specifically to the L-ori fragment (Figure 5A). in the presence of an excess of the pUC18 vector (154-fold as nucieotide), the complex re-

Cell 326

C

S ,

B I

1

S

a LMR RI L oric

gid

Fr VI

C R2R3 R4

15

mid

d

16

17

18

19

20



kDa 97-

Figure

gid +VW

1. Physical

L-ori -VW

R-ori (271)

66-

-

Map of the oriC Region

45-

The 638 bp Clal restriction fragment from plasmid pBSoriC contains the minimal 245 bp oriC, its flanking regions, and vector pBluescript sequence at the extreme left (Buhk and Messer, 1983; Zyskind et al., 1983; Bramhill and Kornberg, 1988b): L, M, and Rare the left, middle, and right 19mers, respectively; Rl, R2, R3, and R4 are the 9-mers. Restriction sites are: 8, BamHI; C, Clal; and S, Sau981.

31-

21 -

Fraction

Q $y$

II-3 (pg)

14-

Figure

3. SDS-Polyacrylamide

Gel Electrophoresis

of 33 kd Protein

Fractions were analyzed on a 12% SDS-polyacrylamide gel and visualized by silver staining: fractions II-3 to fraction V, 800 U of each; fraction VI (Mono S column fractions 15-20) 1 trl of each. Fraction Ii-3 and fraction Ill were precipitated with 10% TCA and redissolved in gel loading buffer. Molecular weight markers (in kd) were phosphorylase 8, 97; bovine serum albumin, 88; ovalbumin, 45; carbonic anhydrase, 31; soybean trypsin inhibitor, 21; and lysozyme, 14. Figure 2. Identification of a Protein taining the 13-mer Region

Binding to the L-ori Fragment

Con-

Plasmid pBSoriC isolated from E. coli GM271 (c/cm-d) was digested with Clal. The resulting 638 bp fragment (Figure 1) was purified and digested with Sau981. Gel shift assays with fraction II-3 (Table 1) were performed as described in Experimental Procedures.

The particular sequences of L-ori bound by the 33 kd protein were revealed by footprinting based on DNAase I protection (Figure 6). The protein symmetrically protected 55 nucleotides on each strand, including the three 13mers and short flanking sequences on each side. The T residue of the 5’-GATC-3’ in the middle 13-mer (Figure 7) was not protected from DNAase I (Figure 6). Protection of regions to the right and left of the 13-mers was inconsistent and probably artifactual. Protection of 50% to all of the 19mers (15-30 fmol) was obtained with 5-10 ng (150-300 fmol of a monomer) of the protein. These calculations suggest a ratio of 10 monomers per fragment. The higher value of 24 determined in the gel shift assay (Figure 5) may be caused by the lower concentration of fragments and the presence of a huge excess of poly(dl)poly(dC) introduced as competitor DNA (Experimental Procedures).

tarded by 1.25 ng of 33 kd protein (Figure 5A) was reduced by 10%. Further cleavage of the cloned L-ori fragment by BamHl yielded three fragment8 (Figure 1): a 91 bp fragment containing the 18mers, a 51 bp fragment to the left, and a 68 bp fragment to the right of the 91 bp fragment. Only the 91 bp fragment containing the 13-mers was bound by the protein. Based on the amount of fragment shifted, the binding ratio of the protein to each corresponded to about 24 monomers. For example, the shift of about 0.75 fmol of the L-ori fragment required about 18 fmol of monomers.

Table 1. Purification

of the L-ori Binding

Protein

Volume

I.

Lysate 11-l. Ammonium sulfate 11-2. Ammonium sulfate k-3. Ammonium sulfate III. Heparin-agarose IV. Fast-flow Q Sepharose V. Phosphocellulose VI. Mono S The L-ori binding

protein

880 190 50 35 132 14 0.92 0.8

was purified

from

(ml)

Protein

(mg)

17,600 4,980 1,940 1,140 46.2 2.0 0.17 0.10

E. coli W3110

Activity

(U x 10m3)

3,520 9,860 4,070 2,960 1,570 980 370 265

as described

in Experimental

Specific 0.2 1.9 2.1 2.6 34 490 2,180 2,650 Procedures

Activity

(U x lo-Ymg)

Yield (%)

100 42 31 18 10

3.8 2.8

Inhibitor 327

of Minichromosome

Initiation

A 33 kDa PROTEIN

B

(ng)

0

10

20

40

*.

z& r 3

10 40 0 20 ..i”--.

2.2 l.O-

.:,

g@-“-

9-mer

Figure

4. Gel Fitration

of the 33 kd Protein

13-mers

The protein (fraction VI, 11,600 U) in 0.1 ml of buffer B containing 0.15 M KCI was applied to a Superose 12 gel filtration column (FPLC) and &ted with buffer B containing 0.15 M KCI at a flow rate of 0.3 mllmin. Fractions (0.3 ml) were collected and diluted B-fold in buffer B containing 0.15 M KCI; the L-ori fragment binding activities in 0.5 ul were determined as in Figure 2. Molecular weight markers were blue dextran (BD), ferritin (440 kd), catalase (236 kd), aldolase (156 kd), bovine serum albumin (66 kd). ovalbumin (45 kd), and cytochrome c (12 kd).

RI [r

1 M 1 r* ..“xl ..*s1R

I

L

M

L

-#

-

L

1RI

The 33 kd Protein Inhibits Replication of the or/C Piasmid Replication of oriC plasmids can be reconstituted from purified proteins in the absence of RNA polymerase (Ogawa et al., 1985) but transcriptional activitation by RNA poly merase is required at elevated levels of HU protein (van der Ende et al., 1985; Baker and Kornberg, 1988). With each of these replication systems, without or with RNA polymerase, the 33 kd protein was profoundly inhibitory. Elution of the inhibitory activity from the Mono S column (fraction VI) coincided precisely with that of 13-mer binding activity and protein (Figure 8). To inhibit the replication of 83 fmol of pBSoriC by 60% (i.e., 50 fmol), 450 fmol of the 33 kd protein was required. A ratio of 18 monomers can be calculated, a value near those observed for the binding of the 13-mer fragment (Figures 4 and 5).

al., 1984) the 13-mer region is opened and then admits the entry of dnaB helicase to form the prepriming complex. The 33 kd protein can block formation of the open complex (Figure 9A), but has no effect once this stage has been achieved (Figures 9B and 9C).

Inhibition by the 33 kd Protein Precedes Formation of the Open Complex Initiation of oriC replication can be staged as a succession of three complexes: initial, open, and prepriming (Sekimizu et al., 1988; Bramhill and Kornberg, 198813). Following the initial binding of oriC by dnaA protein (Fuller et

The 33 kd Protein Prevents Opening of the 13-mer Region As Demonstrated by Sensitivity to Pi Endonuclease Cleavage Melting of aduplex region can be probed by single-strandspecific endonucleases, such as Pl (Kowalski, 1984). Pl endonuclease had revealed the opening of the 19mer re-

A 33 kDa PROTEIN

R-ori

(ng)

-

L-ori gid -

0

+ Q I+ c, 0’ 0’ 4’ ‘L’

B 0

b 91 66 51 -

&.

.. ;isc..

-

Figure 6. The 33 kd Protein Protects Digestion

the 13mer

Region from DNAase

I

A “P-labeled Hindlll-EcoRI restriction fragment of plasmid pXMA2 wasdigested with (A) Smal and (B) Pstl. DNAase I digestions were performed as described in Experimental Procedures. Maxam-Gilbert sequencing of the same fragments localized the protected region.

%I a 4? G, 0 0’ 0’ h’ CL; +

Figure 5. Preferential tein to a Fragment Region

Binding of the 33 kd ProContaining the 13-mer

The purified protein was used in the gel shift assay with (A) Sau961 cleavage of the Clal restriction fragment (636 bp); or(B) BamHl cleavage of the Xbal restriction fragment (210 bp) purified from plasmid pXMA2 (Experimental Procedures and Figure 1).

Cdl 326

Figure 7. Symmetric tein to the 13-mers

Binding

of the 33 kd Pro-

The nucleotides protected by the 33 kd protein from DNAase I digestion, as observed in Figure 6. are bracketed. The three Id-mers (L, M, and R from left to right) are underlined. Nucleotide numbering starts from the 5’end of the oriC sequence.

gion at the initiation stage of oriC plasmid (Bramhill and Kornberg, 1988b). Because cleavage throughout the plasmid creates a background noise level, a primer extension (see Experimental Procedures) was employed

replication significant rather high technique to monitor

= E 2 z H ii 6 5

0.4

0.2

0 800

,

1

breaks localized in the oriC region. In brief, a reaction mixture at a given stage was digested briefly with Pl nuclease, after which the reaction was stopped and the DNA denatured with sodium hydroxide. A 32P-labeled synthetic oligonucleotide (16-mer) complementary to a sequence to the left of the oriC region was annealed and served as a primer for extension by the DNA polymerase I large fragment. Synthesis terminated wherever the template had been cleaved by Pl nuclease (Figure 10). Little endonuclease cleavage was observed in the 13mer region in the absence of dnaA protein, but was prominent in the 60 nucleotide 18mer region when dnaA protein was present (Figure lo), indicative of formation of the open complex. In addition, opening in the vicinity of the 9-mer Rl was observed, indicating a previously undetected conformational change in oriC (Bramhill and Kornberg, 1988b); an opening this small would not be expected to provide enough room for the entry of dnaB helicase to form prepriming complexes. When the 33 kd protein was added before dnaA protein action, cleavages in the 13mers and Rl region were prevented (Figure 10). However, the 33 kd protein had no effect if added after the dnaA protein had acted (Figure 10). Thus, inferences from the effects of the 33 kd protein observed in the staged reactions (Figure 9) are confirmed directly by this endonuclease probing. Discussion

o-

14

16

18

20

22

FRACTIONS Figure

6. The 33 kd Protein

Inhibits

oriC Plasmid

Replication

The Mono S column fractions (Table 1) were assayed for (A) protein concentration and salt gradient, (6) L-ori fragment binding activity by gel shift, (C) inhibition of solo primase oriC plasmid replication (Experimental Procedures using 0.125 ul of each fraction), and (D) inhibition of RNA polymerase-plus-primase reconstituted oriC plasmid replication (Experimental Procedures using 0.125 PI of each fraction).

Three tandem repeats of a 13-mer are highly conserved in the chromosomal replication origins of gram-negative bacteria (Zyskind et al., 1983). Located next to the sites of binding by dnaA protein (the initiator protein), the 13-mer region becomes the locus for melting the duplex to admit the dnaB helicases that enlarge the opening and set the stage for priming of bidirectional replication (Bramhill and Kornberg, 1988b). The cell cycle in E. coli is regulated at the stage of initiation (von Meyenburg and Hansen, 1987; Skarstad et al., 1986) and it is at this point that one might expect positive and negative effecters to exert their actions. By the use of a gel shift assay, we have discovered and purified a 33 kd E. coli protein that binds the 13-mer region with high specificity and blocks the opening of this region. Once the 13-mers are opened, the 33 kd protein has no effect on the subsequent events in replication of the oriC plasmid. Thus, we find in our oriC replication system, reconstituted with purified proteins, that the 13-mer region is the locus of the initial melting of the duplex by the action of dnaA protein and a site for binding of a protein that blocks this early event.

Inhibitor 329

of Minichromosome

Initiation

BEFORE

33 kDa dnaA 33 kDa

dnaA

9-mer

-

Rl

-

- - 18377575 - - -

62

62 62 75 - -

ng

C

R’ LL -

-\. \

M

13-mers

1

\ ‘\,BEFORE \\

dnaA

L

\

1

[

Lanes

I

‘,\ L,IBEFOAE -5

25

PREPRIMING -em,

--_.

--s_

-*

123456

78

Figure 10. The 33 kd Protein Blocks Open Complex Formation As Judged by Pl Nuclease Cleavage Reaction mixtures for open complex formation were incubated at 3PC for 20 min. All lanes except lane 1 contained 8.4 ng of protein HU. In lane 6, the 33 kd protein was added after a 10 min incubation that was then continued 10 min more; in lane 7, the dnaA protein was added after a 10 min incubation that was then continued for 10 min more. Pl nuclease cleavage was performed as described in Experimental Procedures. The Pl nuclease cleavage sites were localized by dideoxy sequencing of plasmid pBSoriC with the same primer used in the primer extension.

t 0

62.5 33 kDa

PROTEIN

125 (ng)

Figure 9. The 33 kd Protein Blocks Open Complex Formation in Staged Reactions The protein was added before or after dnaA protein in each of several staged reactions of oriC plasmid replicatron as described in Experimental Procedures. (A) Initial complex formation; (6) open complex formation; and (C) prepriming complex formation.

fold or more, suggestive of a negative effector role for the protein in vivo. Manipulation of a unique genetic locus, identified by hybridization with oligonucleotides based on an amino acid sequence of the 33 kd protein, should also contribute to an understanding of how this protein participates in the regulation of chromosome initiation. Experimental

In current studies, certain base pair substitutions in the leftmost 18mer that have little effect on initiation of replication by the purified enzyme system nevertheless profoundly alter the binding and inhibitory activities of the 33 kd protein. For example, inversion of AT base pairs (retaining their number and position in the 13-mer) virtually eliminated binding by the 33 kd protein. Remarkably, the copy number of such mutant oriC plasmids was increased lo-

Procedures

Reagents Sources were: ATP GTP CTP, UTP CNBr, and heparin (sodium salt grade I), Sigma; dNTPs, poly(dl)-poly(dC), HEPES, Fast-flow Q Sepharose, FPLC Mono S HR 5/5. and Superose 12 HR 10/30, Pharmacia LKB Biotechnology, Inc.; Pll-phosphocellulose, Whatman; BioGel A-15m (agarose) and Bradford reagent, Bio-Rad; [ae3*P]dTTP (800 Cilmmol) and (Y-~~P]ATP (6000 Cilmmol). Amersham Corp.; silver staining kit, ICN Inc; and ArdarmineZyeast extract, Champlain Inc. Buffer A: 25 mM HEPES-KOH (pH 7.6). 250 mM KCI, 20 mM spermidine-HCI, 1 mM EDTA, and 2 mM DTT. Buffer 6: 25 mM HEPES-KOH (pH 7.6) 0.1 mM EDTA, 2 mM OTT, and 15% (v/v) glycerol.

Cell 330

Proteins Replication proteins were purified as previously described (Kaguni and Kornberg, 1984) except for dnaA protein (Hwang et al., 1990), DNA polymerase III (Maki and Kornberg. 1988), and the p subunit of DNA polymerase Ill (Maki and Kornberg, 1988). Restriction endonucleases. T4 polynucleotide kinase, and DNA ligase were purchased from New England BioLabs; calf intestinal alkaline phosphatase was from Boehringer Mannheim; DNAase I was from BRL; gel filtration molecular weight markers, endonuclease Pl, and DNA polymerase I large fragment were from Pharmacia LKB Biotechnology, Inc.; bovine serum albumin (fraction V) was from Sigma; and SDS-polyacrylamide gel electrophoresis molecular weight markers were from Bio-Rad. Bacterial Strains and Plasmid DNAs E. coli W3110 (h-, /N[rrmD-rrnE]I) was used for purification of the 33 kd protein. DH5a ((p80d lacZAM15 endAl recA7 hsdRi7 Irk- mk+] supf44 N-7 i;- gyrA96 relA7 4/acZYA-argf]U769) was used as a host for plasmid preparations. GM271 (ara-14 leu66 fhuA37 lacy7 fsx-78 supf44 galK2 galT22 h- dun-6 hisG4 rfbD7 rpsL736 xyl-5 mtl-7 thi-7 hsdR2 mcrA- mem-) was the host for plasmid pBSoriC DNA prepared for cleavage by Sau961 restriction enzyme. Unless indicated, plasmid DNAs were isolated from DH5a and manipulated as described (Sambrook et al., 1989). Plasmid pBSoriC was previously described (Bramhill and Kornberg, 1988b). Plasmid pXMA2 was constructed by filling the ends of the L-ori fragment (198 bp, Figure 1) with DNA polymerase I large fragment, then cloning the resulting blunt fragment into the Xbal site of plasmid pUC18 using the linker 5’-TGCTCTAGAGCA-3’. The 5’end of the 13-mer L in the plasmid pXMA2 is closest to the Sal1 site of the polylinker. Gel Shift Assay The indicated DNA fragments were dephosphorylated with calf intestinal alkaline phosphatase and 5’end-labeled with [y3*P]ATP using T4 polynucleotide kinase. The gel shift reaction mixture (20 ~1) contained 10 mM Tris-HCI (pH 7.5), 1 mM EDTA, 75 mM KCI, 2 mM DTT, 10% (v/v) glycerol, 2 pg of poly(dl)-poly(dC), 1.5 fmol of 32P-labeled fragments, and the indicated amounts of protein. Reactions were incubated at 30°C for 30 min and subjected to electrophoresis through a 5% polyacrylamide gel at 100 V for l-2 hr in 45 mM Tris-borate (pH 8.3) and 1 mM EDTA. The gel was dried and visualized by autoradiography. The radiolabeled fragments were cut out from the dried gel and quantitated in a liquid scintillation counter. Purification of the 33 kd Protein E. coli W3110 was grown in a Chemap 300 liter fermentor at 37% in 200 liters of a medium containing 2.2 kg of Bacto Trypton, 4.5 kg of Ardamine 2 yeast extract, 2 kg of glucose, 0.8 kg of KH2P04, and 3.3 kg of K2HP04 (pH 7.4) to an ODGoo value of 15. The pH was maintained and three 2 kg additions of glucose were made during the run. The cells were harvested by centrifugation in a Sharples, resuspended to an OD,joo value of 600 in 50 mM Tris-HCI (pH 7.5) and 10% (w/v) sucrose, and then frozen in liquid nitrogen. Thawed cell paste (700 g) was diluted to 1.4 liters with 25 mM HEPES-KOH (pH 7.6), 1 mM EDTA. and 2 mM DTT then additions were made of 3 M KCI to 0.25 M, 1 M spermidine-HCI to 20 mM, and 20 mg/ml lysozyme to 0.4 mglml. The suspension was incubated at 37°C for 3 min followed by further incubation on Ice for 30 min, frozen in liquid nitrogen, and thawed at 8% lo lyse the cells. The thawed sample was centrifuged for 20 min at 40,000 rpm In a Beckman Ti45 rotor. Operations at this step and beyond were at O”C-4% unless noted. The supernatant (fraction I) was precipitated by addition of solid ammonium sulfate (0.24 g per ml of supernatant) with stirring. After an additional 30 min of stirring, the suspension was centrifuged for 20 min at 13,000 rpm in a Sorvall GSA rotor, the precipitate was resuspended in 140 ml of buffer A (fraction II-l), then 29.4 g of ammonium sulfate was added, stirred, and centrifuged as described above. The precipitate was resuspended in 30 ml of buffer A (fraction 11-2). and 5.7 g of ammonium sulfate was added, stirred, and centrifuged as described above. The resulting precipitate was resuspended with buffer B, cleared by centrifugation (fraction II-3), diluted to a conductivity equivalent lo buffer B containing 50 mM KCI, and applied to a heparin-agarose column (bed volume, 110 ml; Davison et al., 1979) equilibrated in buffer B containing 50 mM KCI.

The column was washed with 400 ml of buffer B containing 50 mM KCI and the protein eluted with a linear gradtent of 1 .l liters of 50-500 mM KCI in buffer B. Fractions with L-ori fragment bindlng activity. eluted in a peak between 100 and 150 mM KCI, were pooled (fraction Ill), diluted with buffer B to a conductlvlty equivalent 10 60 mM KCI In buffer B, loaded onto a Fast-flow Q Sepharose column (bed volume, 10 ml) equilibrated with 50 mM KCI in buffer B, washed with 50 ml of 50 mM KCI in buffer B, and eluded with a linear gradient of 100 ml of 50-600 mM KCI in buffer B. The active fractions, eluted in a peak between 100 and 170 mM KCI. were pooled (fraction IV), diluted with buffer B to a conductivity equlvalent to 70 mM KCI in buffer 8, loaded onto a phosphocellulose column (P-11; bed volume, 1 ml), equilibrated with 50 mM KCI In buffer 8, washed with 3 ml of 50 mM KCI in buffer B, and eluted with 20 ml of 50 mM-1M KCI in buffer B. The active fractions were pooled (fraction V), diluted to a conductivity equivalent to 70 mM KCI in buffer 8, applied to a Mono S HR 5/5 column (FPLC) equilibrated with 50 mM KCI in buffer B, and washed with 3 ml of 50 mM KCI in buffer B. The L-on fragment binding activity was eluded with a ltnear gradient of 10 ml of 50 mM to 1.25 M KCI In buffer B and collected as 0.3 ml fractions. The L-on fragment binding activity was eluted in a single peak at about 0.18 M KCI (fraction VI) Unless indicated, the Mono S column fraction 17 was used for the reported experiments. Protein concentration in the Mono S column fraction 17 was determined by the Bradford method and confirmed by amino acid analysis. One unit of 33 kd protein activity shifts one-fourth of the input L-ori fragment (1.5 fmol) DNAase I Footprinting The Hindlll-EcoRI restriction fragment (260 bp) from plasmid pXMA2 was isolated, dephosphorylated with calf intestinal alkaline phosphatase, and labeled with (v-~*P]ATP using T4 polynucleotide kinase. The 5’ end-labeled fragment was cleaved with Smal or Pstl and used for DNAase I footprinting. The footprinting reactions included 10 mM Tris-HCI (pH 7.6), 50 mM KCI, 10 mM MgCIZ, 2 mM DTT, 10% (v/v) glycerol, 30 fmol of fragment, and the indicated amounts of 33 kd protein. After incubation at 30% for 10 min, 2 ng of DNAase I in 1 ~1 of Hz0 was added, incubated at 23°C for I min, and stopped by addition of 40 ~1 of 20 mM EDTA, 7% SDS, 0.2 M NaCI, and 250 wglml yeast tRNA. The mixture was precipitated with ethanol, washed with 70% ethanol, and subjected to electrophoresis through a 6% polyacrylamide buffer gradient sequencing gel containing 7 M urea. Inhibition of Reconstituted oriC Plasmid Replication by the 33 kd Protein The standard reaction (15 ~1) containing 40 mM HEPES-KOH (pH 7.6). 50 mM potassium glutamate, 0.3 mM EDTA, 17% (v/v) glycerol, 200 ng of plasmid pBSoriC, and the indicated Mono S column fractions was assembled at 0% and incubated at 30% for 15 min; subsequently, a mixture (10 PI) that sustains the solo primase or the RNA polymeraseplus-primase reconstituted oriC plasmid replication (Ogawa et al., 1985; van der Ende et al., 1985) was added. The solo primase mixture (10 ~1) contained 40 mM HEPES-KOH (pH 7.6); 25 mM magnesium acetate; 5 mM ATP; 1.25 mM each CTP, UTP, and GTP; 250 NM each dATP, dCTP and dGTP; 250 PM [a-32P]dTTP (-50 cpmlpmol); 300 ng of gyrase A subunit; 150 ng of gyrase B subunit; 8.4 ng of protein HU; 75 ng of dnaA protein; 60 ng of dnaB protein; 20 ng of dnaC protein; 400 ng of SSB protein; 10 ng of primase; 70 ng of DNA polymerase Ill* ; and 75 ng of a subunit of DNA polymerase Ill. The RNA poly merase-plus-primase mixture contained 140 ng of protein HU. 0.2 ng of RNAase H, and 160 ng of RNA polymerase in addition to the components in the solo primase mixture. The DNA synthesis reaction was incubated at 30°C for 10 min for the solo primase reaction or 20 min for the RNA polymerase-plus-primase reaction and treated as described (Kaguni and Kornberg, 1984). Staged Reactions of Initiation of oriC Plasmid Replication The initial-complex-formation mixture (15 &I) contained 40 mM HEPESKOH (pH 7.6), 0.3 mglml bovine serum albumin, 50 mM potassium glutamate, 0.3 mM EDTA, 17% (v/v) glycerol, 0.6 mM magnesium acetate, 10 pM ATF’, and 75 ng of dnaA protein (or the indicated amount of 33 kd protein) and was incubated at 0°C for 10 min followed by the addition of the indicated amount of 33 kd protein (or 75 ng of dnaA pro-

Inhibitor 331

of Minichromosome

Initiation

tein). The mixture was further incubated at O°C for 10 min. The omitted components required for solo primase reconstituted DNA synthesis (10 ~1) were added and incubated at 3oOC for 5 min. DNA synthesis was measured as described (Kaguni and Kornberg, 1984). The open-complex-formation mixture (15 ul) containing the initial complex formation mixture, 6.4 ng of protein HU, and 4 mM ATP was incubated at 3PC for 10 min, followed by the addition of the indicated amount of 33 kd protein (or 75 ng of dnaA protein), and further incubated at 3PC for 10 min; the next stages were performed as described above. The prepriming-complex-formation mixture (15 ul) containing the open complex formation mixture, 60 ng of dnaB protein, and 20 ng of dnaC protein was incubated at 3pC for 10 min followed by the addition of the indicated amount of 33 kd protein (or 75 ng of dnaA protein); the next stages were performed as described above. Pl Nuclease Cleavage and Primer Extension Open-complex-formation mixture (25 ul) was incubated at 3PC for 20 min; Pl nuclease (0.45 U in 1.5 ul of 30 mM sodium acetate, pH 5.5) was added and incubated at 37°C for 30 s. The cleavage was stopped by the addition of 27 ul of 25 mM EDTA, 0.4 M NaOH and incubated at room temperature for 10 min followed by the addition of 6 MI of 3 M sodium acetate (pH 4.8). Proteins were removed by phenol-chloroform extraction (1 vol/l vol). DNA was precipitated with ethanol, washed with 70% ethanol, and resuspended in 10 ul of Klenow buffer (50 mM Tris-HCI [pH 7.21. 10 mM MgCIs. and 2 mM DTT) containing 0.2 pmol of the 32P-5’ end-labeled synthetic oligonucleotide 5’-TTGAAGCCCGGGCCGT-3’, complementary to the region of the Smal site to the left of oriC (Buhk and Messer, 1983) located 59 bp away from the left-most 19mer. The resuspended mixture was incubated at 90DC for 3 min. shifted to 65’C for 3 min, then allowed to cool slowly to 35OC. Klenow buffer (10 ~1) containing dATP dTTP, dCTP and dGTP, each at 2.5 mM, was added, and the annealed primer was extended with 1.4 U of DNA polymerase I large fragment at 3PC for 10 min. The reaction was stopped by the addition of 10 ~1 of 60 mM EDTA and 1 M sodium acetate (pH 5.5) precipitated with ethanol, washed with 70% ethanol, and subjected to electrophoresis through a 6% polyacrylamide sequencing gel containing 7 M urea. Acknowledgments This research was supported by grants from 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 16 USC Section 1734 solely to indicate this fact. Received

July 10, 1990; revised

August

2, 1990

References Baker, T. A., and Kornberg. A. (1966). Transcriptional activation of initiation of replication from the E. coli chromosomal origin: an RNA-DNA hybrid near oriC. Cell 55, 113-123. Bramhill, D., and Kornberg, A. (1966a). of DNA replication. Cell 54, 915-918.

A model for initiation

at origins

Bramhill, D., and Kornberg, A. (1988b). Duplex opening by dnaA protein at novel sequences in initiation of replication at the origin of the E. coli chromosome. Cell 52, 743-755. Buhk, H.-J., and Messer, W. (1983). The replication Escherichia coli: nucleotide sequence and functional 265-279.

origin region of units. Gene 24,

Davison, B. L., Leighton, T., and Rabinowitz, J. C. (1979). Purification of Bacillus subtilis RNA polymerase with heparin-agarose. In vitro transcription of rp29 DNA. J. Biol. Chem. 254, 9220-9226. Fuller, R. S., Funnell. 8. E.. and Kornberg, A. (1984). The dnaA protein complex with the E. coli chromosomal replication origin (oriC) and other DNA sites. Cell 38, 889-900. Hwang. D. S., Crooke, E., and Kornberg, A. (1990). Aggregated dnaA protein is dissociated and activated for DNA replication by phospholipase or dnaK protein. J. Biol. Chem., in press.

Kaguni, J. M., and Kornberg, A. (1984). Replication gin (oriC) of the E. coli chromosome reconstituted zymes. Cell 38, 163-190. Kornberg, A. (1982). Supplement W. H. Freeman and Co.).

to DNA Replication

imtiated at the oriwith purified en(San Francisco:

Kowalski, D. (1984). Changes in site specificity of single-strand-specific endonucleases on supercoiled PM2 DNA with temperature and ionic environment. Nucl. Acids Res. 12, 7071-7086. Lebner-Olesen, A., Skarstad, K., Hansen, F. G., von Meyenburg, K., and Boye, E. (1969). The dnaA protein determines the initiation mass of Escherichia coli K-12. Cell 57, 661-669. Maki, S., and Kornberg, A. (1986). DNA polymerase Ill holoenzyme of Escherichia coli. II. A novel complex including they subunit essential for processive synthesis. J. Biol. Chem. 263, 6555-6560. Ogawa, T., Baker, T A., van der Ende, A., and Kornberg, A. (1985). Initiation of enzymatic replication at the origin of the Escherichia coli chromosome: contributions of RNA polymerase and primase. Proc. Natl. Acad. Sci. USA 82, 3562-3566. Ogden, G. B., Pratt, M. J., and Schaechter, M. (1986). The replicative origin of the E. coli chromosome binds to cell membranes only when hemimethylated. Cell 54, 127-135. Russell, D. W., and Zinder, N. D. (1967). Hemimethylation DNA replication in E. coli. Cell 50, 1071-1079.

prevents

Sambrook, J., Fritsch, E. F., and Maniatis. T. (1969). Molecular Cloning: A Laboratory Manual. Second Edition. (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory). Sekimizu. K., Bramhill. D., and Kornberg, A. (1988). Sequential early stages in the in vitro initiation of replication at the origin of the Escherichia coli chromosome. J. Biol. Chem. 263, 7124-7130. Skarstad, K., Boye, E., and Steen, H. B. (1986). Timing of initiation of chromosome replication in individual Escherichia coli cells. EMBO J. 5, 17ll-l7l7. Skarstad, K., Baker, T. A., and Kornberg, A. (1990). Strand separation required for initiation of replication at the chromosomal origin of E. coli is facilitated by a distant RNA-DNA hybrid. EMBO J. 9. 2341-2348. van der Ende, A., Baker, T. A., Ogawa, T., and Kornberg, A. (1985). Initiation of enzymatic replication at the origin of the Escherichia coli chromosome: primase as the sole priming enzyme. Proc. Natl. Acad. Sci. USA 82, 3954-3958. von Meyenburg. K., and Hansen, F. G. (1987). Regulation of chromosome replication. In Escherichia coli and Salmonella typhimurium. Cellular and Molecular Biology, Vol. 2, F. C. Neidhardt, ed. (Washing ton, DC: American Society for Microbiology), pp. 1555-1577. Yung, B. Y.-M., and Kornberg, A. (1988). Membrane attachment activates dnaA protein, the initiaiion protein of chromosome replication in Escherichia coli. Proc. Natl. Acad. Sci. USA 85, 7202-7205. Zyskind. J. W., Cleary, J. M., Brusilow, W. S. A., Harding, N. E., and Smith, D. W. (1983). Chromosomal replication origin from the marine bacterium Vibrio harveyi functions in Escherichia coli: oriC consensus sequence. Proc. Natl. Acad. Sci. USA 80, 1164-1168.

A novel protein binds a key origin sequence to block replication of an E. coli minichromosome.

A sequence of three tandem repeats of a 13-mer in the replication origin (oriC) of E. coli is the highly conserved site of opening of the duplex for i...
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