Sequence features of the replication terminus of the Bacillus subtilis chromosome

Abstract

The sequence of 1267 nucleotides spanning the replication terminus, terC, of the Bacillus subtils 1s168 chromosome has been determined. The site of arrest of the clockwise fork, which defines terC, has been localized to a 30-nucleotide portion (approximately) within this sequence. The arrest site occurs 1n an A+T-r1ch
region between two open reading frames and very close to one of two imperfect inverted repeats (47–48 nucleotides each) which
are separated by 59 nucleotides. The closeness of approach of the arrested clockwise fork to the first Imperfect Inverted
repeat encountered in this region raises the possibility of a role for the Inverted repeats in the mechanism of fork arrest.

Full text

Volume
15
Number
20
1987
Nucleic
Acids
Research
Sequence
features
of
the
replication
terminus
of
the
Bacilus
subtis
chrowosome
C.M.Carrigan,
J.A.Haarsma,
M.T.Smith
and
R.G.Wake
Departnent
of
Biochemistry,
University
of
Sydney,
NSW,
Australia
Received
August
12,
1987;
Revised
and
Accepted
September
25,
1987
ABSTRACT
The
sequence
of
1267
nucleotides
spanning
the
replication
terminus,
terC,
of
the
Bacillus
subtilis
168
chromosome
has
been
determined.
The
site
of
ar-
rest
of
the
clockwise
fork,
which
defines
terC,
has
been
localized
to
a
30-
nucleotide
portion
(approximately)
within
this
sequence.
The
arrest
site
occurs
in
an
A+T-rich
region
between
two
open
reading
frames
and
very
close
to
one
of
two
imperfect
inverted
repeats
(47-48
nucleotides
each)
which
are
separated
by
59
nucleotides.
The
closeness
of
approach
of
the
arrested
clockwise
fork
to
the
first
imperfect
inverted
repeat
encountered
in
this
region
raises
the
possibility
of
a
role
for
the
inverted
repeats
in
the
mech-
anism
of
fork
arrest.
INTRODUCTION
Termination
of
chromosome
replication
in
Bacillus
subtilis
168
occurs
at
a
site,
terC,
which
is
approximately
opposite
the
origin
of
replication,
oriC
(1,2).
The
clockwise
replication
fork
reaches
this
site
first
and
is
blocked,
or
severely
impeded,
in
its
movement
(3).
The
anticlockwise
fork
arrives
a
few
minutes
later
to
presumably
fuse
with
the
arrested
fork
and
so
complete
termination. terC
has
been
localized
to
a
particular
1.95
kb
PstI
-
EcoRI
segment of
DNA,
which
has
also
been
cloned;
and
the
site
of
fork
arrest
has
been
estimated
to
lie
approximately
1.2
kb
from
the
EcoRI
end
of
this
seg-
ment
(4).
In
this
paper
we
present
the
nucleotide
sequence
of
approximately
1.3
kb
of
DNA
spanning
terC
and
examine
it
for
structural
features.
Analysis
of
single
strands
derived
from
the
arrested
clockwise
fork
has
fixed
more
pre-
cisely
the
location
of
the
fork
junction
within
the
sequence.
The
most
strik-
ing
feature
of
the
sequence
in
the
vicinity
of
the
site
of
fork
arrest
is
an
A+T-rich
region
containing
two
imperfect
inverted
repeats.
This
region
is
located
between
two
open
reading
frames
(ORFs)
and
the
possibility
that
it
has
a
special
role
in
the
termination
process
is
discussed.
©
I
R
L
Press
Limited,
Oxford,
England.
Nucleic
Acids
Research
Volume
15
Number
20
1987
8501

Nucleic
Acids
Research
MATERIALS
AND
METHODS
Bacterial
strains
and plasmids
B.subtilis
strain
GSY1127,
a
class
II
stable
merodiploid
(hisH2
ilvC/
ilvC+)
was
obtained
from
C.
Anagnostopoulos.
Plasmid
pWS10,
which
contains
the
B.subtilis
terC
region,
was
constructed
in
this
laboratory
(4).
DNA
preparations
For
analysis
of
forked
DNA,
or
its
single
strand
products,
BamHI
digested
DNA
was
obtained
from
exponentially
growing
GSY1127
using
the
'osmolysate'
procedure
described
previously
(3)
but
at
a
three
fold
cell
concentration.
To
obtain
BamHI
+
EcoRI
digests
for
single
strand
analysis
in
alkaline
gels
the
BamHI
osmolysate
was
firstly
fractionated
in
a
preparative
CsCl
density
gradient
and
the
DNA
band
concentrated
ten
fold
by
pressure
dialysis
against
10
mM
Tris
HCO
(pH
8)
+
1
mM
EDTA.
It
was
then
digested
with
EcoRI
under
standard
conditions.
DNA
from
spores
of
GSY1127
was
obtained
as
described
previously
(3).
DNA
sequencing
and
data
analysis
DNA
sequencing
was
performed
by
the
dideoxynucleotide
chain
termination
method
(5).
To
obtain
most
(-
90%)
of
the
sequence
suitable DNA
fragments
were
sub-cloned
into
M13mplO
or
M13mpll
vectors
and
sequenced
according
to
the
procedure
outlined
by
Amersham
International,
U.K.
Part
of
the
sequence
was
obtained
by
dideoxy
sequencing
of
double
strand
DNA
cloned
in
the
Blue-
script
M13
vectors
according
to
the
instructions
provided
by
Stratagene
(San
Diego,
U.S.A.).
The
complete
sequence
reported
here
was
done
in
both
directions.
DNA
sequences
were
analysed
for
various
features
with
the
SEQNCE
general
purpose
analysis
program
(Delaney
Software
Ltd.,
Vancouver,
Canada).
Second-
ary
structure
was
analysed
with
the
ZUKER
program
(PCFOLD:
version
3.0,
adapted
for
IBM
PC
by
D.
Brunelle)
provided
by
the
Molecular
Biology
Computer
Research
Resource
(Dana
Farber
Cancer
Institute,
Harvard
Medical
School).
Protein
sequences
were
compared
with
the
Genbank
and
EMBL
nucleic
acid
data-
bases
translated
in
all
6
reading
frames
using
the
program
of
M.
Kanehisa
provided
by
the
CSIRO
Division
of
Molecular
Biology.
Agarose
gel
electrophoresis,
transfer
of
DNA
to
membranes
and
hybridization
Electrophoresis
of
BamHI
+
EcoRI
digests
of
DNA
were
carried
out
in
2%
alkaline
agarose
gels
(6).
Procedures
for
transfer
of
DNA
to
nylon
membranes,
hybridization
with
32P-labelled
cloned
DNA
and
autoradiography
were
as
de-
scribed
previously
(7).
8502

Nucleic
Acids
Research
P.
Pv
Pv
E
10.131
1.01
o
0.86
0.90
1.11
H
I-4
IlrC
wgion
SEQUENCED
CWCKWISE
FRK
FEg.
1.
Features
of
the
B.subtilis
terC
-
containing
PstI
-
EcoRI
insert
of
pWS10.
The
top
section
shows
a
restriction
map
of
the
insert;
the
terC
region
is
located
approximately
1.2
kb
from
the
EcoRI
end.
The
clockwise-moving
rep-
lication
fork
generated
at
the
origin
of
the
chromosome
enters
this
segment
from
the
EcoRI
end
and
stops
at
terC
(bottom
section).
The
heavy
line
(middle
section),
covering
1.3
kb
and
spanning
terC,
was
sequenced.
The
arrowed
lines
above
and
below
the
sequenced
portion
show
the
directions
of
dideoxy
sequen-
cing
through
the
terC
region.
P,
PstI;
Pv,
PvuII;
H,
HindIII;
E,
EcoRI.
All
sizes
are
in
kb.
The
overall
size
of
this
segment
of
DNA
has
been
estimated
at
1.95
kb,
while
the
sum of
the
smaller
portions
of
it
give
a
value
closer
to
2.0
kb.
RESULTS
Nucleotide
sequence
Figure
1
(top
section)
shows
a
restriction
map of
the
terC
-
containing
B.subtilis
insert
of
the
plasmid
pWS1O.
The
clockwise
replication
fork
enters
this
segment
as
it
resides
in
the
bacterial
chromosome
from
the
right
(or
EcoRI
end)
and
is
arrested
(bottom
section)
at
around
the
position
indicated,
terC,
approximately
1.2
kb
from
the
EcoRI
end.
The
portion
shown
as
a
heavy
line
just
below
the
map
was
sequenced.
The
sequence
of
1267
nucleotides
is
shown
in
Figure
2
(the
clockwise
fork
enters
this
sequence
from
the
bottom).
It
contains
two
ORFs
reading
in
the
PstI
-o
EcoRI
direction
(opposite
to
movement
of
the
clockwise
fork).
They
are
shown
diagramatically
in
Figure
3.
ORF1
(177
amino
acids)
is
incomplete
and
is
followed
by
a
possible
transcrip-
tion
termination
site.
ORF2
(122
amino
acids)
is
complete.
It
is
preceded
closely
by
the
consensus
Shine-Dalgarno
(SD)
sequence
AAGGAG
which
is
comple-
mentary
to
the
3'end
of
B.subtilis
16S
RNA
(see
ref.
8),
and
is
followed
by
a
possible
transcription
termination
site.
No
attempt
has
been
made
to
identify
possible
transcription
start
sites
associated
with
ORF2.
There
is
an
addi-
tional
complete
ORF
(73
amino
acids)
reading
opposite
to
ORF1
and
ORF2.
It
8503

Nucleic
Acids
Research
12
24
38
48
60
72
A
D
V
K
D
A
D
Q
V
N
Q
A
V
A
Q
V
K
E
Q
L
G
D
I
CTGCAGATGTAAAACATGCCGATCAGGTTAACCAAGCTGTAGCTCAAGTGAAGGAACAGCTCGGTGATAT
CG
IVi
84
96
108
120
132
144
D
I
L
I
N
N
A
G
1
8
K
F
G
C
F
L
D
L
S
A
D
E
W
E
ATATCCTCATTAATAATGCCGGCATCAGCAAATTTGGCGGTTTCTTAGATCTGTCAGCTGATGAGTGGGAAA
158
188
18O
192
204
216
N
I
I
Q
V
N
L
H
G
V
Y H
V
T
R
A
V
L
P
E
H
I
E
R
ATATTATTCAAGTCAACCTAATGGGTGTGTACCATGTCACTCGCGCOGGOGCTTCCGGAAATGATCGAACUCA
228 240
252
284
278
288
K
A
G
D
I
I
N
I
S S
T
A
G
Q
R
G
A
A
V
T
S
A
Y
S
AAGCCGGAGACATCATTAATATTTCATCTACAGCGGGCCAAAGAGGAGCTGCTGTAACAAGTGCTTACAGCG
300
312
324
338
346
360
A
S
K
F
A
V
L
G
L
T
I
S
L
H
Q
E
V
R
K
H
N
I
R
V
CTTCTAAATTTGCCGTTCTCGGGTTAACAGAGTCTCTTATGCAAGAAGTGAGAAAACATAATATCAGAGTCA
372
384
398
408
420
432
S
A
L T
P
8
T
V
A
8 D
K
8
I
E
L
N
L T D
GN
P
E
CCGCGTTAACGCCGACCACTGTCGCTAGTGATATGTCTATCGAATTGAACTTAACAGACGGTAATCCTGAAA
444
458
488
480
492
504
K
V
N
Q
P
E
D L
A
E
Y
M
V
A
Q
L
K
L
D
P
R
I
F
I
AAGTTATGCAGCCTGAGGATCTTGCTGAGTACATGGTGGCACAACTGAAATTAGATCCTCGTATTTTCATCA
518
528
540
552
564
576
K
T
A
G
L
V
8
T
N P
AAACAGCGGGATTATGCTCAACAAATCCTTAAAAATGAAAACCTCTCTTTCGACAGGTTTTTTTATTTGAAT
588
800
812
824
636
648
GAAATCCGTACCGGTAAAATGAGATATGTAAACCCTGGCAATCGTTTAAATTGAAGATAGCAGTAAATGCAG
880
872
884
696
708
720
CCTATAATAGAACTAAGAAAACTATGTACCAAATGTTCAGTCGAAATTTATTTTTTCCGCTACACCTATAAT
732
744
1
758
768
780
792
CAGTAAACATGAAATAACTGGACTATCAGTCTTTAATATAAAGAAGGAAAACAATAAAAGAAAATTGAATAT
804
818
828
840
852
2
864
H
K E
I
K
R
S
S
T
G
TTAGTACATAGTGTTGTCAGTGACAGAAAfMEGCCATATGATGAAAGAAGAAAAAAGGAGTTCAACAGG
SD
876
888
900
912
924
936
F
L
V
K
Q
R
A
F
L
K
L
Y
H
I
T
H
T
E
Q
E
R L
Y
G
CTTTTTAGTGAAACAGCGCGCATTTTTGAAGCITTATATGATAACGATGACAGAGCAAGAGAGACTCTATCG
948
980
Hlnd¶72
984
996
1008
L
K
L
L
E
V
LRS
E
F
K
E
I
G
F
K
P
N
IJ
T
E
V
Y
GTTAAAGCTGCTTGAACTACTTCGGTCTGAATTTAAAGAGATTGGTTTTAAACCAAATCATACAGAAGTATA
1020
1032
1044
1056
1068
1080
R
S
L
H
E
L
L
D
D
G
I
L
K
Q
I
K
V
K
K
E
G
A
K
L
CCGGTCTTTGCATGAGCTTCTTGATGACGGOATACTAAAACAAATTAAAGTAAAAAAAGAAOGGGCTAAGCT
1092
1104
1118
1120
1140
1152
Q
E
V
V
L
Y
Q
F
K
D
Y
E
A
A
K
L
Y
K
K
Q
L
K
V
E
CCAGGAAGTCGTCCTCTATCAATTTAAAGATTACGAAGCTGCCAAGCTATATAAAAAACAGCTOAAGGTAGA
1184
1176
1188
1200
1212
1224
L
D
R
C
K
K
L
I
I
K
A
L
S
D
N
F
GCTGGATCGCTGTAAAAAACTGATTGAAAAAGCTCCTCTAGATAATTTTTAATAGAAACACCCGCC
1238
1248
1280
AGCAGTGO2AAG2gagTG
TCTGCTTTTCATTATACATATT
Fig.
2.
Nucleotide
sequence
of
approximately
1.3
kb
of
DNA
spanning
terC.
The
sequence
extends
from
the
PstI
end
of
the
insert
of
pWS10
and
covers
1267
nucleotides
(heavy
line
in
Fig.
1).
The
boxed
sequence
is
a
potential
ribo-
some-binding
site
(SD
sequence);
and
the
double-underlining
indicates
poten-
tial
transcription
termination
sites.
Two
ORFs
are
defined
by the
amino
acid
sequences
shown.
The
heavy
arrows,
labelled
I
and
II,
define
two
imperfect
inverted
repeats
located
between
the
ORFs.
The
PstI
and
HindIII
sites
(see
Fig.
1)
are
identified.
8504

Nucleic
Acids
Research
p
H
E
L
A
J.
.
I
.
1
200
400
600
800
1000
1200
-2000
ORF1
ORF2
80
0
60-
at40
Fig.
3.
Sequence
features
of
the
1267
nucleotide
segment
of DNA
spanning
terC.
The
top
section
shows
a
scale
(nucleotide
number)
of
the
sequenced
re-
gion
which
extends
from
the
PstI
site
(position
1)
and
through
the
HindIII
site
(895);
terC
is
located
around
position
800
in
the
sequence.
The
middle
section
shows
the
regions
(shaded)
covered
by
the
two
ORFs;
ORF1
is
incom-
plete.
The
arrowed
lines
(I
and
II)
between
ORF1
and
ORF2
define
the
inverted
repeats.
The
bottom
section
shows
the
A+T
distribution
through
the
sequence;
the
dotted
line
is
at
the
level
of
the
A+T
content
of
total
B.subtilis
DNA,
P,
PstI;
H,
HindIII;
E,
EcoRI.
occurs
within
ORF1
but
there
is
no
associated
consensus
SD
sequence
within
a
distance
of
20
nucleotides
from
the
putative
initiation
codon.
The
leftwards
extremity
of
ORF2
extends
60
nucleotides
to
the
left
of
the
HindIII
site
shown
in
Figures
1
and
3.
This
places
it
close
to
the
arrest
site,
terC.
The
non
translatable
region
of
301
nucleotides
between
ORF1
and
ORF2
could
define
terC
and
it
exhibits
some
special
features.
It
is
relativ-
ely
rich
in
A+T
(Fig.
3,
bottom
section).
More
striking
is
the
presence
of
two
imperfect
inverted
repeats
within
the
660-813
portion
of
the
sequence.
Each
of
the
inverted
repeats
(labelled
I
and
II)
extends
for
47-48
nucleoti-
des.
They
show
77%
homology
to
one
another
(Fig.
4)
and
are
separated
by
59
nucleotides.
Refined
sequence
location
of
the
arrested
fork
The
earlier
localization
of
terC
to
a
region
approximately
1.2
kb
to
the
left
of
the
EcoRI
site
of
the
pWS10
insert
(see
Fig.
1)
was
based
upon the
size
of
the
double
strand
'arm'
released
from
the EcoRI
+
PstI-derived
arres-
ted
fork
either
during
the
isolation
of
the DNA
or
after
S1
nuclease
treat-
ment
(4).
A
more
reliable
and
refined
estimate
for
location
of
the
fork
8505

Nucleic
Acids
Research
660
706
I
5'
p
jAC
TATGTAcAAAWEGfjcGAAATTTAT
TTTCC
3'
3
PTACAT
CTTTAAATA
AAAGG
5'
813
766
a
5'
ijGhjCjRC
TATGTJT
IAA
T
TCTTtrTTC
AT7iGffiTTTCC
H
3'
7G3
GATACAT
TT
AAGAAAATA
GG|
5'
Fig.
4.
Homology
within
the
inverted
repeats
identified
as
I
and
II
in
Figs.
2
and
3.
The
boxed
segments
are
regions
of
perfect
homology.
junction
has
been
obtained
from
the
size
of
the
single
strands
derived
from
the
forked
molecule
present
in
an
EcoRI
+
BamHI
digest.
The
first
BamHI
site
to
the
left
of
the
EcoRI
site
shown
in
Figure
1
is
10.9
kb
distant
from
it.
Thus,
the
arrested
fork
in
such
a
digest
would
yield
single
strands
of
appro-
ximately
1.2
and
10.9
kb.
Figure
5
compares
the
single
strand
forms
of
DNA
in
EcoRI
+
BamHI
digests
of
DNA
from
exponentially
growing
cells
of
the
merodip-
loid
strain
GSY1127
(see
ref.
7)
and
from
spores
of
the
same
strain.
Spore
DNA
contains
completed
non-replicating
chromosomes
and
is
therefore devoid
of
forked
molecules.
The
DNA
from
exponential
cells
where
the
arrested
fork
is
present
in
a
portion
of
the
chromosomes
(lane
2)
shows
single
strand
mat-
erial
in
the
1.2
kb
size
range
(labelled
X)
which
is
absent
from
the
spore
DNA
as
expected.
It
covers
the
range
1.12-1.21
kb
and
appears
to
comprise
two
poorly
resolved
species.
Lane
1
in
Figure
5
contains
the
1.11
kb
EcoRI
-
HindIII
segment
of
the
pWS10
insert
(see
Fig.
1).
The
smaller
of
the
two
poorly
resolved
species
is
marginally
larger
than
this
segment.
The
use
of
appropriate
digests
of
pWS10
as
standards,
sometimes
in
the
same
lane
as
the
DNA
to
be
analysed,
established
the
smaller
of
the
two
single
strand
species
to
be
10-40
nucleotides
longer
than
the
EcoRI
-
HindIII
portion
of
the
pWS10
insert
(Fig.
1),
and the
larger
to
be
70-100
nucleotides
longer
than
it.
This
means
that
the
junction
of
the
arrested
fork
extends
at
the
most
70-100
nuc-
leotides
upstream
of
the
HindIII
site
i.e.
to
the
795-825
region
of
the
sequ-
ence,
and
in
the
vicinity
of
inverted
repeat
II
(see
Fig.
2).
In
Figure
5
the
larger
of
the
unique
species,
X,
in
lane
2
is
the
more
prominent.
In
other
experiments,
using
different
DNA
preparations,
the
rel-
ative
amounts
of
the
two
species
were
not
as
markedly
different.
It
should
also
be
pointed
out
that
the
material
spreading
downwards
from
the
10.9
kb
species
(very
obvious
here
because
of
the
heavy
exposure
for
autoradiography)
frequently
showed
banding
within
it,
even
in
spore
DNA.
This
banding
was
mostly
at
positions
above
2.0
kb.
It
probably
reflects
over-digestion
of
the
8506

Nucleic
Acids
Research
1
2
3
10.9-
4.50-
9
2.07-
1.66-
X
1.11-
*
w
0.86-
Fig.
5.
Single
strand
composition
of
the
arrested
fork
localized
within
the
EcoRI
-
BamHI
segment
of
the
B.subtilis
chromosome.
EcoRI
+
BamHI
digests
of
highly
purified DNA
from
exponentially
growing
cells
of
GSY1127
containing
the
arrested
fork
(lane
2)
and
spore
DNA
(lane
3)
were
fractionated
in
a
2%
agarose
gel
in
alkali.
The
DNA
was
transferred
to
a
nylon
membrane,
hy-
bridized
with
32P-labelled
pWS10
DNA
and
autoradiographed.
Lane
1
shows
one
of
the
lanes
containing
size
standards,
in
this
case
an
EcoRI
+
HindIII
digest
of
pWS10.
Material
in
the
1.2
kb
region
(labelledVT
is
present
in
lane
2
and
absent
from
lane
3.
Sizes
are
in
kb.
DNA
and
is
certainly
unrelated
to
fork
arrest
at
terC.
It
has
been
shown
that
deletion
of
a
substantial chromosomal
segment
to
the
left
of
and
up
to
the
PstI
site
in
Figure
1
has
no
noticeable
effect
on
fork
arrest
(unpublished
data).
Thus,
the
single
strands
from
the
'arm'
region
of
an
EcoRI-derived
arrested
fork
could
not
be
longer
than
2.0
kb.
DISCUSSION
Termination
of
chromosome
replication
in
B.subtilis
involves
the
arrest
of
the
clockwise
moving
fork
at
a
unique
site,
terC.
Knowledge
of
the
nucleotide
sequence
spanning
terC
is
obviously
important
in
understanding
the
molecular
mechanism
of
fork
arrest.
While
the
minimum
sequence
needed
for
this
aspect
of
termination
has
not
yet
been
established
it
is
likely
that
the
sequence
close
to
and
just
ahead
of
the
arrested
fork
plays
a
crucial
role.
The
1267
nucleotide
sequence
described
here
spans
terC.
While
the
newly
synthesized
8507

Nucleic
Acids
Research
strands
within
the
arrested
fork
are
heterogeneous
in
size,
the
longest
ones
(possibly
leading
strands)
extend
from
the
EcoRI
site
into
a
region
between
two
ORFs
(Fig.
3)
and
terminate
very
close
(within
20 nucleotides)
to
the
first
of
two
47-48
nucleotide
imperfect
inverted
repeats
that
is
encountered
in
the
direction
of
fork
movement
(II
in
Figs
2
and
3).
The
present
data
do
not
indicate
whether
the
longest
strands
actually
stop
short
of
this
inverted
repeat
i.e.
before
position
813
in
Figure
2,
or
enter
it.
The
other
inverted
repeat,
I,
is
59
nucleotides
distant,
and
both
are
located
within
a
DNA
seg-
ment
that
is
relatively
A+T
rich.
The
closeness
of
approach
of
the
arrested
fork
to
the
first
inverted
re-
peat
that
it
encounters
points
to
a
possible
role
for
it
in
arresting
fork
movement.
The
single
strand
components
of
the
region
between
the two
ORFs,
in
which
both
inverted
repeats
are
located,
can
be
folded
with
the
ZUKER
computer
program
into
a
stable
secondary
structure
within
which
the
paired
inverted
repeats
are
a
prominent
feature.
However,
while
in
vitro
synthesis
by
mammalian
DNA
polymerase
a
is
arrested
at
stable
secondary
structures
(hairpins)
formed
by
palindromic
sequences
within
single
strand
templates,
it
appears
that
such
structures
do
not
form
in
vivo
(9).
The
sequencing
experi-
ments
here
showed
that
the
Klenow
fragment
of
DNA
polymerase
I
had
no
diffi-
culty
in
replicating
the
terC
-
containing
single
strand
templates
in
both
orientations
(see
Fig.
1).
The
question
arises,
whether
there
is
some
feature
of
chromosomal
organisation
in
the
vicinity
of
terC
and/or
the
replication
machinery
itself
that
causes
a
stable
secondary
structure,
incorporating
the
imperfect
inverted
repeats,
to
form.
Of
course,
it
is
possible
that
the
in-
verted
repeats
function
to
block
fork
movement
through
a
mechanism
not
invol-
ving
secondary
structure
formation
within
the
separated
template
strands.
They
might
even
function
independently
of
one
another,
each
arresting
a
fork
moving
in
one
particular
direction.
It
is
appropriate
to
point
out
that
the
nucleotide
sequence
surrounding
the
replication
terminus
of
the
Escherichia
coli
plasmid
R6K
was
described
some
time
ago
and
found
not
to
contain
any
2-fold
rotational
symmetry
(10).
But
use
of
the
ZUKER
program
has
enabled
the
detection
of
two
20-nucleotide
imperfect
inverted
repeats
(90%
homology)
separated
by
73
nucleotides.
The
explanation
favoured
for
arrest
of
the
fork
at
the
R6K
terminus
was
interac-
tion
of
a
protein
with
the
terminus
sequence
(10)
and
this
must
be
considered
a
possibility
for
B.subtilis.
At
present
there
is
no
information
on
the
pos-
sible
expression
of
the
complete
open
reading
frame,
ORF2.
Also,
the
possib-
ility
that
its
putative
protein
product
has
a
role
in
termination
cannot
be
8508

Nucleic
Acids
Research
ruled
out.
A
search
of
the
Genbank
and
EMBL
nucleic
acid
data
bases
for
a
translatable
protein
(all
reading
frames)
related
to
the
product
of
ORF2
was
negative.
Current
work
is
aimed
at
establishing
the
minimal
sequence
required
for
fork
arrest
and
the
precise
sequence
extremities
of
the
leading
and
lagging
strands
within
the
arrested
fork.
Such
information
will
help
to
clarify
the
possible
role
of
the
inverted
repeat
region
in
the
termination
of
replication
process.
ACKNOWLEDGEMENTS
We
thank
Dr
A.
Reisner
for
advice
on
the
computer
analysis
of DNA
sequen-
ces.
This
work
was
supported
by
the
Australian
Research
Grants
Scheme
and
the
University
of
Sydney
Cancer
Research
Fund.
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8509