Downloaded from symposium.cshlp.org on March 5, 2016 - Published by Cold Spring Harbor Laboratory Press
Nucleotide Sequence of the Origin for
Bacteriophage M13 DNA Replication
S. V. SUGGS AND D. S. RAY
Molecular Biology Institute and Department of Biology, University of California, Los Angeles, California 90024
MATERIALS A N D M E T H O D S
M13, fd, and fl are three very closely related
filamentous bacteriophages that belong to the Ff group
(for review, see Ray 1977). These bacteriophages
contain a single strand of DNA that replicates in three
stages. Initially, the infecting viral strand is converted
to duplex or replicative-form DNA (SS~RF) (Ray et
al. 1966; Pratt and Erdahl 1968), which subsequently
replicates to form a pool of RF DNA molecules. In
the final stage of Ff DNA replication, the pool of
RF DNA molecules serves as precursor for the asymmetric synthesis of progeny single-stranded DNA
(RF~SS) (Ray 1969; Lin and Pratt 1972).
The origin of complementary-strand synthesis and
that of viral-strand synthesis have been localized. The
synthesis of the complementary strand in the in vitro
SS~RF DNA reaction is initiated at a unique site by
RNA polymerase (Tabak et al. 1974). The nucleotide
sequence of the RNA that primes this synthesis has
been determined (Geider et al. 1978). Synthesis of the
complementary strand initiates in the same region
during RF DNA replication in vivo as it does during
SS~RF (Horiuchi and Zinder 1976; van den Hondel
1976), proceeding unidirectionally clockwise on the
genetic map.
The origin of viral-strand synthesis during both RF
DNA replication and single-strand synthesis has been
localized in the same segment of the genome as the
complementary-strand origin (Horiuchi and Zinder
1976; Suggs and Ray 1977). Synthesis is unidirectional
and proceeds counterclockwise on the genetic map
from a region located between genes II and IV that
does not code for any known protein (Vovis et al.
1975).
As a first step towards a further understanding of
the mechanisms regulating viral DNA synthesis, we
have determined the nucleotide sequence of this intergenic region in M13 DNA that contains the sites of
initiation and termination of both the viral and complementary strands. In addition to these sites involved
in DNA replication, the intergenic region also contains
a promoter of transcription (Seeburg et al. 1977) just
preceding gene II, a viral gene required for both RF
DNA replication and SS DNA synthesis. Comparison
of the nucleotide sequence of the intergenic space of
M13 DNA to the analogous sequences of fd and fl
DNA reveals several sites within this region at which
base changes can be tolerated.
Phage and bacterial strains. Escherichia coli K37
(Hfr supD) was used as the host for M13 wild type and
M13 am2H2c2. M13 am2H2c2 is a clear-plaque variant that arises spontaneously in cultures of M13
am2H2-infected cells. The site of the c2 mutation has
been mapped in Haemophilus parainfluenzae restriction endonuclease (HpalI) fragment C by markerrescue experiments (M. Farber and D. S. Ray, unpubl.),
and the am2H2 mutation has been mapped in HpaII
fragment D (van den Hondel et al. 1975). The HpalI C
and D fragments are not in the region that has been
subjected to sequence analysis.
Media. E. coli cultures were grown in M9 medium
(Anderson 1946) containing 0.4% glucose, 0.5%
casamino acids, and vitamin B1 at 2/zg/ml.
Haemophilus strains were grown in BHI medium
containing 1 mg/ml hemin and 2/zg/ml DPN. Thermus
aquaticus YTI (TaqI) cultures were grown in Castenholz-TYE medium (Brock and Freeze 1969).
Chemicals. Carrier-free 32p was purchased from
International Chemical and Nuclear Corporation.
Enzymes. Restriction endonucleases from Haemophilus aegyptius (HaelII), Haemophilus influenzae
Rf (HinfI), and Haemophilus haemolyticus (HhaI)
were isolated by the following procedure. Cells were
grown to approximately 5 x 108 ml in a BHI-heminDPN medium at 37~ The cells were collected by
centrifugation, resuspended in 0.01 M Tris (pH "7.8)
containing 0.01M /3-mercaptoethanol, and lysed by
sonication. Cellular debris was removed by centrifugation. Nucleic acids were removed by a two-phase
system of polyethylene glycol-dextran (Okazaki and
Kornberg 1964). The resulting enzyme solution was
desalted by Sephadex G-25 gel filtration or by dialysis
and applied to a phosphocellulose column (Whatman
P l l ) equilibrated with 0.01 M Tris (pH 7.4)containing
0.01 M /3-mercaptoethanol, 0.0001 u EDTA, and 10%
glycerol (Buffer A). The enzyme was eluted with a
linear gradient of KCI from 0 M to 1 M in Buffer A. The
active fractions were pooled and concentrated using a
Millipore immersible molecular separator. Glycerol
was added to 50% and the enzyme was stored at
-20~
In the case of HinfI, the enzyme was further
purified after phosphocellulose chromatography. The
379
Downloaded from symposium.cshlp.org on March 5, 2016 - Published by Cold Spring Harbor Laboratory Press
380
SUGGS AND RAY
active fractions were dialyzed into 0.025 M KPO4 (pH
7.4) containing 0.0001 M EDTA, 0.007 M fl-mercaptoethanol (Buffer B), and the sample was applied to a
hydroxylapatite column (Bio-Rad, Bio-Gel HTP)
equilibrated in Buffer B. The enzyme was eluted with
a linear gradient of KPO4 (pH 7.4) from 0.025 M to
0.5 M in Buffer B.
Restriction endonuclease TaqI was isolated by the
following procedure. Cells were grown to log phase in
Castenholz-TYE medium at 70~
The cells were
collected by centrifugation, resuspended in 0.01 M Tris
(pH 7.9) containing 0.01M /3-mercaptoethanol, and
lysed by sonication. Cellular debris was removed by
centrifugation and the DNA was precipitated by adding streptomycin sulfate to 2%. The resulting precipitate was removed by centrifugation. The sample was
dialyzed into 0.01 M KPO4 (pH 7.6)containing 0.01 M
/3-mercaptoethanol, 0.0001 M EDTA, and 10%
glycerol (Buffer C) and applied to a phosphocellulose
column equilibrated with Buffer C. The enzyme was
eluted with a linear gradient of KC1 from 0 M to 0.5 M
in Buffer C. Active fractions were pooled, glycerol
added to 50%, and the enzyme stored at -20~
Restriction endonuclease HpalI was purchased from
New England BioLabs. Arthrobacter luteus restriction
endonuclease (AluI)was purchased from Bethesda
Research Laboratories. Polynucleotide kinase from
T4-infected E. coli was purchased from P. L. Biochemicals. Bacterial alkaline phosphatase ( B A P F ) w a s
purchased from Worthington Biochemical Corporation.
Preparation of M13 RFI DNA. M13 RFI DNA was
prepared by the following procedure. A culture of K37
was grown in M9 medium at 37~ to a cell density of
2 • 108/ml. M13 wild type or M13 am2H2c2 was added
at a multiplicity of 20 and growth was continued for
1-4 hours. The infected cells were collected by centrifugation and lysed as described by Katz et al. (1973).
To the cleared lysate was added 0.95 g/ml cesium
chloride (CsCI) and 100/zg/ml ethidium bromide
0EtdBr). The solution was centrifuged to equilibrium
in a Beckman Ti60 rotor at 40,000 rpm for 60 hours.
The lower band material (RFI DNA) was collected by
side puncture with a needle and syringe. The RFI
DNA was extracted three times with isobutyl alcohol
and dialyzed extensively against 0.01 i Tris (pH 8)
containing 0.001 M E D T A (TE).
Digestion of DNA by restriction end~
HaelII, HhaI, HinfI, HpalI, and AluI reactions contained 7 mM Tris (pH 7.4), 7 mM MgCI2, 7 mM fl-mercaptoethanol, 7 mM NaCI, and gelatin at 100/.tg/ml and were
incubated at 37~
TaqI reactions contained 10mM
HEPES buffer, 25 mM (NH4)2 SOn, 6 mM fl-mercaptoethanol, and 6 mM MgC12 and were incubated at
65-70~
F.lectrophoresis of DNA fragments. Restriction
fragments were separated on 5% polyacrylamide gels
(acrylamide:bisacrylamide ratio of 29:1) run at
200-250 V. The electrophoresis buffer was that described by Greene et al. (1974). The base-specific
cleavage products of the sequencing reactions were
separated on 12%, 20%, and 25% polyacrylamide gels
containing 7 M urea. The 12% gels were run at 500 V,
the 20% gels at 800 V, and the 25% gels at 1000 V.
Purification of restriction fragments.
M13 RFI
was cleaved with restriction endonuclease
HaelII, HinfI, or HpalI. The digests were then treated
with BAPF in the same buffer at 37~ After phosphatase treatment, bromphenol blue and glycerol
were added to 0.05% and 5%, respectively. The
digests were applied to 5% polyacrylamide gels
(40 x 20 x 0.3 cm or 20 x 20 x 0.3 cm) and subjected
to electrophoresis at 200-250 V. After electrophoresis,
the DNA bands were stained with 2/.~g/ml EtdBr and
visualized by ultraviolet (UV)irradiation. The bands
were cut out and the DNA was eluted as described by
Maxam and Gilbert (1977).
DNA
[y-3ZP]ATP exchange synthesis. [y-32p]ATP was
prepared as reported previously by Maxam and Gilbert (1977).
Kinase treatment. The kinase reaction conditions
were as reported previously by Maxam and Gilbert
(1977) except that only the HaelII fragments were
denatured prior to the kinase reaction.
M13 SS DNA-ceUulose hybridization. M13 SS DNA
was covalently linked to m-aminobenzyloxymethyl cellulose as described by Noyes and Stark (1975). Approximately 35/zg SS DNA was bound per milligram
of cellulose. The hybridization reaction contained
1-5/~g fragment, a 200-400 M excess of M13 SS DNA
(linked to cellulose) to fragment, 50% formamide,
0.75 M NaCI, and 0.075 M sodium citrate. The reaction
was heated to 75~ for 2 minutes and then incubated
for 18-24 hours at 42~ in a shaking water bath. After
hybridization, the reaction mix was centrifuged at
12,000g for 5 minutes at room temperature. The pellet,
containing the SS DNA cellulose hybridized to the
fragment, was washed three times at room temperature with 50% formamide, 0.75 M NaC1, and 0.075 M
sodium citrate. The supernatants from the initial centrifugation and the three washes were pooled, and the
DNA was precipitated with 0.1 volume 3 M NaC1 and 2
volumes 95% ethanol. This material was resuspended
in TE and subjected to electrophoresis through a 5%
polyacrylamide gel (59:1 ratio of acrylamide :bisacrylamide). The major band of the single-stranded fragment was eluted as described previously (Maxam and
Gilbert 1977) and represents the viral strand of the
hybridized fragment.
The SS DNA-cellulose pellet from the previous
centrifugations was washed three times at room temperature in 0.3 M NaCI and 0.03 M sodium citrate. The
pellet was resuspended in 99% formamide and heated
to 70~ and immediately centrifuged at 12,000g for 5
minutes at room temperature. This wash step was
repeated twice. The supernatants from the 99% form-
Downloaded from symposium.cshlp.org on March 5, 2016 - Published by Cold Spring Harbor Laboratory Press
M13 REPLICATION O R I G I N
amide washes were pooled and the DNA precipitated with ethanol. This material represents the complementary strand of the fragment.
Base-specific cleavage. The chemical reactions that
produce base-specific cleavages of DNA chains were
developed previously by Maxam and Gilbert (1977). In
the earliest experiment sequencing HpalI fragment F,
only four reactions were used: strong guanine/weak
adenine cleavage; strong adenine/weak guanine cleavage; c~,tosine cleavage; and cleavage at cytosine and
thymine. In all the subsequent experiments, a fifth
reaction was also performed: strong adenine/weak
cytosine cleavage.
Computer analysis of nucleotide sequence. The
computer method of Studnicka et al. (in prep.) was
used to analyze the nucleotide sequence for potential
secondary structures. This method uses the general
approach developed by Tinoco et al. (1971). The
nucleotide sequence is compared against itself in all
possible reading frames. A table of pairing regions is
established and the regions are assigned values of free
energies of base-pair formation based on the bonding
energies summarized by Salser (1977). The pairing
regions are then branch-migrated so that when two
regions partially overlap, portions of both regions may
be used in a structure. Using the table of pairing
regions, the computer generates the energetically most
favorable structures and computes the corresponding
free energies of formation.
The computer method of Korn et al. (1977) was
used to compare the M13 DNA sequence to the
nucleotide sequences near the replication origins of
several prokaryotic DNA species.
RESULTS
General Scheme for Sequence Analysis
The sites at which several restriction endonucleases
cleave M13 RF DNA have been mapped (van den
Hondel et al. 1975; Konings and Schoenmakers 1978).
Figure 1 is the restriction map determined from the
sequence analysis of the region between genes II and
381
IV. The two HpalI sites shown in this map delimit
HpalI fragment F, which has been shown to contain
the origins of replication of both the complementary
and viral strands (Tabak et al. 1974; Suggs and Ray
1977; van den Hondel 1976).
The restriction fragments that were subjected to
sequence analysis are indicated by the arrows in the
lower part of Figure 1. The sequence of nucleotides in
the intergenic region was determined for both strands
of the DNA except for a 60-nucleotide stretch near the
gene-IV boundary.
For the sequence analysis, the 5' ends of the restriction fragments were labeled in kinase reactions. Table
1 lists the restriction fragments that were kinaselabeled and the techniques that were used for separating the labeled 5' ends of each fragment. In most
instances, a kinase-labeled restriction fragment was
cleaved with a second restriction endonuclease to
separate the two labeled 5' ends. Separation of the
cleavage products was achieved by polyacrylamide gel
electrophoresis. In one instance, the labeled strands of
a restriction fragment were separated by hybridization
to M13 SS DNA cellulose.
The kinase-labeled fragments were subjected to
chemical reactions to produce base-specific cleavages
as developed by Maxam and Gilbert (1977). In most
instances, five reactions were performed: strong
guanine/weak adenine cleavage; strong adenine/weak
guanine cleavage; strong adenine/weak cytosine
cleavage; cytosine cleavage; and cleavage at cytosine
and thymine. The reaction products were subjected to
electrophoresis on polyacrylamide-urea gels. Figure 2
shows autoradiographs of sequence gels used to determine the nucleotide sequence of the region containing
the complementary-strand origin. On the left of each
gel is the deduced sequence. The sequence of the
region between genes II and IV of M13 DNA is shown
in Figure 3. The gene boundaries are those determined
by Schaller et al. (1978) for fd DNA.
Analysis of the Nucleotide Sequence
The nucleotide sequence was analyzed for unusual
base composition. The base composition of M13 SS
Figure 1. Physical map of the intergenic region of
Gene TV
Intergenic Region
Gene 11'
M13 DNA. At the top is shown the location of the
physical structures in this region in relation to the
hiirpin ~
J
AT rich
adjacent genes. The striped boxes represent hair~\\\\\\\~1
I~\\\\\~1 ~
~
li~i~i~i~i~i~i~i~ii!~iiiii:.!i~i~i:::iii]
pins; the cross-hatched box represents a pyrimidine
trace 20 nucleotides in length; the dotted box
20- mer
ori- RNA
Pribnow bo~ m- RNA
represents the AT-rich segment; and the solid box
represents the "Pribnow" sequence (Pribnow 1975).
Included on this map are the start point for com'~ I=I
I=I H
H
l~l
H~
plementary-strand synthesis (ori-RNA) (Geider et
e: ~
al. 1978) and the initiation site for gene-II mRNA
I I
I
I
I I
I I
I
(Schaller and Takanami, 1978). Below the physical
structures are the sites of restriction-endonuclease
N
cleavage as located by the sequence analysis. The
arrows at the bottom indicate the fragments that
were subjected to sequence analysis; the star on
the end of an arrow indicates the kinase-labeled 5' end of a strand and the length of the arrow indicates the length of sequence
determined. The region between genes II and IV is 504 nucleotides long.
Downloaded from symposium.cshlp.org on March 5, 2016 - Published by Cold Spring Harbor Laboratory Press
382
SUGGS AND RAY
Table 1. Restriction Fragments Subjected to Sequence Analysis
Restriction
fragment labeled
by kinase
treatment
HaelII
HaelII
HaelII
HinfI
HinfI
HpalI
HpalI
D
E
G
F
H
A
F
Number of nucleotides
sequenced (5'--+3'order)
Method for separating the two
5' ends of the kinase-labeled
fragment
viral
strand
digestion with HinfI
digestion with/-/haI
hybridization to M13 SS DNA-cellulose
digestion with TaqI
digestion with AluI
digestion with HaeIII
partial digestion with HaeIII
DNA is 41% GC (Salivar et al. 1964). The base
composition of the entire intergenic space is 45% GC;
however, within this segment are regions rich in GC
base pairs and regions rich in AT base pairs. The
GC-rich and AT-rich regions are indicated in Figure 4.
The first 74 nucteotides of the intergenic space (nucleotides 18-91) comprise a region that is 72% GC.
This region contains self-complementary sequences
that form a large hairpin (see below). There are large
blocks of AT base pairs in the right-hand half of the
intergenic space near gene II. A region of 104 nucleotides that contains the initiation site for gene-II
(b)
O.A A+GA>C C C,T
.
.
.
.
complementary
strand
384-529
235-120
245-360
1-70
310-395
135-250
275-225
529-480
125-1
505-370
mRNA (nucleotides 387-490) is 79% AT base pairs.
The function of these AT-rich segments is unknown.
Also contained in the intergenic space is an unusually
long stretch of pyrimidine residues (the 20-mer shawn
in Fig. 1). This is the long pyrimidine tract that has
been observed in the depurination analysis of Ff SS
DNA (Ling, 1972; Tate and Petersen 1974).
The nucleotide sequence of the intergenic region
was analyzed for possible protein-coding sequences. In
vitro experiments indicate that the entire M13 genome
is transcribed into RNA (Okamoto et al. 1969; Chan
et al. 1975). In vivo, the complementary strand of M13
(c) ....
G>AA+GA~'GC C§
(d)
A+GG,A C C+T
Figure 2. Autoradiographs of sequence gels of the region containing the origin for complementary-strand synthesis. The Hinf F,
Hae E, Hae G and Hpa F restriction fragments were end-labeled with [a-32p]ATP and T4 polynucleotide kinase. The two
labeled ends of each fragment were purified as described in Table 1. The labeled fragments were subjected to the base-specific
cleavage reactions described by Maxam and Gilbert (1977). The reaction products were subjected to electrophoresis of 20%
polyacrylamide-7 M urea gels. (a) Hinf fragment F complementary strand; (b) Hae fragment E complementary strand; (c) Hae
fragment G viral strand" and (d) Hpa fragment F viral strand.
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M1S REPLICATION ORIGIN
_..
.$8S
CA
gene IV
f~,
1 TAGTAC~CGCCCTGTAGICGG CGCATTAAGC GCGGCGGGTG TGGTGGTTAC GCGCGCGTGA CCGCTACACT TGCTCGCGCC CTAGCGCCCG CTCCTTTCGC
T
A
f
+
i01 TTTCTTCCCTTCCTTTCTCG CCACGTTCGC CGG~TCCC CGTCAAGCTC TAAATCGGGGGCTCCCTTTA GGGTTCCGAT TTAGTGCTTTACGGCACCTC
del eted
TC
It
201 C4kCCCCAAAAAACTTGATTT GGGTGATGGT TCACGTAGTG GGCCATCGCC CTGATAGACGGTTTTTCGCC CTTTGACGTTGGAGTCCACG TTCTTTAATA
A
CAA
A
C
AT
T AT
T T C
ff+
f
f
ff
+ ff
f f f
301 GTGGACTCTTGTTCCAAACT GGAACAACACTCAACCCTAT CTCGGGCTAT TCTTTTGATT TATAAGGGAT TTTGCCGATT TCGGCCTATT GGTTAAAAAA
401
A
T
A
C
+
+
+
f
T~GCTGATT T / ~ C ~ T
A
~
f
TTAACGC6/~ TTTT.~C.~M, ATATT/~CGT TTAC.~TTTA AATATTTGCT TATAC/~TCT TCCTGTTTTT GGGGCTTTTC
501 TGATTATCAACCGGGGTACA TATGATTGAC
gene IT
Figure 3. Nucleotide sequence of the untranslated region between genes II and IV of phage M13 DNA. The nucleotide
sequence of the viral strand is shown oriented in the 5'-*3' direction. Included in this figure are the gene boundaries as
determined for fd DNA (Schaller and Takanami 1978) and the sites at which the fd (Schaller and Takanami 1978) and fl
(Ravetch et al. 1977) DNA sequences differ. The fd DNA changes are shown above the sequence; the fl DNA changes are
shown below the sequence.
RF DNA serves as the template for transcription
(Jacob and Hofschneider 1969). Thus the intergenic
region is transcribed into RNA having the same sequence as the viral strand of the DNA. In the nucleotide
sequence of the RNA transcribed from the intergenic
space, there are two AUG triplets (positions 226 and
400) and eight GUG triplets (positions 38, 40, 43, 57,
184, 223, 238, and 301) that could potentially code for
the initiation of protein synthesis. All of these potential initiation codons are preceded by sequences 3 to 7
nucleotides in length that resemble known ribosomebinding sites (Steitz et al. 1977); however, all but one
of the potential initiation codons are located in regions
that would be base-paired in an mRNA molecule (see
below), and it is improbable that an AUG or GUG
triplet in the stem of a hairpin structure could initiate
i9
gene ~
protein synthesis. The only potential initiation codon
not located in the stem of a hairpin is the AUG triplet
at position 400. It is not likely that protein synthesis
initiates at this position, since this would result in the
synthesis of a dipeptide. The possibility exists that
protein synthesis is initiated Within gene IV in a
reading phase other than that of the gene-IV protein.
The TAG triplet at positions 1-3 would terminate
translation in one alternate reading phase. The TAA
triplet at positions -41 to -39 (data not shown)would
terminate translation in the other alternate reading
frame. It should be stressed that the exact structural
requirements of a translational initiation site are not
known; for example, Steege (1977)has presented
evidence that protein synthesis can start at codons
other than AUG and GUG. Thus, given this proviso,
st
tat
'.-' - I G I ~ ; GCGCC C I G I ~ GGCG C ~ C
GCGGCG G C ~ G G ~ , ~
GCGCGCGIG~CG C I ~ I ~ C G C
GCCC ~ C GCCCGCICI C G : l l : . C I
ClICGC:~CI
GCCGGC
GCG G G C G I G I G C ~ G I ~ G C G ~ ~ C G G C C G
I C I G C G C G G G [ ~ C CGC~ I , : G C G C CGCCCIcIcc~IGCGCGCGCIC~GC~ ~ G C G C G G ~
15!
9
9
I
:
.
:
....
:
...........
45t
.....
.... ....
~
9
GG~CC~GGcci~
:'
..........
Frlbnow
.....
..i ....
I
c(;cc / { : ! ~ I G ~ C CIC~ C ~ ~ C [ I C / I G ~
r
~:;cc , : G m m G c m m c ~ ; ~ G I G ~ c l i : m m G , ~ c ~ G ~ c c c ~ J I c ~ ~ c c c / r
sequence
; 501 ~:i ....
................ ,,,,,~,,;:,.... ........
gene~ m R N A
~
~
..............
rl:Dosome
gene
binding
s i fe
_ _
Figure 4. AT-rich regions in the intergenic space of M13 DNA. The nucleotide sequence of both strands of the M13 intergenic
space are shown with the AT base pairs drawn in shaded boxes. Included are the gene boundaries and the Pribnow sequence,
initiation site, and ribosome-binding site for gene-II mRNA as proposed for fd DNA (Seeburg et al. 1977; Schaller and
Takanami 1978).
I
Downloaded from symposium.cshlp.org on March 5, 2016 - Published by Cold Spring Harbor Laboratory Press
384
SUGGS AND RAY
cleotides of gene IV and the first 36 nucleotides of gene
II. (Not all of the sequence is shown.) A comparison of
the M13 D N A sequence to the fd D N A sequence
reveals a single A->G change at position -23. This
change occurs in the third position of a codon and
does not result in a change in the amino acid sequence
of the gene-IV protein.
The nucleotide sequence of the M13 intergenic
space was analyzed for possible secondary structures
by the computer method of Studnicka et al. (in prep.)
as described in Materials and Methods. Three large
hairpins and numerous small hairpins were predicted;
the predicted structures of the energetically most
favorable hairpins are shown in Figure 5. The first
large hairpin occurs immediately after the end of gene
IV in a GC-rich region. This hairpin is followed by a
pyrimidine-rich sequence including the 20-mer mentioned above. Immediately after the pyrimidine-rich
segment are two large hairpins that have been implicated in initiation of complementary-strand synthesis
(see Discussion). In addition, there are two small
hairpins, one of which may function in viral-strand
synthesis (see Discussion). When the remainder of the
nucleotide sequence of the intergenic space was analyzed for secondary structures, no clearly stable structures were predicted.
The computer method of Korn et al. (1977) was
used to compare the nucleotide sequence of the M13
intergenic space to the following nucleotide sequences:
the replication origins of plasmid ColE1 (Tomizawa et
al. 1977), bacteriophage A (Denniston-Thompson et al.
1977), and E. coli (Messer et al.; Hirota et al.; both
we conclude that the region of 504 nucleotides between genes II and IV is not translated into protein.
The entire fd genome has been sequenced (Schaller
and Takanami 1978), as has a portion of the fl
intergenic space equivalent to nucleotides 127-234
(Ravetch et al. 1977). The M13 D N A sequence was
compared with these sequences and was found to be
very similar. The differences between the nucleotide
sequences are indicated in Figure 3. Between the M13
and fd D N A sequences there are 24 base substitutions
(5% of the nucleotides in this region), whereas between the M13 and fl D N A sequences there is one
base substitution and a deletion of 2 nucleotides (3%
differences). Most of the differences between the M13
and fd D N A sequences are clustered in two small
regions (334-346 and 368-389). In the region in which
all three phage genomes have been sequenced, there is
no position at which the sequence is not common to at
least two of the phages. With respect to the restriction
map of this portion of the genome, four base substitutions produce three major differences between the
M13 D N A map and the fd D N A map: (1) The base
change at position 75 creates a HaeII site in fd D N A
(nucleotides 75-80)very close to another HaeII site
(nucleotides 83-88); (2) the base substitution at position 162 creates a Bam I site in the fd intergenic space
(nucleotides 160-165); and (3) the base change at
position 346 creates one HaeIII site in fd D N A
(nucleotides 344-347), and the base changes at positions 383 and 386 eliminate another HaeIII site in fd
D N A (nucleotides 383-386). In addition to the intergenic space, we have sequenced the last 56 nu-
GC
C
G
G-C
SZ/C-G
A-T
T.G
T-A
G-C
G-C
T.G
G-C
G.T
T-A
O-C
T-A
G-C
G.T
G-T
C-O
TT
T
T
CTC-G T
G
GGG-CT
G-C
C-G
T-A
A-T
A-T
A-T
151-'-T-A
C-G
C-G"~A
G-C
C-G
T
TAAG-CCc
.
9
m
.
I
TAC-GAT
G-C
C-G
G-C
G-C..
C_GI"
. c-r .
A
ln
.l
I
.
.
.
T
It
de1 e
#
~
"
T
C-G
G-C
A-T
A-T
C
T
T-A
G-C
C-G
C-G
A-T
C-G
T'G
T'G
G-C
G--C
T-A
A-T
G-C
A T -G
9 G-C
" G-C
TT'rG-cTGAT
A
A
G TC-GG... G
T A-T L.~
T C A-T
,~ ,~ A-T
201 " ~ ' ~ A-T
272
I
~
__
I
TcTTTAA
TGc-GAT
A-T
C-G
C-G
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T ACACTCA - 3 '
Complernentory
Strond Origin
Figure 5. Secondary structure in the intergenic region of M13 DNA. The most stable secondary structures of the viral strand of
the M13 intergenic space near gene IV is shown as determined by the computer method of G. Studnicka et al. (in prep.).
Included are the sites at which M13 differs from fd (solid arrows) and fl (dashed arrows), the gene-IV boundary as determined
for fd (Schaller and Takanami 1978), and the site of initiation (nucleotide 272) and direction of synthesis of the or/RNA (Geider
et al. 1978).
Downloaded from symposium.cshlp.org on March 5, 2016 - Published by Cold Spring Harbor Laboratory Press
M13 REPLICATION ORIGIN
this volume); the complementary-strand origin of G4
DNA (Fiddes et al. 1978); the viral strand origins of
~bX174 DNA and G4 DNA (ibid.); and the recognition
site for DNA gyrase (Gellert et al., this volume). No
significant homologies were found in any of the above
comparisons.
DISCUSSION
The mechanism of Ff complementary-strand synthesis has been well defined, mainly by use of the in
vitro SS--,RF reaction. Replication intiates at a unique
site on the phage DNA (Tabak et al. 1974). The
initiation event involves synthesis of an RNA primer
by rifampicin-sensitive RNA polymerase (Brutlag et
al. 1971; Wickner et al. 1972). In the presence of E.
coli DNA-binding protein (DBP), RNA polymerase
binds to phage DNA and protects a unique region of
the DNA (or/) from nuclease digestion (Schaller et al.
1976). The enhanced mobility of the single-stranded ori
fragment during gel electrophoresis and the fragment's
resistance to single-strand-specific nucleases (Schaller
et al. 1974), reveal its partial doubled-strand character.
The ori DNA fragment corresponds to two large hairpins
between nucleotides 140 and 266, which are shown in
Figure 5. The nucleotide sequence of the RNA primer
for the in vitro SS--,RF reaction has been determined
(Geider et al. 1978). The RNA initiates at nucleotide 272
and is approximately 30 nucleotides long.
These data have led to a model for initiation of
synthesis of the complementary strand in vitro (Geider
et al. 1978). Initially, E. coli DBP coats the singlestrand regions of the phage DNA. The large hairpin
structures may not be coated with DBP, thus making
the ori DNA region accessible to RNA polymerase.
RNA polymerase binds to the or/DNA fragment and
synthesis proceeds into the base-paired region. The
hairpin is denatured as it is transcribed, which may
allow DBP to coat the opposite strand of the hairpin.
When RNA polymerase reaches the loop at the top of
the hairpin, the opposite strand of the hairpin, which is
ahead of the polymerase, may be coated with DBP;
thus, RNA polymerase is displaced from the DNA,
terminating primer synthesis. DNA polymerase III
holoenzyme initiates synthesis at the 3' OH of the
RNA chain and continues around the circular template. Unexplained by this model is the mechanism in
this system that enables RNA polymerase to protect
from nuclease digestion a region that is downstream
from the site at which synthesis initiates (see Fig. 3 of
Gray et al. 1978). On fully double-stranded templates,
RNA polymerase protects from nuclease digestion a
region that encompasses the initiation site (see Fig. 4
of Pribnow 1975). Also in need of explanation are the
results of experiments by Barnes (1978) in which he
inserted a portion of the histidine operon of Salmonella typhimurium into M13 DNA at the HaelII
site at position 242-243. The insertion destroys most of
the secondary structure of the hairpin that is the
template for synthesis of the RNA primer. Mapping of
385
more insertion sites should help to more clearly define
the origin of complementary-strand synthesis.
With respect to the structure of the or/ DNA
fragment, which is the binding site for RNA polymerase in this system, a comparison of the M13 and fd
DNA sequences of the approximately 150 nucleotides
in this region reveals four positions at which base
substitutions can be tolerated. One of the base
changes significantly alters the predicted secondary
structure of this region. Gray et al. (1978) drew the fd
ori DNA as a structure in which the initiation site is
contained in a base-paired segment between the two
large hairpins. According to the base-pairing rules
employed in our study, this pairing region would be
weakly stable in fd DNA; however, due to the base
change at position 205 in the M13 DNA sequence, this
pairing region would be unfavored in M13 DNA.
Whatever the means by which RNA polymerase initiates synthesis at this site, the mechanism probably
does not involve a template in which the site of
initiation is base-paired in a double-stranded structure.
A comparison of the M13 and fl DNA sequences
near the complementary-strand origin reveals two additional sites of sequence divergence, one of which
alters the predicted base-pairing of this region. The
base substitution at nucleotide position 220 of fl allows
the formation of four additional base pairs in the stem
of the large hairpin between nucleotides 208-266,
according to the base-pairing rules employed in our
study (Salser 1977). The effects, if any, of this base
substitution on the function of the ori DNA region are
not known.
Both the synthesis of the complementary strand of
M13 and the replication of the ColE1 plasmid are
sensitive to rifampicin (Brutlag et al. 1971; Wickner et
al. 1972; Clewell 1972), indicating that the two systems
require RNA polymerase. Tomizawa et al. (1977) have
determined the nucleotide sequence of the replication
origin of ColE1 and we have compared this sequence
to the sequence of the M13 complementary-strand
origin. No significant sequence homology was found;
however, the two replication origins are similar in that
both have pyrimidine-rich segments and large hairpin
structures (S. V. Suggs, unpubl.). Perhaps RNA
polymerase does not recognize a specific nucleotide
sequence when priming DNA synthesis but rather
recognizes some structural feature of the DNA.
The site of termination of a round of complementary-strand synthesis has been localized by gap-filling
of the RFII DNA product of the in vitro SS~RF
reaction on polA1 cell extracts. The site of the discontinuity in t h e RFII DNA is in HpalI fragment F
(Tabak et al. 1974), very close to the HaelII site at
positions 242-243 (J. M. Cleary, unpubl.). These data
indicate that complementary-strand synthesis terminates near or at the point of transition from RNA
primer to DNA, which Geider et al. (1978) localized at
nucleotide 242.
The mechanism of viral-strand synthesis is not as
well understood as the mechanism of complementary-
Downloaded from symposium.cshlp.org on March 5, 2016 - Published by Cold Spring Harbor Laboratory Press
386
SUGGS AND RAY
strand synthesis; this is mainly due to the difficulties
involved in developing an in vitro RF~SS system. At
present, the rolling-circle model (Gilbert and Dressier
1968) seems to more accurately fit the data than do
other models. Accordingly, the initial event in a round
of viral-strand synthesis would be the nicking of the
viral strand of an RFI (superhelical, covalently closed,
circular, duplex DNA) molecule at the site of initiation. A requirement for supercoiled RFI DNA is
indicated by the sensitivity of the in vitro RF--->SS
reaction to antibiotics that inhibit DNA gyrase
(Staudenbauer et al. 1978). Gene-II protein of the
phage probably performs the nicking function. It is
known that in vivo gene-II product allows accumulation of RFII (circular, duplex DNA in which at least
one strand is not continuous) nicked specifically in the
viral strand (Fidanifin and Ray 1972; Lin and Pratt
1972). Very recently, the site at which purified gene-II
protein nicks the fd viral strand in vitro has been
localized at position 296-297 (Geider and Meyer, this
volume). The 3' OH at this site might serve as a primer
for DNA polymerase III holoenzyme to initiate synthesis. After a round of synthesis by DNA polymerase III
holoenzyme, the gene-II protein might again nick at its
specific site to generate an RFII DNA molecule and a
viral single strand. It is not now known if the gene-II
protein has any of the novel properties displayed by the
analogous SX protein, the cistron-A protein (Eisenberg
et al. 1977).
If the gene-II protein both nicks the viral strand and
subsequently ligates the single-strand product, then
the origin and terminus of the viral strand are at
precisely the same location. The experiments analyzing RF DNA made in vivo late in infection have
localized the terminus of viral-strand synthesis. Two
experimental techniques have been used: gap-filling of
pulse-labeled RFII DNA (Suggs and Ray 1977)and
determination of the distribution of a short pulse label
in RFI DNA (Horiuchi and Zinder 1976; Suggs and
Ray 1977). These experiments place the site of termination of viral-strand synthesis near the center of the
intergenic space. More recent experiments, in which
the HpalI F fragment of M13 RFI DNA pulse-labeled
late in infection was cleaved with restriction endonucleases HaelII and HinfI, show that termination occurs
very close to the HinfI site at position 304-305 (S. V.
Suggs, unpubl.). This result places the terminus near or
at the gene-II nicking site. The proximity of the terminus
and the gene-II nicking site is consistent with a replication model in which nicking occurs at the
origin-terminus.
In the 70-nucleotide segment encompassing the
viral-strand origin-terminus, the M13 and fd DNA
sequences are completely identical. This finding may
imply a stringent requirement for specific nucleotide
sequences at the viral-strand origin.
It has been. proposed that Ff DNA replication
involves only two reactions: SS~RF and RF--->SS
(Horiuchi and Zinder 1976). According to this
hypothesis, RF DNA replication would be a composite
of these two reactions, each reaction occurring independently of the other. This model is compatible with the
relative locations of the two origins. In the RF-->SS
reaction, the last segment to be replicated is the region of
the complementary-strand origin; thus complementarystrand synthesis could not initiate until after completion
of the entire viral-strand template. A similar argument
can be made for the requirement of a completed
complementary strand for initiation of viral-strand
synthesis. The requirement that a round of complementary-strand synthesis be completed prior to initiation of
viral-strand synthesis is also indicated by the sensitivity
of the RF-->SS reaction to antibiotics that inhibit DNA
gyrase (Staudenbauer et al. 1978).
If Ff duplex DNA replication employs no replication systems other than those of the SS-->RF and
RF-->SS reactions, then the apparent requirements for
the dnaB (Olsen et al. 1972) and dnaG (Ray et al.
1975; Dasgupta and Mitra 1976) gene products for in
vivo RF DNA replication must be explained. Staudenbauer et al. (1978) have attempted to reconcile these in
vivo results with the two-step model for RF DNA
replication by proposing that (at the nonpermissive
temperature) dnaB and dnaG mutants accumulate
single-strand regions in the host chromosome as a
result of abortive replication and that the single-strand
regions adsorb E. coli DBP to such an extent that Ff
DNA replication is inhibited. It is hoped that the
development of a soluble in vitro RF DNA replication
system for Ff DNA will resolve this question.
Included within the Ff intergenic space, in addition
to the origins for DNA replication, is a promoter for
transcription. The location of this promoter (described
as G1 [Okamoto et al. 1969, 1975], G0.08 [Konings
and Schoenmakers 1978], and P[II] [Schaller and
Takanami 1978]) has been deduced from the results of
several experiments: (1) mapping of DNA fragments
which bind RNA polymerase (Seeburg and Schaller
1975; Seeburg et al. 1977); (2) sizing of in vitro
transcripts of whole RF DNA or of restriction fragments (Okamoto et al. 1969, 1975; Seeburg and Schaller 1975); and (3) analyzing protein products made in a
coupled transcription-translation system using restriction fragments as templates (Horiuchi et al. 1978). The
promoter is located within a region rich in AT base
pairs (see Fig. 4). We propose that the AT-rich region
is involved in the regulation of gene-II expression by
gene V. The results from two experiments suggest that
gene V is a negative control element for gene II: (1) In
vivo, considerably more gene-II protein can be isolated from cells infected with gene-V amber mutants
than can be isolated from wild-type infected cells
(Meyer and Geider 1978), and (2) in a coupled transcription-translation system, the addition of gene-V
protein to the reaction decreases the amount of geneII protein produced (Horiuchi et al. 1978).
To explain these results, we propose the following
model. Late in infection, a large pool of gene-V
protein accumulates within the cell (Oey and Knippers
1972; Alberts and Frey 1972). Gene-V protein, which
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M13 R E P L I C A T I O N O R I G I N
binds specifically to single-stranded D N A (Oey and
Knippers 1972; Alberts and Frey 1972), might invade
the supercoiled R F D N A in the AT-rich region
around the gene-II promoter. Morrow and Berg (1973)
have shown that a single-strand-binding protein, the
gene-32 protein of bacteriophage T4, can bind to
simian virus 40 (SV40)supercoiled D N A at a unique
site that may be rich in A T base pairs. The binding of
gene-V protein to the AT-rich region of M13 RFI
D N A might prevent binding of R N A polymerase at the
gene-II p r o m o t e r site or inhibit progression through this
region of R N A polymerase molecules bound at promoters upstream. Thus, we propose that in the former case,
the AT-rich region is an operator and the gene-V protein
is a repressor and that in the latter case the AT-rich
region is an attenuator. We are currently investigating
this possibility; preliminary results have shown that
gene-V protein does bind to M13 D N A supercoils.
Segments of the Ff intergenic space may be involved
in functions other than those discussed above. This
region may contain a site at which morphogenesis
initiates or a site for rho-dependent termination of
transcription (Staudenbauer et al. 1978). At present,
there is no known function for the 20-nucleotide-long
pyrimidine tract or for the very large, GC-rich hairpin
at the gene-IV boundary of the intergenic space (see
Fig. 5). The pyrimidine tract may be involved in
initiating complementary-strand synthesis on the basis
of similarities between the M13 and ColE1 origins (see
above); however, the large, GC-rich hairpin is probably not involved in complementary-strand synthesis,
since it is not protected from nuclease digestion by
R N A polymerase (Schaller et al. 1976). Further studies
should elucidate the contributions of the structural
elements of the Ff intergenic space to the various
functions of this region of the genome.
Acknowledgments
This research was supported by grants from the
National Institutes of Health (AI 1 0 7 5 2 ) a n d the
National Science Foundation (PCM 76-02709). S. V.
S. was supported by a training grant from the National Cancer Institute (CA-09056).
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Nucleotide Sequence of the Origin for Bacteriophage
M13 DNA Replication
S. V. Suggs and D. S. Ray
Cold Spring Harb Symp Quant Biol 1979 43: 379-388
Access the most recent version at doi:10.1101/SQB.1979.043.01.044
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