MICROBIOLOGY
REVIEWS
ELSEVIER
FEMS Microbiology Reviews 17 (1995~ 99-107
Bacteriophage P4 DNA replication
Giinter Ziegelin, Erich Lanka *
Max-Planck-lnstitut fiir Molekulare Genetik, Abteilung Schuster, lhnestrasse 73, Dahlem, D-14195 Berlin, Germany
Abstract
Replication of satellite phage P4 of Escherichia coil is dependent on three phage-encoded elements: the origin (ori), a
cis replication element (crr), and the product of the a gene, g p a . In vitro P4 replication is origin-specific resulting in
monomeric form I DNA. D N A synthesis requires chromosomally encoded proteins DNA polymerase III holoenzyme, SSB,
DNA gyrase and probably topoisomerase I; host-encoded initiation and priming functions are dispensable. The ot protein is
multifunctional in P4 replication, combining three activities in a single polypeptide chain. First, the protein complexes
specifically with type I repeats at ori and crr. Second, the helicase activity associated with gpt~ unwinds DNA with 3' --~ 5'
polarity. Third, the primase activity results in the synthesis of RNA primers. Defined sequence motifs in g p a correlate with
the helicase and primase activities which are arranged in distinct, separable domains. Primase activity is associated with the
N-terminal half of the protein, o r i / c r r binding with the C-terminal portion. A model for the initiation mechanism of P4
replication which resembles that of mammalian simian virus 40 is discussed.
Keywords: P4 gpct; DNA replication initiation; Origin binding protein; DNA helicase; DNA primase
1. Introduction
Bacteriophage P4 is a small naturally defective
temperate virus of Escherichia coli (for recent reviews see [1,2]). Phage P4 propagation can only
occur in the presence of phage P2 genes which
provide structural and functional components for encapsidation. The P4 genome consists of an 11 624-bp
double-stranded, linear DNA possessing 19-bp single-stranded cohesive ends (for capsid structure and
D N A paul':aging see Lindqvist and Naderi [44]). The
substrate far packaging is negatively supercoiled circular DNA (form I).
* Corresponding author. Tel.: +49 (30) 8413 1242; Fax: +49
(30) 8413 1393
Two forms of lysogeny are known for P4: (i)
integration into the host chromosome preferentially
at the P4 att site located at the 3' end of the leuX
gene of E. coli C or K12. Integration of the P4 DNA
via site-specific recombination is catalysed by the P4
integrase (In0, which may also mediate excision
[3,4]; (ii) as a high copy number plasmid. Lysogenisation resulting in the plasmid state occurs only in a
small fraction (approx. 0.3%) of P4 lysogens (for
regulation and maintenance of the P4-1ysogenie state
see Dehb and colleagues [45]).
Replication of P4 DNA is independent of helper
phage functions. In vivo D N A synthesis starts at an
unique origin ( o r i ) in a bidirectional 0-type manner
[5]. In addition to the origin, a second intergenic
region, the cis replicating region (crr) is required
for replication [6]. A single P4-encoded protein, the
product of the ot gene ( g p a ) is sufficient for P4-
0168-6445/95/$29.00 © 1995 Federation of European Microbiological Societies. All rights reserved
SSDI 0168-6445(94)00080-8
G. Ziegelin, E. Lanka / FEMS Microbiology Reviews 17 (1995) 99-107
I00
E,.~b. replicat/on
s"
4p
~q,q
~
"dk
supples~~u
Fig. 1. Genetic map of the bacteriophageP4. The map has been
drawn accuratelyfrom the completenueleotidesequenceof the P4
genome [7]. Elements involved in replication are indicated by
shading, the relevant gene starts are marked by arrowheads.
Genetic elements of P4 required for the various phage-encoded
functions are given outside of the circle.
specific replication (Fig. 1). Replication during the
plasmid state or during the lytie cycle is gpte-dependent. Host replication proteins thought to be required
for P4 DNA synthesis in vivo include DnaE (the key
subunit ot of DNA polymerase III holoenzyme), and
SSB. Proteins DnaA, DnaB, DnaC, DnaG, Rep and
RNA polymerase are dispensable. These early observations made in the Calendar laboratory in the midseventies, suggested P4 replication to be unique
among temperate phages in terms of recruiting only
a few host replication proteins [8], i.e. the elongation
machinery.
2. In vitro replication of P4 D N A
Phage P4 DNA is replicated in cell-free extracts
of Escherichia coli in the presence of partially purified P4 tz protein [9]. By using a modified in vitro
replication assay, which essentially consisted of exchanging the buffer system and introducing an
ATP-regenerating system, we have further character-
ized this process [10]. Analysis by agarose gel electrophoresis and autoradiography of in vitro replicated
molecules demonstrated that the system yields supercoiled monomeric DNA as the main product. Electron microscopic analysis of in vitro generated intermediates indicates that DNA synthesis initiates
mainly at ori, the origin of replication used in vivo
[5]. Replication proceeds from this origin bidireetionally, resulting in 0-type molecules. In contrast to the
in vivo situation, no extensive single-stranded regions were found in these intermediates in vitro. The
initiation proteins of the host, DnaB and DnaG, and
the chaperones DnaJ and DnaK are not required for
P4 replication, since polyelonal antibodies against
those polypeptides do not inhibit the process. The
reaction is inhibited by antibodies against the SSB
protein, and by ara-CTP, a specific inhibitor of DNA
polymerase III holoenzyme (Table 1). Consistent
with previous reports, P4 in vitro replication is independent of transcription by host RNA polymerase,
because the system is rifampicin-resistant. Novobioein, a DNA gyrase inhibitor, strongly inhibits P4
DNA synthesis, indicating that form I DNA is the
preferred substrate [10].
In summary, P4-specific DNA synthesis L'~ vitro
depends on P4 gpoe, DNA polymerase III holoenzyme, SSB and DNA gyrase. Topoisomerase I may
be needed for the maintenance of a certain degree of
negative superhelicity.
Table 1
Requirements for P4 DNA synthesis in vitro
Omission ( - - ) / a d d i t i o n ( + )
Yield (%)
Complete a
- P4DNA
- gpa
- Energy mix b
- PEG (6000)
4- ddTl'P ( 3 0 / x M )
+ A.ra-CTP (400/,~M) c
+ Rifampiein ( 2 0 / z g / m l )
+ Novobiocin ( 1 0 / ~ g / m l ) d
100
<5
<5
"< 5
82
10
30
95
< 5
a Approximately 50 pmol of dTMP incorporation depending on
extracts,
b Supply of ATP and an ATP-regenerating system.
c Ara-CTP, cytosine ~-D-arabinofuranoside 5'-triphosphate.
a Assayed at 35°C.
lot
G. Ziegelln, E. Lanka / F E M S Microbiology Reviews 17 (1995) 99-107
3. A multifunctional replication
combines three activities
protein: g p a
ca
gpa binding
In vivo and in vitro studies suggested gpc~ to be a
multifunctional replication protein active in initiation
and elongation of P4 D N A synthesis. Thus, potential
activities in D N A binding, unwinding and priming
were assayed systematically with purified ot protein.
Direction of
replicatio~
Fig. 2, Replication origin of phage P4. The 360-bp origin fragment is shown (P4 coordinates 9107 [Sspi]-9467 [BstNl]).
A/T-r/ch ( > 70%) segments are shaded. Arrowheads represent
sequence repetitions, Protection of ori DNA by gpc~ against
DNase i cleavage is summarized by brackets.
Purified gp(x recognizes specifically ori and e r r
fragments as demonstrated by DNA-protein complex retention on gels and DNase I footprinting
which showed that only type I repeats are protected.
Protection of type II repeats by purified gp a has not
been found (Fig. 2; [11]). Specific complexes were
also visualized in the electron microscope. In addition, looping between ori and e r r was observed in
the presence of gp ~ in supercoiled and linear DNA
(Fig. 3). Complexes visualized in the electron microscope are normally quite large and are comparable to
the size of RNA polymerase-promoter DNA complexes suggesting that more than one or two gp,a,
monomers are present.
5. Helicase activity of gp o~
The independence o f P4 replication from the
replicative heliease DnaB or Rep protein suggested
that g p a might possess unwinding activity which
was indeed the case [11]. The helicase activity is
accompanied by a NTPase activity that was found to
be associated with purified gp a . The NTPase activity of g p a is stimulated by single-stranded DNA. In
addition, a nucleotide binding site has been found in
the primary structure of g p ~ (vide infra). Substitu-
m,,o,
-
type I
TGTTCACC
~oo b 2 . -
4. gpez is a specific oH-and err-binding protein
L-[
type II
YCAYTTAAAG
,,,
f,O ggl
a
4
'~
;-~'="~'~...........
i~ 3.-::..,..~i:~-~.....-,~~.-..-.=~:_,.:~
.-;:
i1~
:,
7,
0
:~
1000 bp
0
,.
n
,-
~
-
A
Fig. 3. Electron microscopy of gp(z-P4 DNA complexes. The complexes were formed by incubating supercoiled P4 DNA with gpa.
Following fixation with glutaraldehyde, the DNA was cut with Pvul and prepared for electron microscopy as described [12]. The histogram
depicts the frequency distribution of protein bound to ori and err. Within the upper bar representing the P4 genome of 11624 bp, ori, err
and the a gene are marked. Insets: representative electron microgmphs of looped gpo~-P4 DNA complexes, the bar represents 1 kb.
102
G. Ziegelin, E. Lanka / FEMS Microbiology Reviews' 17 (1995) 99-107
tion of amino acid residues in this nucleotide binding
fold resulted in helicase-null mutations. The heliease
activity unwinds DNA in the presence of NTPs in a
3 ' ~ 5' direction with respect to the strand it has
bound [11]. A tailed helicase substrate mimicking
some of the structt~ral features of a replication fork,
is unwound several fold more efficiently compared
to an untailed one. This observation gave a clue for
the role of g p a as a replicative DNA helicase. The
results indicate that g p a enters the duplex region
more efficiently when it encounters a fork rather
than a fully base-paired region. All hydrolysable
nucleoside 5' tdphosphates function as substrates
except UTP and dTYP. The reaction requires Mg 2+
or Mn 2+ ions. Kinetic studies with a short-tailed
substrate (single-stranded M13 m p l 8 DNA to which
a 45-mer oligodeoxynucleotide is hybridized via 22
complementary base pairs at its 3' end) demonstrated
that a lag phase of several minutes at 37°C is required until a displaced product was detectable. Thus,
the rate-limiting step in this reaction may be the
formation" of an active enzyme-substrate complex,
involving the oligomerization of gp o~ monomers on
the template.
6. Primase activity of gpo~
Barrett et al. [13] have described a rifampicin-resistant RNA polymerase activity of g p ~ on several
synthetic DNA templates. Since P4 replicates in
dnaGts mutants at non-permissive temperatures, the
phage was thought to be independent of cellular
priming systems [8]. Thus, it was proposed that gp~x
possesses primase activity. Initiation of complementary strand synthesis on phage fd D N A in the presence of gpot demonstrated a priming activity [14].
The reaction was rifampiein-resistant and dependent
on N T P s / M g 2+. We have also shown that purified
gpot synthesizes oligoribonucleotides of defined sequence on single-stranded D N A [11]. The synthesized oligoribonueleotides are complementary to the
template DNA. Di- to pentanucleotides are generated
on viral fd DNA, with the predominant product
being the dimer pppApG on which most of the
longer products are based. Using synthetic
oligodeoxynucleotides of defined sequence as templates, synthesis of pppApG was also detectable
suggesting that a two-base template 3'-d(TC) is nec-
Table 2
Prokaryotie DNA primases, their template requirements and
primers synthesized
Source of
rNMP product
DNA
dNMPtemplate
PhageP4
pppAfiH '
Spa
3 ~]TCN...
E, cob
DnaG.
pppAGG/A.,,
3 '[iTCC/T. . .
Phage T7
pppACCC]A
ReFerence
II
15, 16, 17;
~4
3'i~G~/r
3, ~rGTG
pppACAC
18, 19
Phage T4
~,]36118p41
pp~_/~C(rl')z.: z
3 '~TG (N)z.3
pp.p_GC(1'1')z.3
3 '~jl2G(~) t. 3
2o, 21
Piasmid RP4
TraC
(p)CA...
3 ' f i t . +.
(p)GC...
3 ' CT.,.
22
Plasmid Collb-P9
SO8
(p)CA...
3 ' GT...
(p)GC...
3 ' CT...
22
The shaded nueleotide marks a template recognition site for the
enzyme.
essary and sufficient to support the synthesis of
pppApG by gpo~. Requirements for several prokaryotic primases (Table 2) indicate that in all cases short
oligoribonucleotides are synthesized in a templatedependent manner. In most cases, the 5' nucleotide
of the primers consists of a 5' triphosphate adenosyl
residue. DnaG protein, the E. coli D N A primase,
and gpo~ seem to belong to the same class. The
primer sequence is similar and both are elongated b y
D N A polymerase III holoenzyme. However, suppression of E. coli dnaG mutants in the presence of
the a gene has not been observed (unpublished, R.
Calendar and E.L.). The same observation was made
when the heliease activity of gp ~ was inactivated b y
mutation or the ability to specifically bind D N A was
absent. The remaining primase activity did not suppress dnaG mutants, perhaps because gp a is unable
to interact specifically with other E. coli initiation
proteins, The only primases known to suppress the
thermosensitive phenotype of dnaG mutants are
plasmid-encoded enzymes [23].
7. gpo~ shares sequence motifs with D N A primases and helicases
gpot consists of 777 amino acid residues (subunit
M r = 84 900) with a net charge of + 2 and a calculated isoelectric point of 7.8. Computer algorithms
G, Ziegelin, E. Lanka / FEMS Microbiology Reviews 17 (1995) 99-107
revealed that a region of about 120 amino acid
residues shares significant sequence similarity with
plasmid-eneoded D N A primases (approxinmtely 35%
identity and 55% similarity; [14]). Several self-transmissible plasmids within incompatibility groups lneI,
lneP, etc. [22] encode primases belonging into a
class of enzymes which are known to be involved in
conjugative D N A transfer [24,25]. A motif of six
amino acids (EGYATA) has been shown by mutational analysis to be essential for primase activity in
two of these enzymes -the RP4-specified primase
and gpc~ [14]. A rudimentary form of this motif has
been detected in the sequences of prokaryotic primases (Fig. 4; [26-28]). A second domain of g p a
may be involved in primase activity since it consists
o f a potential Zn 2+ -ion binding site (Fig. 5). In the
gp4 of phage T7 which possesses primase and helicase activity, the absence of these residues in the
corresponding domain abolished primase activity
completely [29]. The cysteine-rich motif in T7 gp4
has been shown to bind Z n z÷ [30]. It has been
proposed that this region of T7 gp4 may be involved
in the recognition and selection of priming sites on
the template D N A [29]. Another interesting domain
of gp a aligns well with the nucleotide binding fold
P4 gp=
201
C 0 , } ~ 80g
U. ~P/OnaS
255
251
Ft. prow. DnaG
S. ~/~hO,aG
243
252
Bu.aphLDnaG
RSFI010 RepB
"17 gp4
T3 glO4
"i'4 gp61
Consensus
EG QNQ AG~W~..
T.'~/HHLTTGE
LGTPI~qGQ~.~'T_~S
~HF-~TGL P
QQDNAEP~
. ~ : ~ A Q Y
I S S SYKKN~F~.~HOA.
Q Q Y S A E P Q ~ / ~ Q Y
........ ~ ~ ~ ' g Q Y N I
R L H I R K Q ~ ~ V S S .
•G I N
GFN
•D I N
EY
DVK
463
1.44 K H L h r ~ G G ~ ~ ~ L Q D C K
143 K.HLWS G G K ~ ~
~ , ~ : ~ L
QDCK
=o.,22 'L,'E
RVI<.DG D ~ ~
S~'F'~IEN G m"A Z T
bhhhh~YhDhh
13
Fig. 4. Amino acid s e q u e n c e alignment of a common motif among
prokaryotic D N A primases. Numbers indicate the sequence positions of the first amino acid residues shown. Conserved amino
acid positions are shown with a black background. Positions
which are identical or similar in at least seven or four sequences
are underlayed in dark or light grey, respectively. Consensus
sequence: b, basic residue; h, hydrophobie residues, respectively.
GenBank/EMBL accession numbers of sequences: phage 1)4
gpte, A26869; RP4 TraC2, X59793; R751 TraC4, X59794; CollbP9 Sog, A42815; E. coli DnaG, J01687; S. typhimurinm; DnaG,
B23985; R. prowazekii DnaG, $27660; Bu. aphidicola DnaG,
PCl137; B. subtilis DnaE, A22282; RSF1010 RepB, JH0126;
phage T7 gp4, A04314; phage T3 gp4, S07508; phage T4 gp61,
A94456.
P4 gP0t
"1"4 ;pill
35
87
•r7 ~
.w
T"J DP4
E. o d l DnaG
'15
40
8.~¢L Dna~
B. eubt Drag
E. ~ll RecR
B. su~ RecR
40
E
¢~'1 UwA
40
57
67
253
763
Consensus
103
~
RF ......
R~DDREGRG~
D Q N K A R G W ~ Y G D I ~ - N E G N I.H
GN . . . . . . . . SLFSDGHT~.
GN ........ SWSDGHEt~.
S . . . . . . . ~TVNGEKQF~
S ....... ~VNGEKQP~E
S ....... ~SVS PDKQ'r~
QEV ...............
QDP ...............
j~.q~l, mEr~EpRr.. ~Fm~PAGA.
IE~QGDGVIKVEMH..
~LPDIYVP..
ip~s
g-
Fig. 5. Amino acid sequence alignment of a potential gn 2÷
binding motif in gpa~ and other prokaryotic DNA primases and
repair proteins. Numbers indicate the sequence positions of the
first amino acid residues shown. Cysteine/histidine residues conserved in all sequences have a black background, Identical, aromatic and similar residues conserved are underlayed in dark,
medium and light grey, respectively. For GenBank/EMBL
database accession numbers of the primases, see legend to Fig. 4;
E. coli UvrA, M13495; E. coli RecR, P12727; B. subtilis RecR,
P24277.
of different D N A helicases [31,14]. The amino acid
sequence o f this portion of gp oL resembles an A-type
nueleotide binding site (pos. 501-508: GPGGSG K S ) found in other helicases [32]. The motif is
believed to form a structure that orients the phosphates of incoming nueleotides in the nucleotide
binding fold [33,34].
A search for motifs known to be involved in D N A
recognition was unsuccessful indicating, that, as for
most replication initiator proteins, the g p a pr,~?einDNA interaction(s) needs further explanation.
8. D o m a i n structure o f g p ~
The sequence comparison studies suggested that
g p a may be organized in separable domains corresponding to the three different functions of the protein. This hypothesis was tested by a deletion study
resulting in truncated proteins and by site-directed
mutagenesis to generate missense mutations that
would knock out or alter either the primase or helicase activity of the complete protein. A primase-null
mutation (E2140; [14]) retains full helicase activity
and specific DNA-binding ability. A similar observation was made with helicase-null mutations (K507Q,
K507T) which retained both full primase activity and
unchanged DNA-binding property in vitro. A double
mutant (E214Q, K507T) without either primase or
G. Ziegelin, E. Lanka / FEMS Microbiology Reviews 17 (1995) 99-107
104
ZnZ+
:,
bigZ+
IerP
-EGYArA,
-q
A2 :
100 aa
('~peA)
i
-- A4
~
~
i
! NTPase
:::::::i
[~ ....
°"__:"..~'~sorf/crr iecog~tion
]
:-::7:
HeUcase
Pxiraase
Fig. 6, Domain structure of gpot, The upper bar represents Spa,
the wh.L'e segment shows the region exhibiting amino acid similarities betweea Spot and primases of Incl/IncP plasmids. The
locations of the potential Zn z+ binding motif, the EGYATA
motif and the type A nucleotide binding site are indicated. Mg 2+
defines a short stretch related to the Asp-Asp motif observed in
different DNA and RNA polymerases [39l. The bars mark truncated polypeptides fimctional in either primase activity (A2) or
o r i / c r r recognition and probably NTPase/helicase activity (A4).
helicase activity retained specific DNA recognition
ability indicating that the gp c~ domains can function
independently in vitro. These observations were consistent with the activities found in truncated gpot
polypeptides.
The N-terminal gpccA2 polypeptide did not bind
ori or err specifically. However, DNA binding by
the C-terminal half of gpotA4 (410 amino acids) was
indistinguishable from that of the wild-type protein
(to be published elsewhere). Thus, DNA primase and
the specific DNA recognition ability were separable,
the N-terminal half of the protein containing primase
activity [14] and the C-terminal half the DNA binding property (Fig. 6). The specific primase activity of
the 373-amino acid polypeptide g p a A 2 was reduced
about 200-fold as compared to the wild-type protein,
suggesting either the presence of inactive molecules
in the preparation or that an additional part of the
protein is needed for the full activity. Therefore, the
absenee of NTPase activity in gpc~A2 may explain
the reduction of the specific activity of the primase
since NTP hydrolysis is known to be involved in
unidirectional translocation along a single-stranded
DNA template. Thus, the "scanning' of the template
during the search for primer sites by gpc~A2 might
take place distributively rather than processively.
9. M o d e l o f initiation o f P 4 D N A replication
gpot combines three activities on one polypeptide
chain: (i) specific interaction with ori and err; (ii)
strand separation of partial duplex DNA with 3' ~ 5'
directionality; (iii) template-directed synthesis of
short oligoribonucleotides. The a protein unwinds
DNA with 3'---> 5' polarity which is in contrast to
other well characterized prokaryotic replication systems. However, the structure of in vitro replicating
DNA molecules of the mammalian simian virus 40
(SV40) resembles that of P4 intermediates [35]. The
well characterized SV40 large T antigen which is the
only virus-encoded protein essential for virus DNA
replication combines two replication properties on a
single polypeptide chain: specific origin recognition
and 3' --->5' helicase activity [36-38]. Therefore, the
replication mode of P4 and SV40 may share fundamental similarities in the initiation and elongation
stages. The reaction may include four major consecutive steps (Fig. 7): (i) recognition and binding of the
initiator protein to the origin; (ii) local unwinding of
the ori region; (iii) initiation of DNA synthesis by
off-Recognition
.~r lll|iititl|iil
ttiititlllttl~t
*tlliiililll
t tit Ittlttt
~l|litlltllllllilillill|lili~rfllllll!
ttlitltltltlt
till
off
~
Unw'indmg
~.
5VllUl|ll|lllllllllltll
~lllllllllllflillllllll
IlalltilltllllllUlllll
||lll~llllllllll|l]ll[
~~_~..
Priming
~
Elongation
31
~gpa
11111
O SSB
O
DNk-Gyi-ase
Poll/[
Fig. 7. Proposed model for P4 DNA replication initiation. Sec text
for details.
(3. Ziegelin, E. Lanka / FEMS Microbiology Reviews 17 (1995) 99-107
formation of RNA primers; (iv) elongation of nascent
chains by DNA polymerase, and to complete the
round of replication, termination and maturation of
the daughter molecules.
The initial binding of gp a to the origin appears to
be rather complex. Footprinting analysis indicates
that g p a binds to all six type I repeats (TGTTCACC) located within 104 bp of the origin (Fig. 2).
It is conceivable that gpc~ forms a nueleoprotein
complex containing several g p a monomers. Complexes detected in the electron microscope support
this hypothesis (vide supra). The P4 origin includes
two segments of a markedly high A / T content ( >
70%; Fig. 2) within the type I iteron domain and on
one side of the type II repeats. These segments may
melt locally, as generally observed for A / T rich,
iteron containing regions in other replication origins
[40]. Initial unwinding may occur at the type II
repeats. Then, g p a separates duplex DNA using its
intrinsic DNA helicase activity fuelled by (d)NTP
hydrolysis, translocating in the 3' -~ 5' direction on
the template for the leading strand, ahead of the
polymerase. E. coli SSB may function to stabilize
the unwound strands. Following the initial unwinding of the duplex at ori, short pppApG(pN) RNA
primers are synthesized as a result of the primase
activity associated with gp c~. Thus far, the nature of
the signals for primer synthesis remains unknown
(Table 2). The existence of a specific recognition
sequence, as found for the phage T7 and T4 primases
[17,21] cannot be ruled out at present. The short
primers would then be elongated b y E. coli DNA
polymerase Ill holoenzyme. Because of the relatively
short primers, it is conceivable that, like T7 gp4 [41],
gpc~ is also involved in stabilizing the primers on the
template, thereby facilitating recognition of the free
base-paired 3'-OH by the DNA polymerase.
The formation of the 'P4 pdmosome' diverges
from most other known prokaryotic systems, because
it includes the involvement of the e r r element which
is essential in vivo and in vitro [6]. It has been shown
that either of the 120-bp long repeats suffices for c r r
function; although crr cannot function by itself as an
origin of replication. In the case of R6K, a cis-acting
region was suggested to deliver the plasmid-encoded
replication initiator protein ¢r to the R6K origin, to
which free gpcr binds only poorly [42]. The P4 ot
protein, however, binds strongly to both ori and crr.
105
Thus, looping observed between ori and e r r in vitro
in the presence of gpot suggests a direct involvement
of e r r in I'4 replication, perhaps by rearranging the
gp c~-ori complex to make it competent for initiation
of bidirectional replication.
10. Concluding remarks
The actual replication mode of P4 is open to
question. Our data suggest that both daughter strands,
once initiated at ori, are synthesized continuously in
the 5'---, 3' direction until the site of replication
termination is reached. Such a displacement mechanism was proposed for plasmid RSF1010, which,
like phage P4, replicates independently of host-encoded initiation and priming factors [43]. Another
unanswered question is whether the same g p a
molecules involved in origin recognition and local
unwinding also participate in primer synthesis and
strand separation during movement of the replication
forks. Further intriguing questions concern the function of e r r and its possible involvement in initiation
of P4 replication, and the role of the ori type II
repeats.
Acknowledgements
We are grateful to Heinz Schuster for generous
support and stimulating discussions. We thank Laura
S. Frost for constructive criticism on the manuscript.
We further thank Rudi Lurz and Beate Dobfinski for
contributing the electron microscopic data. This work
was supported by the Deutsche Forschengsgemeinschaft (grant La 627/3-1).
References
[1] Lindqvist, B.H., Dehb, G. and Calendar, R. (1993) Mechanisms of genome propagation and helper exploitation by
satellite phage P4. Microbiol. Rev. 57, 683-702.
[2] Bertani, L.E. and Six, E.W. (1988) The P2-1ilce phages and
their parasite, P4. In: The Bacteriophages (Calendar, R., Ed.),
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