THE JOURNAL
Q 1992 by The

OF BIOLOGICAL CHEMISTRY

American Society for Biochemistry and Molecular Biology,

Inc.

Vol. 267, No. 13, Issue of May 5, PP. 8778-8784 1992
            Printed in fi.S.~.

Features of Replication Fork Blockage by the Escherichia coli
Terminus-binding Protein*

(Received for publication, July 15, 1991)

Eui Hum Lee and Arthur Kornberg
From the Department of Biochembtry, Stanford University School of Medicine, Stanford, California 94305-5306

Blockage of the progress of a DNA replication fork
in Escherichia coli can be ascribed to an inhibition of
helicase action at the orientation-specific binding of a
termination sequence (ter) by the ter-binding protein
(Lee, E. H., Kornberg, A., Hidaka, M., Kobayashi, T.,
and Horiuchi, T. (1989) Proc. Natl. Acd. Sci. U. S. A.
86, 9104-9108). These observations have been ex-
tended to include the PriA helicase, thus confirming
that blockage is general for helicases. The site of arrest
of synthesis by a replication fork is at the very first
nucleotide of the 22-base pair E. coli-terB sequence.
Strand displacement by DNA polymerases is also in-
hibited, but is less profound and is orientation-specific.
The ter sequences of plasmids R1-terR and -terL and
of plasmids R6K and RlOO have been compared with
those of E. coli-terA and -terB.

In the replication of the circular Escherichia coli chromo-
some, two forks move bidirectionaily from the unique origin
(oriC) and terminate in a region that contains five terminus
(ter) sites that are inverted repeats of a 22-bp' sequence (1,
2). Binding of ter by the E. coli terminus-binding protein
(TBP), encoded by the tau (or tus) gene, is essential (3-5).
One orientation of the bound sequences (terB and terC) blocks
the clockwise (CW)-moving fork, whereas the other sequences
(terA, terD, and terE) block the counterclockwise (CCW) fork
(2).
Homologous functional and structural ter sites have also
been demonstrated in E. coli plasmids R6K (6, 7), R1 (7, S),
and RlOO (8) and in Bacillus subtilis (9, 10). Orientation-
specific blockage of the replication forks observed in vivo (3,
4,7,8) has been observed with a reconstitutedpurified enzyme
system for replication of the minichromosomal oriC plasmid
(11). The component in the system most immediately affected
is the DnaB helicase (12, 13). However, the obstruction to
helicase action appears to be general inasmuch as two other
helicases are also blocked in an orientation-specific manner
(11).
This study was undertaken to explore other features of the
TBP-ter complex (its capacity to block the action of the PriA
helicase and strand displacement by DNA polymerases), to
determine precisely the location of the blockage in or near
the bound ter sequence, and to compare the relative blocking
strengths of the various ter sequences.

* This work was supported by Grants GM07581 and AGO2908 from
the National Institutes of Health. The costs of publication of this
article were defrayed in part by the payment of page charges. This
article must therefore be hereby marked ``aduertkement" in accord-
ance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: bp, base pair(s); TBP, terminus-
binding protein; CW, clockwise; CCW, counterclockwise; SDS, so-
dium dodecyl sulfate; Hepes, 4-(2-hydroxyethyl)-l-piperazineeth-
anesulfonic acid; kb, kilobase(s).

EXPERIMENTAL PROCEDURES

Strains, Enzymes, and Other Reagents-E. coli strains used were
DH5a (F-, @801acZA115, endA1, recA1, hrs-, hsm+, supE44, thi-1,
gyrA, A-, A(lacZYA-ArgF), U169) and JM83 (aru, A(lac-proAB), rpsL,
@801acZAl15). Restriction endonucleases were AccI, ClaI, DruI,
EcoRV, NaeI, PstI, Smd, SfzT, SacI, SacII, and SnaBI; and other
enzymes used were T4 DNA ligase, T4 DNA polymerase, T4 poly-
nucleotide kinase, and DNA polymerase I (large fragment), all from
New England BioLabs, Inc. Calf intestine alkaline phosphatase was
from Boehringer Mannheim. T7 DNA polymerase was from U. S.
Biochemical Corp. T5 DNA polymerase was a gift from Dr. R. K.
Fujimura (Oak Ridge National Laboratory). The enzymes used in the
oriC reconstitution assay were as described (11, 12, 14). Other re-
agents were unlabeled deoxynucleoside and ribonucleoside triphos-
phates (Pharmacia LKB Biotechnology Inc.(, [Y-~'P]ATP (6000 Ci/
mmol, 1 Ci = 38 GBq) and [a-32P]dTTP (800 Ci/mmol) (Amersham
Corp.), and bovine serum albumin (Pentex). TBP buffer contained
550 mM NaC1, 50 mM Tris-HC1 (pH 7.5), 20% (v/v) glycerol, 1 mM
EDTA, and 1 mM dithiothreitol.
Plnsmids, Phage DNA, and Synthetic Oligonucleotides-Plasmids
pTZ18R and pTZ19R were from Pharmacia. pTBlOl contains a 678-
bp HincII-PstI fragment spanning oriC (positions -189 to +489) of
pCM959 (15) cloned in the pBluescript vector (Stratagene). Plasmid
pKHG300 (obtained from Dr. Horiuchi, Kyushu University, Fukuoka,
Japan) is a pUC9-based vector carrying the tau gene and overproduces
TBP >2000-fold (3). M13mp18 and M13mp19 replicative form DNAs
were from New England BioLabs, Inc. Ml3mpl8.ter CCW and
MlSmpl9.ter CW DNAs were as described (11). Synthetic oligomers
were synthesized by Alan Smith in the Protein and Nucleic Acid
Facility at Stanford University.
In Vitro oriC Reconstitution Assay: Coupled and Staged-The cou-
pled reconstituted oriC DNA replication reaction was essentially as
described (11). In brief, the reaction mixture (40 pl) contained 30 mM
Hepes/KOH (pH 7.6), 8 mM magnesium acetate, 2 mM ATP, template
DNA (600 pmol as nucleotide), 0.3 mM each CTP, GTP, and UTP,
0.1 mM each MTP, dCTP, dGTP, and dTTP, 16 nM [(Y-~'P]TTP
(100-300 cpm/pmol), 150 mM potassium glutamate, 80 ng of DnaA,
900 ng of E. coli single-stranded DNA-binding protein, 8 ng of DnaB,
27 ng of DnaC, 50 ng of gyrase A, 30 ng of gyrase B, 8 ng of HU
protein, 17 ng of primase, 100 ng of DNA polymerase I11 holoenzyme,
and 26 ng of the 8-subunit of DNA polymerase I11 holoenzyme. After
incubation at 30 "C for 30 min, reactions were stopped by adding 100
p1 of 100 mM pyrophosphate containing 20 pg/ml carrier DNA (calf
thymus). The extent of DNA synthesis was measured by liquid
scintillation counting after precipitation with 10% (w/v) trichloroa-
cetic acid and filtration on Whatman GF/C glass filters.
Progress of bidirectional DNA replication was assayed in a staged
reaction. The prepriming complex was formed in 20 p1 containing 40
mM Hepes/KOH (pH 7.6), 15% (w/v) glycerol, 200 pg/ml bovine
serum albumin, 0.007% Brij 58, 0.25 mM EDTA, 200 mM potassium
glutamate, 3.3 mM ATP, template DNA (600 pmol as nucleotide), 8
ng of HU protein, 8 ng of DnaB, 27 ng of DnaC, and 80 ng of DnaA.
After 20 min at 37 `C followed by 2 min at 18 "C, 20 ng of TBP (or
TBP buffer as a control) was added and incubated for 1 min. The
omitted replication components (primase, gyrases A, and B, polp-
erase I11 holoenzyme, @-subunits, E. coli single-stranded DNA-bind-
ing protein, GTP, CTP, UTP, dATP, dCTP, dGTP, dTTP, and [a-
32P]dTTP), in a volume of 10 p1 that also contained magnesium
acetate at 10 mM and potassium glutamate at 200 mM, were added.
Incubation was continued for 2,4, and 6 min; and the reactions were
stopped by adding EDTA to 20 mM. A small portion of each sample

8778

Replication Fork Blockage in E. coli

8779

was used to determine DNA synthesis as described above. The re-
mainders of the samples were extracted twice with phenol and twice
with chloroform and isoamyl alcohol, (24:l) and precipitated with
ethanol. The DNA pellets were redissolved in buffer containing 10
mM Tris-HC1 (pH 8.0), 0.2 mM EDTA and digested with the desired
restriction endonucleases following the manufacturer's instructions.
One unit of DNA replication activity promotes the incorporation of
1 pmol of nucleotide/min at 30 "C.
purification of TBP-All operations were at 4 "C. E. coli strain
JM83 (pKHG300) (3) was grown in 1.5 liters of L-broth to optical
density (AGoonm) of 0.5, harvested by centrifugation in a Beckman JA-
10 rotor for 10 min at 6000 rpm, and frozen ii liquid nitrogen; frozen
cells were resuspended in 20 ml of lysis buffer containing 50 mM
Tris-HCl (pH 7.5), 10% sucrose, 5 mM dithiothreitol, 10 mM EDTA,
100 mM NaCl, 20 mM spermidine HCl (pH 7.5), (NHJ2S04 to 5%
saturation, and 4 mg of lysozyme and were incubated on ice for 45
min. Cells were incubated at 37 "C for 5 min (inverting tubes once
every minute), incubated on ice for 10 min, and centrifuged in a
Somall SS-34 rotor for 1 h at 14,000 rpm (Fraction I, 20 ml).
Ammonium sulfate (0.313 g/ml) was added during a 30-min period to
Fraction I; stirring was continued for another 30 min. The precipitate
was collected by centrifugation in a Sorvall SS-34 rotor for 20 min at
18,000 rpm and resuspended in 4 ml of buffer A (50 mM Tris-HC1
:pH 7.5), 20 mM NaC1, 20% glycerol, 1 mM dithiothreitol, and 1 mM
EDTA) (Fraction 11, 4.2 ml). Fraction I1 was dialyzed against buffer
A overnight and applied at 7.2 ml/h to a 13-ml column (2.6 cm high,
2.5-cm diameter) of DEAE-Sephacel equilibrated with buffer A. Flow-
through fractions that contained TBP were collected (Fraction 111,
21.8 ml). Fraction I11 was applied at 7.2 ml/h to a 15-ml heparin-
agarose column (3.0 cm high, 2.5-cm diameter) equilibrated with
buffer A. The column was washed with 30 ml of buffer A plus 280
mM NaCl prior to elution using a linear gradient of 280-980 mM
NaCl in buffer A (45 ml). The peak of TBP activity collected in 12-
ml fractions eluted between 500 and 700 mM NaC1. Protein concen-
trations were measured by the Bradford assay (28). One unit of TBP
causes a 50% inhibition of replication of the oriC-ter CW.CCW
plasmid (Fig. IC) in uiuo in 1 min at 30 `C. The purification data are
shown in Table I.
Purified TBP is near homogeneity (>95%) as judged from COO-
massie Blue staining of an SDS-polyacrylamide electrophoresis gel
(16) and binds to the ter site in a 1:l molar stoichiometry ratio when
assayed by oriC reconstitution in uitro. Purified TBP does not inhibit
DNA replication of oriC plasmids lacking ter sites even at a 30-fold
molar excess.
Preparation of DNA Polymerase Substrates-A 64-mer oligonucle-
otide (40 ng) containing the complementary ter sequence was 5'-end-
labeled with 32P using T4 polynucleotide kinase (17) and mixed with
M13mp18. ter CCW or M13mp19. ter CW single-stranded DNA (10

in 100 pl of annealing buffer containing 10 mM Tris-HC1 (pH
8.0), 100 mM NaC1, and 1 mM EDTA. The mixtures were heated at
100 `C for 2 min, cooled slowly to 50 "C, and incubated for 1 h and
then cooled slowly to room temperature. The template, with the
Oligomer annealed to it, was purified by a Bio-Gel A-5 spun column
(171, extracted with phenol and chloroform, and precipitated with
ethanol. Annealing of the substrates was confirmed by electrophoresis
on a 0.8% agarose gel followed by autoradiography. Similarly, a 30-
mer (or 28-mer) oligonucleotide containing the ter sequence was
annealed to M13mp18.ter CCW (or Ml3mpl9-ter CW) DNA as
described above.

Assay for Strand Displacement of DNA Polymerases-The reaction
mixture (30 pl), containing 10 mM Tris-HC1 (pH 7.5), 50 mM NaC1,
O.1pg of DNA substrate (with annealed 5'-3ZP-end-labeled ter oligo-
mer, 6000 cpm), and 0.2 pg of M13 sequencing primer (17-mer), was
Incubated at 37 "C for 30 min and cooled to room temperature. The
reaction volume was brought to 100 p1 by adding 50 mM Tris-HC1
(pH 7.51, 10 mM MgS04, 1 mM dithiothreitol, 50 pg/ml bovine serum

   and 250 p~ each dATP, dCTP, dTTP, and dGTP. Samples
were divided into two portions. To one was added 30 ng of TBP; to
the other was added TBP buffer as a control. After incubation at
30."c for 1 min, DNA polymerase was added; and at indicated time

  Portions of each sample were transferred to an equal volume
Of "S~OP" solution containing 60 mM EDTA, 1% SDS, 20% glycerol,
0.02% bromphenol blue, and 0.02% xylene cyanol. Samples were

      on a nondenaturing 7% polyacrylamide gel, dried,
and autoradiographed. Slices of the dried gel that contained undis-
'laced (substrate) and displaced (oligomer) bands were cut out; their
'adloactivity was measured in a liquid scintillation counter; and the
Percentage of displaced oligonucleotide was calculated. The DNA

oriC-terCW oriC-terCWCCW oriGterCWCCW
E.coli-ter A RGK-terRterL E.coli-terB.terB
E.wMerB RI-terRterL

RGK-terR
RGK-ferL

RI-terR
RI-terL
~~o~tem
RlOOterL
BSubIih-terfI

FIG. 1. Construction of oriC-ter plasmids. Pairs of comple-
mentary synthetic oligomers were annealed to generate ter duplexes
with cohesive ends for ClaI and EcoRV restriction endonucleases. (i)
E. coli-terA, 5`-ATCAATTAGTATGTTGTAACTAAAGTAT-3' (28
mer) and 5'-CGATACTTTAGTTACAACATACTAATTGAT-3'
(30-mer); (ii) R100-terR, 5'-ATCATTATGAATGTTGTAACTAC-
TTCAT-3' (28-mer) and 5'-CGATGAAGTAGTTACAACATTCA-
TAATGAT-3` (30-mer); (iii) R100-terL, 5'-ATCTGTCTGAGTGT-
TGTAACTAAAGCAT-3' (28-mer) and 5'-CGATGCTTTAGTT-
ACAACACTCAGACAGAT-3` (30-mer); and (iu) B. subtilis-terII, 5`-
ATCATTGAATATTTAGTACATAGTGTAT-3' (28-mer) and 5'-C-
GATACACTATGTACTAAATATTCAATGAT-3' (30-mer). To gen-
erate a unique ClaI restriction site in pTBlOl DNA (15), the addi-
tional ClaI site (at nucleotide 764) was inactivated by T4 DNA
polymerase treatment after partial digestion of the plasmid DNA
with ClaI. a, the ter sequences listed were inserted into a plasmid
vector cleaved with ClaI and EcoRV restriction enzymes to generate
the oriC-ter CW plasmids. Other oriC-ter plasmids were constructed
by inserting the ter DNA fragments into pTBlOl DNA; E. coli-terB,
204-bp AccI-PstI DNA fragment of the oriC-ter CW plasmid (11);
R6K-terR, 74-bp AluI-HaeIII DNA fragment of pUC-R6K-ter (26);
R6K-terL, 142-bp AluI-HaeIII DNA fragment of pUC-RGK-ter, R1-
terR, 195-bp DraI-Sfir DNA fragment of pHM6050 (27); and R1-terL,
105-bp Sfir DNA fragment of pHM6050 (27). The above DNA frag-
ments containing ter sites were blunt-ended with T4 DNA polymerase
and inserted into the EcoRV site of pTBlOl DNA to generate oriC-
ter CW plasmids. b, the R6K-ter sites (terR and terL) containing the
216-bp AluI DNA fragment of the pUC-R6K-ter plasmid (26) and the
R1-ter sites (terR and terL) containing the 554-bp DraI-EcoRV DNA
fragment of pHM6050 (27) were isolated, blunt-ended, and inserted
into the NueI site of pTBlOl to generate oriC-ter CW. CCW plasmids.
c, the oriC-ter CW. CCW (E. coli-terB-terB) plasmid was constructed
by inserting the synthetic ter oligomers used for construction of oriC-
ter CCW plasmids into the oriC-ter CW plasmid (11) that was cleaved
with Sac1 and Sac11 restriction enzymes. The T-like symbol represents
a ter sequence oriented such that the head of the T should block the
progress of the replication fork.

   TABLE I
Purification of TBP

Total activity Specific activity Yield

protein

Steps

mg units x IOd units x 10-5/mg %

I. Lysate" 210

                 14 1.23 (100)
111. DEAE-Sephacel 11.8 11 9.3 78

11. Ammonium sulfate 113

IV. Heparin-agarose 0.96 8 83 57

a The activity of this fraction could not be determined accurately.

polymerases used were DNA polymerase I (large fragment), (5 units),
T5 DNA polymerase (4.4 units), and T7 DNA polymerase (2 units).
Construction of PriA Helicase Substrates (See Fig. 8)-The 56-mer
phage 6x174 primosome assembly site (pas) recognized by priA
protein (n` protein) was synthesized with an egtension of 4 residues
at the 5'-end to generate the 60-mer: 5'-CCTTGAGGTTATTA-
ACGCCGAAGCGGTAAAAATTTTAATTTTTGCCGCTGAGGG-
GTTGACC-3'. This 60-mer was annealed (by a 4-bp match) and

8780

Replication Fork Blockage in E. coli

ligated to the pair of 36-mer complementary synthetic oligomers
containing the 22-bp E. coli-terB sequence: oligomer 1, 5'-
AAGGGGCACATAATAAGTATGTTGTAACTAAAGTGT-3`; and
oligomer 2, 5'-TCACACACTTTAGTTACAACATACTTAT-
TATGTGCC-3'. To verify the ligation, the 96-mer was isolated by
gel electrophoresis in 7 M urea, 10% polyacrylamide and then an-
nealed with a 5'-3ZP-end-labeled complementary ter 36-mer (oligomer
1) to generate the CCW ter duplex, the sequence of which is oriented
to block the PriA helicase movement from pas. In a similar way, the
oppositely oriented E. coli-terB duplex substrate was prepared from
the pair of synthetic ter oligomers: oligomer 3,5'-AAGGGGCACAT-
CATTTAGTTACAACATACTTATTGT-3'; and oligomer 4, 5'-
TCACACAATAAGTATGTTGTAACTAAAGTATGTGCC-3'. The
T-like symbol (see Fig. 8) enclosing the ter sequence is oriented such
that the head of the T blocks the progress of the replication fork.
Two-dimensional Gel Electrophoresis-In vitro replicated DNA
samples (3000-20,000 cpm) were cut with EcoRV and SnaBI restric-
tion endonucleases and electrophoresed in two dimensions as de-
scribed (18). The first dimension was in 0.4% agaroseafor 18 h at l
V/cm at 4 "C in buffer containing 37 mM Tris borate (pH 8.3), 1 mM
EDTA; DNA size markers (HindIII-digested X DNA) were included.
The second dimension was in 1% agarose for 4 h at 8 V/cm at 4 "C
with 0.3 pg/ml ethidium bromide. Gels were dried, autoradiographed,
and scanned by densitometry.
SDS, Agarose, and Polyacrylamide Gel Electrophoresis-SDS-poly-
acrylamide gel electrophoresis was as described (16). HaeIII and
EcoRV restriction endonuclease digests of replicated DNA and reac-
tion samples from strand displacement of DNA polymerase assays
were brought to 0.5% SDS, 10% glycerol, 0.01% bromphenol blue,
and 0.1% xylene cyano1 and were loaded on 7% polyacrylamide gel
(acrylamide/N,N'-methylenebisacrylamide ratio of 30:l). Gels were
electrophoresed in buffer containing 37 mM Tris borate (pH 8.3), 1
mM EDTA for 4 h at 10 V/cm, dried, autoradiographed, and scanned
by densitometry. Also, slices of dried bands were measured for their
radioactivity in a liquid scintillation counter. Agarose and polyacryl-
amide gel electrophoresis were essentially as described (17).
DNA Sequencing-DNA sequencing was by the dideoxynucleotide
sequencing method (19) using the Sequenase system (U. S. Biochem-
ical Corp.) according to the instructions of the manufacturer. In brief,
the complementary ter oligomers (100-fold molar excess) and se-
quencing primer were annealed to template substrates by boiling for
2 min and cooling to room temperature slowly; a 20-fold molar excess
of TBP was added; and the reaction mixture was incubated at room
temperature for 1 min. The rest of the procedure was as described by
the manufacturer.

RESULTS

Effectiveness of Several ter Sequences in Inhibition of DNA
Replication-Among the 17 identified ter sequences (7-9), the
capacity to block DNA activity in vitro has been reported for
only the E. coli-terB (5, 11) and RGK-terR (20) sequences.
oriC plasmids containing these or seven other ter sequences
(Fig. la) were compared at an equimolar ratio of TBP to the
ter sequence. Those of E. coli and R1 were more effective than
those of R6K and RlOO (Table 11). The dissociation constant
(KO) for E. coli-terB is 10-l' M (5 X lo9 M for R6K-terR) (1).
Possibly the 2-3-fold stronger inhibition of DNA replication
by E. coli-terB compared with RGK-terR (Table 11) reflects
differences in their affinity for TBP. However, Kn values for
the other ter sequences have yet to be determined. The
incomplete blockage of replication can be attributed to the
bidirectional mode of oriC replication (11). Thus, a ter se-
quence oriented to block the CW fork fails to prevent move-
ment of the CCW fork, and vice versa.
We tested B. subtilis terII sequence, mindful of the fact
that the E. coli TBP is twice the size of that from B. subtilis
(14.5 kDa) (21). An inhibition of only 10% was observed at a
concentration 30-fold higher than that of the E. coli TBP
(data not shown).
Limits of Progress of Replication Forks in oriC-ter Plus-
rnids-The extent of the movement of a replication fork with
an oriC-ter plasmid, with or without TBP, was analyzed by
HaeIII and EcoRV restriction enzyme cleavage as previously

           TABLE I1
Comparative effectiveness of several ter sequences
   in the inhibition of DNA replication

Plasmid DNA synthesis, Yll3P"

-

                      %
pBS-oriC 97
E. coli-terA 35
E. coli-terB 23
R6K-terR 60
R6K-terL 64
Rl-terR 33
R1-terL 34
R100-terR 58
R100-terL 57

"For each plasmid, the value for DNA synthesis with TBP is
compared to that without it. Values, determined after incubations of
15 and 30 min, were similar; and the average is given. All the ter
plasmids contained the ter sequence in pBS-oriC (Fig. la). The
coupled reconstitution assay (see "Experimental Procedures") con-
tained 3 ng of TBP and 75 fmol of DNA, as plasmid. The DNA
synthesis of oriC-ter plasmids averaged 200 and 400 pmol for 15- and
30-min incubations, respectively, in the absence of TBP.

600

8 500

- E,

400

cn w
I I-

v)
a z
0 200

300

100

0

0 10 20 30

TIME (min)

FIG. 2. Inhibition of DNA synthesis of oriC-ter CW.CCW
plasmids by TBP-ter complex. The DNA substrate (75 fmol as
circle) was used with or without TBP (30 ng). All plasmids were oriC
with ter CW.CWW sequences, except where indicated (None). The
ter sequences were a pair of E. coli-terB, a pair of R1-terR and -terL,
and a pair of R6K-terR and -terL, as indicated.

described (11). In the absence of TBP, the fragments gener-
ated by digestion of the oriC-ter plasmids indicated a bidirec-
tional mode starting at or near oriC (Fig. 3), a pattern similar
to the symmetrical bidirectional progress for the control oriC
plasmid, whether or not TBP was present (11). With TBP,
replication of an oriC-ter CW plasmid was unidirectional and
blocked in the CW direction; with the oriC-ter CCW plasmid,
movement was blocked in the CCW direction (Fig. 3). In these
replications, full-length DNA appears within 2 min (11, 12).
Thus, there was ample time for a replication fork to proceed
through a CW ter site from the CCW direction, and vice versa.
This problem was circumvented by constructing oriC-ter Cw.
CCW plasmids (Fig. 1, b and c), which blocked replication
fork movements from oriC in both directions.
Replication of a plasmid with ter sequences oriented in
opposite directions on both sides of oriC was almost com-
pletely inhibited (Fig. 2). The three constructions included a
plasmid with two E. coli-terB sequences, one with R1-terB
and -terL, and one with RGK-terR and -terL, each pair SO
oriented as to prevent both CW and CCW movements. Rep-
lication was sharply limited by the bound ter sequences to the
oriC region (Fig. 3).

Replication Fork Blochge in E. coli

110 im

OHC OHC otic

8781

FRAGMENT

OO

10

LABELIN

oticXerCWCCW+TBP

otiCterCCW+TBP

01234 234

LENGTH (kb) LENGTH (kb) LENGTH (kb)

FIG. 3. Progress of replication forks of oriC and oriC-ter plasmids. The oriC in vitro reconstitution
assay was staged, and the replication products were separated on 10% glycerol, 7% polyacrylamide gels and
identified by autoradiography. DNA fragments were generated by Hue111 and EcoRV cleavage of a 4-min DNA
replication product; replication fragments RO-R3 are in the CW order from oriC; fragments LO-LIO are in the
CCW order. TBP (30 ng) was in 10-fold molar excess over plasmid DNA (75 fmol as circle). The autoradiograph
was scanned by densitometry, and the peaks were cut out and weighed. These intensity measurements were
corrected for fragment size, and the percent of the total intensity of the lane in each fragment was calculated. The
positions of minimal oriC and ter (2`-shaped area) are shown. The fragments generated by HaeIII and EcoRV
digestion were as follows: RO, 785 bp; Rl, 250 bp; R2, 926 bp; R3, 216 bp; LO, 785 bp; LI, 108 bp; L2, 9 bp; L3, 448
bp; L4, 484 bp; L5, 76 bp; L6, 85 bp; L7, 374 bp; L.8, 22 bp; L9, 45 bp; and LIO, 204 bp. The DNA size of L3 is 262
bp for oriC-ter CCW and oriC-ter CW.CCW plasmids.

FIRST DIMENSION

(a)

1

SPOT1 - LINEAR

FIG. 4. Analysis of replicated
molecules by two-dimensional gel
electrophoresis. The EcoRV- and
SnaBI-digested replication products
were analyzed as described under "EX-
perimental Procedures." a, the diagram
indicates the sizes and migration pat-
terns expected of the products (18). Spot
I, oriC-containing linear DNAs: 1.14 kb
from oriC, 1.16 kb from oriC-ter cw,
0.92 kg from oriC-ter CCW, and 0.95 kb
from oriC-ter Cw.CCW spot 2, repli-
cation intermediates of spot 1 DNAs
(bubble-shaped molecules); spot 3, 2.87-
kb linear DNAs; spot 4, replication in-
termediates of spot 3 DNAs (doub1e-Y-
and Y-shaped). b, two-dimensional gel
Profile of the replication ,products after
4,min of synthesis in a staged oriC in
vitro assay. TPB, where indicated, was
Present in a 10-fold molar excess relative
to ter.

orlc

.. . -TBP +TBP

  orlGt8tCCW
-TBP +TBP

. -.

.

SPOT2 e BUBBLE

SPOT 3 LINEAR

SPOT 4

Y AND DOUBLE Y

  orlCter CW
-TBP +TBP

oriGterCWCCW
-TBP +TBP

Shapes of Replicated Molecules Analyzed by Two-dimen-
sional Gel Ekctrophores&-The first dimension separates
molecules based on mass and the second, according to shape
(18). In autoradiographs of the two-dimensional gels Of
`WBI- and EcoRV-digested replication products (Fig. 4), two

  of linear DNA molecules were observed from both the
OrfC and OriC-ter plasmids replicated in the absence of TBP:
?C-containing small linear molecules (spot I ) and laf.ge
'lnear molecules (spot 3) (Fig. 4). In addition, spot 2 contains
bubble-shaped molecules, the replication intermediates of the
Small linear molecules; and spot 4 contains the Y- and double

Y-shaped molecules, the replication intermediates of the large
linear molecules. In the presence of TBP, a similar pattern
was obtained with the control (oriC) plasmid. However, with
the ter CW and ter CCW plasmids, spot 2 molecules were
enriched 5-6-fold, and the spot 1 molecules decreased drasti-
cally; with the ter CW.CCW (Fig. IC) plasmid, only spot 2
molecules were detected by autoradiography (Fig. 4). These
results further demonstrate that the` DNA replication forks
were confined between the two ter sites and terminated their
DNA synthesis at the ter sites.

Exact Location of Replication Block-Since replication forks

8782

Replication Fork Blockage in E. coli

do not proceed beyond the ter site, the precise position of the
blockage can be determined in a sequencing gel after cleavage
with an appropriate restriction endonuclease. SmI digestion
of replication products of oriC-ter CWaCCW (Fig. IC) gener-
ated a 120-bp DNA fragment (Fig. 5) that corresponds exactly
to the distance from the SmaI site to the first nucleotide
position of the E. coli-terB (CCW) site. A similar result was
obtained from a digestion with AccI (Fig. 5), which yielded a
137-bp fragment that also corresponds to the distance from
the first nucleotide position of the E. coli-terB (CW) site to
the AccI site (Fig. 5). In addition to these 120- and 137-bp
DNA fragments, others of 106, 463, and 476 bp were seen,
presumably generated by synthesis from initiation sites in
oriC. To confirm that the 120- and 137-bp DNA fragments
are true replication-arrest bands, other restriction enzymes
such as HindIII, BarnHI, AatII, and BglII were used. For
example, HindIII-SmaI double cleavage generated 120-bp
(SmaI-terB (CCW)), 180-bp (HindIII-terB (CW)), 106-bp,
and 290-bp fragments, all of which are readily interpreted as
true DNA replication-arrest bands (data not shown). Also
deduced from several restriction-enzyme analyses and com-
parisons of fragment sizes, two potential initiation sites were
located at positions -70 and -100 downstream from oriC for
the CW direction and 10 or more potential initiation sites
within oriC for CCW synthesis (data not shown).
Effect of TBP-ter Complex on Strand Displacement by DNA
Polymerase-Three DNA polymerases (T5, T7, and E. coli
polymerase I (large fragment)), each lacking a 5` -P 3` exo-
nuclease but able to displace the strand annealed to the
template (22), were examined for their ability to dissociate a

ter sequence bound by TBP. The template-primers (Fig. 6a)
possessed ter sequences oriented to block either the CCW or
CW replication forks. Displacement of the annealed oligonu-
cleotide was measured electrophoretically. In the absence of
TBP, all three DNA polymerases displaced 60-85% of the
annealed oligomers (Fig. 6b). In the presence or TBP, strand
displacement by DNA polymerases was largely inhibited on
both templates (CW and CCW). However, with M13mp18.
ter CCW, in which ter is oriented to block fork movement,
strand displacement was inhibited twice as much as with
M13mp18 ter CW (Fig. 6b). The data also show the inhibition
of strand displacement activity of DNA polymerases when
the ter sequence is correctly oriented and TBP is present.
The location at which the DNA polymerase was blocked by
the TBP-ter complex was readily determined with T7 DNA
polymerase and chain-terminating dideoxynucleoside triphos-
phates. The blockage sites were at the fourth nucleotide from
the 5'-end of ter CCW and at the fifth nucleotide from the
5'-end of ter CW (Fig. 7). Thus, this DNA polymerase pene-
trates several nucleotides into the complex, unlike the com-
plete blockage of the forks observed in replication of the
plasmids (Fig. 5). The greater effectiveness of a ter sequence
against a replication fork is attributable to blockage of the
DnaB helicase and the relative lack of strand-displacing ac-
tivity possessed by the DNA polymerase 111 holoenzyme (23).
Penetration of T7 DNA polymerase 3 bp from one end of the
22-bp ter sequence and 4 bp from the other leaves a 15-bp

M M AccI SmaI

FIG. 5. Site of arrest of DNA replication in oriC-ter CW.
CCW plasmid. The oriC in vitro reconstitution assay was staged
after 6 min of DNA synthesis as described under "Experimental
Procedures." The replication products of an oriC-ter CW. CCW plas-
mid (Fig. IC) were digested with AccI or SmaI and electrophoresed on
a 7 M urea, 8% polyacrylamide sequencing gel (0.4 mm X 60 cm). The
predominant bands observed with SmI were at 120,106, and 470 bp
and with AccI at 137,453, and 465 bp. The 120-bp DNA band from
SmaI and the 137-bp DNA band from AccI were resistant to treatment
with 0.05 M NaOH, but other bands were decreased slightly, suggest-
ing that the 120- and 137-bp DNA bands are not RNA-primed DNA
strands. The two lanes of size markers are indicated (lanes M).
Essentially the same experiment was repeated six times with virtually
identical results. The figure was from overexposed autoradiograph
film to show all possible bands.

DNA DNA

FWymerasa Polymeras9

W

W

TlME (mln)

FIG. 6. Effects of TBP-ter complexes on strand displace-
ment by DNA polymerase. a, the partial duplexes were constructed
from M13mp18. ter CCW and M13mp19. ter CW circles as described
(11) with annealed 64-mer oligomers containing the complementary
ter sequence; a 7-nucleotide tail was present at the 5'-end of the
partial duplexes and a 27-nucleotide tail was at the 3'-end. A 17-mer
sequencing primer was annealed to each plasmid as described under
"Experimental Procedures." b, The assay conditions were as described
under "Experimental Procedures." The products of the DNA polp-
erase reactions with DNA polymerase I (large fragment, 5 units), T5
(4.4 units), and T7 (2 units) were separated by electrophoresis on a
10% glycerol, 7% polyacrylamide gel and detected by autoradiography.
These amounts of the DNA polymerases were based on previom
titrations of their strand displacement activity, TBP, where indicated,
was present in a 20-fold molar excess over ter. Slices of the gel
containing the displaced oligomers were measured for their radioac-
tivity in a liquid scintillation counter. The percentage of displaced
oligomer was based on the total radioactivity (displaced plus undis-
placed oligomers).

Replication Fork Blockage in E. coli

8783

Subs- Yl3mpl9.terCW M13mplB.tsrCCW

TRP! - + -+

--
GATC GATC

..

.-

--
GATC GATC

E.coli-terB

FIG. 7. Site of arrest of replication by TBP-ter complex.
Using the partial duplexes with a sequencing primer (Fig. 6u) and T7
DNA polymerase, dideoxynucleotide sequencing (19) was performed
according to the instructions of U. S. Biochemical Corp. Where
indicated, TBP was present in a 20-fold excess over the ter sequence.
The predominant termination sites are indicated. The T-like symbol
representing the ter (E. coli-terB) sequence is oriented such that the
head of the T should block the progress of a replication fork.

length between the two DNA polymerase termination sites
protected by TBP.
Orientation-specific Blockage of PriA Helicase by TBP-ter
Complex-The orientation-specific inhibition of helicases, in-
cluding DnaB, Rep, and UvrD (helicase 11), by a TBP-ter
complex (11) was tested with PriA protein (24). The strand
displacement activity of PriA protein was almost completely
prevented by the TBP-terE complex and only in the orienta-
tion expected to block the 3' 4 5' movement characteristic
of this helicase (Fig. 8). This finding makes an earlier sugges-
tion that the blockage of DnaB helicase is due to a specific
TBP-helicase interaction (20) even more unlikely.

DISCUSSION

The termination of a replication fork by a protein-bound
sequence has been observed for some circular genomes in vivo
(3,4,7,8) and with a reconstituted system of purified proteins
(5,ll). The inverted repeats of a certain sequence (ter), when
bound by TBP, block helicase actions in an orientation-
specific manner (11, 20). In this study, we have further ex-
amined features of the capacity of a TBP-ter complex to
prevent the separation of strands essential at a replication
fork.
In a previous study (11), we used the oriC plasmid, which
exploits the unique origin of the E. coli chromosome, and
placed the terB sequence of E. coli on either side of it to block
one or the other of the two replication forks. Inasmuch as the
unblocked fork might come full circle, the inhibition of rep-
lication was incomplete. When oppositely oriented ter se-
quences were placed one on each side of oriC, both forks were
Stopped, and replication was confined to the minimal oriC
region (Figs. 2 and 3). As discussed previously (111, the TBP-
ter complex is especially effective in blocking the progress of
the fork in the rolling-circle mode of replication that super-
venes in the reaction. The precise mechanisms involved in
this stage are unclear, as are the means by which the TBP-
ter complex inhibits fork movement, presumably by prevent-
W helicase action on this substrate.

The arrest of the replication fork by a TBP-ter complex
reflected in the migrations of replication intermediates
analyzed by two-dimensional gel electrophoresis. A single
bubble-shaped structure within oriC appeared when synthesis
blocked by complexes oriented in opposite directions on

both sides of oriC (Fig. 4).
Blockage of the replication forks in the reconstituted oriC
   could be attributed to inhibition of the DnaB helicase

S 3

0

40,

E

0 ccw -
   cw -
   cw +

0 25 50 75 100 125

PriA PROTEIN (ne)

FIG. 8. Blockage of PriA helicase by TBP-ter complex. a,
the helicase substrate, oriented to block the CCW movement of a
replication fork, should prevent the action of the PriA helicase that
moves in that direction; the CW substrate should offer no such
impediment. b, assay of the PriA helicase. The reaction mixtures (20
jtl) contained 20 mM Tris-HC1 (pH 7.5), 9 mM MgCL, 4% (w/v)
sucrose, 100 jtg/ml bovine serum albumin, 1 mM ATP, 0.4 jtg of E.
coli single-stranded DNA-binding protein, 1.4 fmol of DNA substrate
(linear oligomer), and 30 fmol of TBP. The amounts of PriA protein
are indicated. The reaction was incubated at 30 "C for 10 min and
terminated by adding 20 mM EDTA, 0.25% SDS. Samples were loaded
on a nondenaturing 10% glycerol, 10% polyacrylamide gel; dried; and
subjected to autoradiography. Slices of the dried gel that contained
the undisplaced (substrate) and displaced (oligonucleotide) bands
were cut out, and their radioactivity was measured in a liquid scintil-
lation counter. The percentage of oligonucleotide displaced is re-
corded.

component of the system (11). However, the inhibition of two
other helicases, Rep and UvrD (helicase 11), by the same
TBP-ter,complex, also in an orientation-dependent way, in-
dicated that the bound ter sequence (rather than specific
TBP-helicase interactions) was responsible. The opposite
conclusion was reached in another study (20), which reported
that a TBP complex with a ter sequence from plasmid R6K
blocked the actions of DnaB, but not those of Rep or helicase
11. The suggestion that this discrepancy might be due to a
tighter association of TBP with the E. coli-terB sequence
compared with that of R6K (1) led us to examine the relative
strengths of a variety of ter sequences. All were found to be
rather similar in their capacity to inhibit a replication fork
(Table 11). The claim that the TBP-ter (R6K) complex fails
to block helicase I1 has since been withdrawn (25). The
generality of the TBP-ter complex inhibition has been ex-
tended to the helicase action of the T antigen of SV40 (25)
and to that of the PriA helicase (Fig. 8).
The capacity of the TBP-ter complex to impede strand
separation was demonstrable with DNA polymerases that
have strand-displacing activity. The DNA polymerases of

8784

Replication Fork Blockage in E. coli

phages T5 and T7 and the large fragment of E. coli polymerase
I were all affected (Fig. 6), but the blockages were less severe
and orientation-specific than those for the helicases.
The exact location at which a replication fork, advanced by
DnaB helicase, is blocked by a TBP-ter complex was placed
at the very first nucleotide on either side of the 22-bp E. coli-
terB sequence (Fig. 5). In the replication of a ColEl-ter
plasmid (5), arrest was observed, as in our studies (Fig. 5), at
the very first nucleotide of the 22-bp terB sequence, but also
at the second nucleotide and prematurely at sites 6 and 47 bp
upstream. Separation of strands by a DNA polymerase was
not only less orientation-dependent, but was also not as
severely restricted; displacements appeared to be limited by a
15-bp region of the complex (Fig. 7). These effects need to be
reconciled with a footprint of DNase I inhibition that showed
that the entire 22-bp sequence was affected by TBP binding.'
Clearly, more extensive and refined analyses of TBP binding
are needed to account for the extent and particularly the
orientation specificity of the binding of a ter sequence.

Acknowledgment-We are grateful to LeRoy L. Bertsch for his

help in preparing the manuscript.

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