In Vivo Replication of Filamentous
Phage DNA
Dan S. Ray
Molecular Biology Institute
and Department of Biology
University of California
Los Angeles, California 90024
Intracellular Forms of Filamentous Phage DNA
T h e first step in the replication of filamentous phage D N A is the conversion
of the infecting viral D N A to a circular, duplex replicative form ( R F ) . This
R F molecule, t e r m e d the parental R F , replicates to produce a pool of
progeny R F molecules. T h e accumulated R F molecules then serve both as
templates for transcription of viral genes and as a source of progeny singlestranded (SS) D N A . T h e latter molecules are produced later in the life cycle
by an asymmetric replication process in which the complementary strand of
an R F molecule serves as a stable, circular template for the r e p e a t e d displacement of viral single strands. T h e duplex replicative form occurs most
often as a covalently closed, superhelical D N A ( R F I ) . Infected cells «lso
contain small, but significant, a m o u n t s of nicked circular R F molecules
which contain one o r more single-strand discontinuities ( R F I I ) and relaxed
circles in which both strands are covalently closed ( R F I V ) . R F I I I , a unitlength, linear R F , appears to occur only as an artifact.
Miniature forms of R F and SS D N A are observed in cells infected with
phage preparations containing " m i n i p h a g e . " Such particles were first
detected in phage preparations obtained after multiple passages beyond the
original single-plaque isolation (Griffith and Kornberg 1974; E n e a and
Z i n d e r 1975). These phage have extensive deletions of the g e n o m e and,
consequently, do not contain any intact genes. Their replication and morphogenesis are d e p e n d e n t on gene functions provided by a helper phage.
T h e miniphages contain only the region a r o u n d the origin of replication and
frequently one or m o r e duplications of a portion of that region.
Double-length SS and R F species, as well as catenated R F D N A , have
been detected in extremely small a m o u n t s (Jaenisch et al. 1 9 6 9 ; W h e e l e r et
al. 1974). T h e origin and significance of these rare forms are unknown.
" M i d i p h a g e " that have somewhat more than a unit-length genome have also
been observed and are described elsewhere in this volume (see W h e e l e r and
Benzinger).
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D. S. Ray
Kinetics and Regulation of Viral DNA Synthesis
T h e replication process of filamentous phage can be divided into three
stages: the first stage, formation of the parental R F (phage -*• R F ) ; the
second stage, replication of the R F ( R F —• R F ) ; and the third stage, asymmetric synthesis of viral single strands ( R F - * SS). T h e first stage of replication occurs so rapidly that no intermediates have yet been observed. The
process does not require the synthesis of viral proteins and is mediated by a
small n u m b e r of host enzymes. Conversion of viral D N A to the parental
replicative form (SS —> R F ) by purified enzymes has been studied extensively (R. B . Wickner et al. 1 9 7 2 ; W . Wickner et al. 1 9 7 2 ; Geider and
K o r n b e r g 1974) and likely reflects the essential elements of the phage - > R F
reaction. The latter reaction has not yet been achieved in a purified system.
A model of the filamentous phage replication process is shown in Figure 1.
M13 — RF
(~0-1min)
RF-SS
RF-*RF
H O min - —)
(~1 - 2 0 min)
Figure 1 The replication cycle of filamentous phage. The three stages of replication
of filamentous phage are indicated schematically along with an indication of the
approximate duration of each stage and the requirements for viral gene products.
(Outer circle) viral strand; (inner circle) complementary strand; (arrowhead) 3'-OH
terminus; (mw) RNA; ( e n ) gene-VIII protein, 8p; (o) gene-V protein, 5p; ( A )
gene-III protein, 3p.
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Filamentous Phage DNA Replication
327
During the first 1 0 - 1 5 minutes of infection the rate of D N A synthesis in
infected cells is severalfold greater t h a n in uninfected cells due to the
synthesis of u p to 100 progeny R F molecules p e r cell ( H o h n et al. 1971). A s
expected for duplex D N A synthesis, radioactive labeling of R F molecules
early in infection labels b o t h strands equally. However, by 25 minutes after
infection synthesis b e c o m e s totally asymmetric (Forsheit et al. 1 9 7 1 ) . Label
is incorporated only into the viral strand of the R F during this stage of the
replication process and flows with high efficiency into progeny single strands
as these are formed. Prelabeled c o m p l e m e n t a r y strands remain covalently
closed a n d retain their radioactivity, which suggests that the complementary
strand serves as a stable template for the r e p e a t e d synthesis of new viral
strands. Preexisting viral strands are displaced as new viral strands are
synthesized ( R a y 1969). T h e single-strand pool that accumulates contains
u p to 2 0 0 viral single strands p e r cell ( R a y et al. 1 9 6 6 ) .
Regulation of viral D N A synthesis is m e d i a t e d by two viral proteins, the
products of genes II and V (Pratt and E r d a h l 1968). T h e gene-II protein is
responsible for the asymmetry of the replication process. Its function is
required for the accumulation of R F I I molecules containing a single discontinuity in the viral strand (Fidanian and Ray 1 9 7 2 ; Lin and Pratt 1 9 7 2 ;
Tseng and Marvin 1 9 7 2 b ) . T h e activity of gene II is essential b o t h for R F
D N A replication and for viral SS D N A synthesis. T h e shift from R F replication to the asymmetric synthesis of viral single strands is d e t e r m i n e d by the
availability of the gene-V protein, a D N A - b i n d i n g protein (Salstrom a n d
Pratt 1 9 7 1 ; Mazur and M o d e l 1 9 7 3 ; Mazur and Z i n d e r 1 9 7 5 ) . Synthesis of
progeny viral single strands increases in parallel with increased synthesis of
the gene-V protein. In the absence of gene-V protein, viral D N A accumulates entirely as R F molecules. T h e functions of b o t h of these regulatory
proteins will be discussed in m o r e detail later in this chapter.
The Phage - » RF Reaction
T h e first stage of filamentous p h a g e replication can b e interrupted by
rifampicin, an inhibitor of R N A polymerase, at a point following a t t a c h m e n t
of R N A polymerase but preceding D N A synthesis (Brutlag et al. 1 9 7 1 ;
M a r c o et al. 1974). This block to the formation of parental R F molecules
must reflect inhibition of the synthesis of an R N A primer for the complementary strand since n o such inhibition is observed in cells treated with
inhibitors of protein synthesis. F u r t h e r m o r e , a r e q u i r e m e n t for all four
ribonucleoside triphosphates (rNTPs) has b e e n observed in vitro in the
rifampiein-sensitive SS —* R F reaction ( G e i d e r and K o r n b e r g 1974). A
noninfectious (eclipsed) phage particle accumulates in the presence of
rifampicin, indicating that infection can proceed u p to the point of attachment of R N A polymerase to the viral D N A at the unique origin of replication, while still preserving the phage structure. However, the sensitivity of
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328
D. S. Ray
the rifampicin-eclipsed phage to D N a s e indicates some alteration of the
virion. T h e exposure of the D N A initiation sequence to the cytoplasm of the
cell while the virion structure is maintained suggests that the RNA-polymerase-bindingsite is adjacent to o r overlaps the binding site for the adsorption protein, the product of gene III. This hypothesis assumes that the viral
D N A has a fixed and unique orientation in the virion. However, pyrimidine
tract analysis of b o t h whole phage and fragments from the adsorbing
end of the virion show no difference, which implies a random orientation of the D N A in the virion (Tate and Petersen 1974). This apparent
conflict might be resolved if the circular D N A were shown to be capable of a
conveyor-belt type of m o v e m e n t within the virion at some point in the
attachment process. Should this prove to be the case, the observed inhibition
of phage attachment by either low t e m p e r a t u r e o r cyanide poisoning (Marco
et al. 1974) might reflect an energy requirement for the m o v e m e n t of the
circular D N A within the filamentous virion.
A n additional role for the gene-III protein (adsorption protein) has been
proposed o n the basis of its observed retention o n the viral D N A even after
conversion to the duplex replicative form (Jazwinski et al. 1973). This
protein is thought t o function as a " p i l o t " that might guide the D N A to some
appropriate host system required for replication of the viral genome. This
hypothesis is w e a k e n e d , however, by the observation (Pratt et al. 1969) that
viral D N A from polyphage particles lacking the adsorption protein is infectious to spheroplasts even though the D N A totally lacks the adsorption
protein.
T h e mechanism by which the complementary strand is synthesized in the
phage —» R F reaction in vivo can be distinguished from that occurring during
R F —*• R F replication by its resistance to nalidixic acid (Fidanian and Ray
1974). T h e complementary strand of parental R F D N A formed in the
presence of this drug is fully functional, as judged by its ability to direct the
synthesis of the gene-II protein. T h e basis for this sharp distinction is
unclear.
RF - * RF Replication
Replication of parental R F D N A to form a pool of R F molecules requires
the function of a single viral gene product, that of gene II (Pratt and Erdahl
1968). Infection in the presence of chloramphenicol, an inhibitor of protein
synthesis, or infection of a nonpermissive host cell with a m b e r mutants
defective in gene II leads to the formation of the parental R F , but further
replication is inhibited (Fidanian and R a y 1972). In addition, there is a
striking reduction in the ratio of R F I I to R F I under these conditions,
which suggests an involvement of gene I I in the formation of the open, circular
R F I I . In contrast, R F D N A replication is also blocked after formation of the
parental R F D N A in cells infected in the presence of nalidixic acid, an
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Filamentous Phage DNA Replication
329
inhibitor of D N A replication, o r in infection of Escherichia coli rep3 cells,
which are defective in small-phage replication beyond the formation of the
parental R F ; but in both these cases gene expression can occur and normal
amounts of R F I I molecules are formed. Analysis of the R F I I molecules
formed u n d e r either of these conditions shows that the newly synthesized
complementary strand is a covalently closed ring, whereas the parental viral
strand contains a single discontinuity (Fidanian and Ray 1972). These
results indicate clearly a r e q u i r e m e n t for expression of gene II for the
formation of R F I I and for the subsequent replication of R F D N A . T h e
gene-II protein appears most likely to be a highly specific nuclease required
for the initiation of a round of replication. T h e corresponding gem-A
protein of ^ X 1 7 4 plays a similar role in <pX replication (Francke and Ray
1972) and has been shown to be a sequence-specific endonuclease.
Except for the gene-II protein, all of the functions required for R F D N A
replication are provided by the host ( D u m a s , this volume). These include
R N A polymerase, D N A polymerases I and III, ligase, a D N A - u n w i n d i n g
enzyme (the rep protein), gyrase, the dnaB protein, and the dnaG primase.
Except for R N A polymerase, these enzymes are also involved in the replication of <p X R F D N A (Wickner; McMacken et al.; Sumida-Yasumoto et al.;
all this volume).
R F D N A replication occurs by a mechanism in which the two strands are
nonequivalent. An asymmetry is introduced initially by the action of the
gene-II protein which leads to the formation of R F molecules that have a
single discontinuity in the viral strand and an intact circular complementary
strand. Subsequent replication of the R F D N A involves rapidly sedimenting
replicative intermediates ( R I ) that have a partially single-stranded structure
(Tseng and Marvin 1972a). Alkaline denaturation of such intermediates
yields viral strands of greater than unit length and both viral- and
complementary-type strands of shorter than unit length (Forsheit and Ray
1 9 7 1 ; Tseng and Marvin 1972a). These results are consistent with a rollingcircle mechanism of replication (Gilbert and Dressier 1969) in which the
o p e n viral strand of R F I I is continuously displaced as a new viral strand is
synthesized. This model predicts that viral strands of greater than unit length
would arise by covalent attachment between the preexisting viral strand and
the nascent viral strand. T h e occasional nicking of the RI at this junction
would produce nascent viral-strand fragments of less than unit length.
T h e products of R F D N A replication are R F I I molecules. Their conversion to superhelical R F I involves a relaxed, but covalently closed, intermediate t e r m e d R F I V (K. Horiuchi and N . D . Zinder, pers. comm.). Conversion of R F I V to R F I appears to be meditated by the host gyrase, since the
conversion is inhibited by coumermycin, a specific inhibitor of gyrase. In the
presence of this drug, R F I V molecules accumulate.
Initiation of the complementary strand most likely occurs by an R N A polymerase-mediated priming, since complementary-strand synthesis is
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D. S. Ray
rapidly inhibited by rifampicin but not by chloramphenicol (Brutlag et al.
1 9 7 1 ; Fidanian and Ray 1974). It has already been mentioned that there is
some difference between complementary-strand synthesis during phage - »
R F synthesis and that during R F - » R F replication, as indicated by the
differential effects of nalidixic acid o n complementary-strand synthesis during these two stages of replication. Synthesis of the complementary strand
during R F - * R F replication is much m o r e sensitive to nalidixic acid than is
synthesis of the viral strand, yet complementary-strand synthesis is resistant
to inhibition by this drug during parental R F D N A formation.
While it is tempting to speculate that viral-strand synthesis is primed by
the 3 ' terminus of the initial R F I I molecule (Fidanian and Ray 1972), there
is as yet no evidence to support this hypothesis. F u r t h e r m o r e , there is a clear
requirement for the dnaG protein in R F D N A replication (Ray et al. 1 9 7 5 ;
Dasgupta and Mitra 1 9 7 6 ) . Since this protein has been found to be involved
in the synthesis of a primer for D N A chain initiation in the G 4 and <pX
replication systems (McMacken et al.; Wickner; both this volume), presumably it functions in a similar capacity in filamentous phage replication. In the
case of G 4 and q>X, however, the dnaG protein primes the synthesis of the
complementary strand during SS
R F synthesis. This is clearly not the case
for the filamentous phages as the dnaG protein is required for R F -> R F
replication but not for SS - » R F synthesis. In addition, as discussed above,
the complementary strand of filamentous phage R F D N A appears to be
primed by R N A polymerase. F u r t h e r m o r e , experiments with both dnaG
and dnaB m u t a n t s indicate that synthesis of b o t h strands is inhibited equally
in these m u t a n t s at a nonpermissive t e m p e r a t u r e ( R a y et al. 1975). These
results indicate that the dnaB and dnaG functions are not required exclusively for complementary-strand synthesis. Like dnaG, the dnaB function is
required only during duplex D N A replication (Olsen et al. 1972). It seems
likely, therefore, that b o t h the dnaG and dnaB proteins may be involved in
some way in the initiation of viral-strand synthesis. However, the possibility
of an indirect effect of these mutations cannot be excluded entirely.
A n o t h e r gene function possibly involved in R F D N A replication is that of
dnaA (Mitra and Stallions 1976). However, the observation that the dnaA
function is no longer required in Hfr strains in which the dnaA mutation is
suppressed as a result of integrative suppression suggests the possibility that
the requirement may be for host replication rather than for the dnaA
product per se. T h e determination of the roles of specific host proteins in R F
D N A replication will require the development of R F D N A replication
systems similar to those already developed for <pX as described elsewhere in
this volume (Eisenberg et al.; Sumida-Yasumoto et al.).
The replicative origins for b o t h the viral and the complementary strand
are located within the intergenic region between genes II and IV (Tabak et
al. 1 9 7 4 ; Horiuchi and Z i n d e r 1976; Suggs and Ray 1977). Both strands
initiate within this region but are synthesized in opposite directions (Fig. 2 ) .
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Filamentous Phage DNA Replication
331
Conscription
Figure 2 Origin and direction of synthesis of the two strands of Ml 3. Both the viral
and the complementary strand initiate within the intergenic space (I.S.) located
between genes II and IV but they are synthesized in opposite directions. The Hpall
cleavage map and its alignment with the genetic map and with other restriction maps
are described by Horiuchi et al., Konings and Schoenmakers, and Schaller et al. (all this
volume).
If complementary-strand synthesis requires a single-stranded template, as in
the SS —» R F reaction, it may be necessary for an entire unit length of viral
D N A to be synthesized prior to initiation of the complementary strand. In
this case, the viral strand could possibly be circularized shortly after initiation of the complementary strand, thus leading to R I containing a circular
viral strand and a nascent complementary strand ( S t a u d e n b a u e r et al., this
volume).
R F D N A replication occurs in association with cellular m e m b r a n e s (Forsheit and Ray 1 9 7 1 ; S t a u d e n b a u e r and Hofschneider 1 9 7 1 ; Kluge et al.
1971). Both parental labeled R F and pulse-labeled R F D N A s can be found
in m e m b r a n e fractions of gently lysed cells. T h e fraction of pulse-labeled R F
D N A associated with the m e m b r a n e is inversely proportional to the pulse
length, which suggests that the R F molecules are synthesized on the m e m brane and then released. Gentle lysis of cells infected with an M l 3 a m 5
m u t a n t results in the observation in the electron microscope of circular R F
molecules attached to the cell envelope (Griffith and K o r n b e r g 1972).
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D. S. Ray
U n d e r the less gentle conditions of osmotic disruption of the infected cells,
the circular R F molecules are found separated from the cell envelope.
RF - > SS Synthesis
T h e transition from duplex D N A replication to asymmetric viral-strand
synthesis is regulated by the accumulation of a DNA-binding protein, the
product of gene V (Salstrom and Pratt 1 9 7 1 ; M a z u r and Model 1 9 7 3 ; Mazur
and Z i n d e r 1 9 7 5 ) . M u t a n t s defective in gene V undergo normal R F D N A
replication but fail to switch over to SS D N A synthesis. This protein has a
very high affinity for SS D N A (Alberts et al. 1 9 7 2 ; O e y and Knippers 1972)
and is thought to affect the transition to SS D N A synthesis by binding to the
nascent viral strands and thereby inhibiting initiation of complementarystrand synthesis. Although there is no direct evidence as yet for the association of gene-V protein with R I , such an interaction would be expected on the
basis of the known properties of the gene-V protein and those of the R I .
Progeny single strands accumulate late in the infection process as D N A protein complexes. By 75 minutes after infection the host cell contains up to
200 progeny single strands (Ray et al. 1966), each of which is coated with
approximately 1300 gene-V proteins (Webster and C a s h m a n 1 9 7 3 ; Pratt et
al. 1974). These complexes have b e e n isolated from infected cells and shown
to be rod-shaped structures 1.1 p.m in length and 16 n m in width. Essentially
all of the SS D N A and at least one-half to two-thirds of the gene-V protein
are contained in such complexes. Neither of the known capsid proteins
appears to be associated with these structures. During morphogenesis, the
gene-V protein is displaced from the viral D N A at the cell surface as the
filamentous virion is extruded out into the medium. These gene-V proteins
can then be recycled, associating with newly formed viral single strands
(Pratt et al. 1 9 7 4 ) . Recycling of the gene-V protein is an active process
requiring viral morphogenesis. Inhibition of phage production immediately
blocks the release of gene-V protein from the complex (Mazur and Zinder
1975).
Direct evidence for the association of gene-V protein with viral single
strands in vivo has been obtained by U V cross-linking of the gene-V protein
to viral D N A late in the infection process (Lica and Ray 1977). These
complexes are readily isolated from detergent-treated lysates by CsCl
equilibrium centrifugation. Only a single tryptic peptide remains associated
with the viral D N A after trypsin t r e a t m e n t of cross-linked complexes,
indicating a specific association of the gene-V protein with the D N A . T h e
cross-link is located close to the carboxyl terminus, between residues 70 and
77.
T h e gene-V protein serves to maintain progeny viral strands in the singlestranded form. Cells infected with a gene-V temperature-sensitive m u t a n t
rapidly convert the pool of viral D N A to a double-stranded form upon a shift
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to the nonpermissive t e m p e r a t u r e (Salstrorn and Pratt 1 9 7 1 ) . T h e ability of
the gene-V protein to prevent complementary-strand synthesis o n the progeny viral strands suggests that it could also function in that capacity o n R I .
Binding of the gene-V protein to nascent viral strands might also provide
protection against exonucleolytic degradation of the viral strand prior to
circularization ( O e y a n d Knippers 1972).
Viral-strand synthesis normally follows the accumulation of a pool of
progeny R F molecules. H o w e v e r , if a pool of gene-V protein is allowed to
accumulate in the absence of R F D N A replication, SS D N A synthesis begins
immediately upon release of the block to D N A synthesis ( M a z u r and Model
1973). U n d e r such conditions the parental viral strand is rapidly transferred
to the pool of progeny SS D N A . T h e accumulation of excess g e n e - V protein
has also been shown to allow chloramphenicol-resistant SS D N A synthesis.
In a normal infection the addition of chloramphenicol leads to a rapid switch
back to duplex D N A replication ( R a y 1970). It therefore a p p e a r s that
viral-strand synthesis is regulated entirely by the availability of gene-V
protein.
W h e t h e r or not the gene-V protein may also play a positive role in SS
D N A synthesis is unclear. It has b e e n observed that SS D N A synthesis is
temperature-sensitive in a dnaB m u t a n t infected with a gene-V ts m u t a n t
( S t a u d e n b a u e r and Hofschneider 1 9 7 3 ) . Since ihednaB function is required
for R F D N A replication but not for SS D N A synthesis (Olsen et al. 1972), it
was concluded that the gene-V protein plays a positive role in viral-strand
synthesis. W h e t h e r or not the altered gene-V protein might b e inhibitory to
SS D N A synthesis at the nonpermissive t e m p e r a t u r e is u n k n o w n . O t h e r
experiments suggest that a distinction must be m a d e between asymmetric
viral-strand synthesis and SS D N A accumulation. It was found that both
strands of R F molecules are synthesized equally in a nonpermissive host
infected with a gene-V a m b e r m u t a n t , yet upon addition of either rifampicin
or nalidixic acid, synthesis b e c a m e entirely asymmetric but n o SS D N A was
formed (Fidanian a n d R a y 1 9 7 4 ) . This asymmetric synthesis of viral strands
without the concomitant accumulation of SS D N A suggests that the function
of the gene-V protein is to protect the viral strand from degradation during
SS D N A synthesis. H o w e v e r , these experiments d o not exclude the possibility that gene-V protein might be required not only for asymmetric viralstrand synthesis but also for stable accumulation of viral SS D N A , but at
different levels. A low level of gene-V protein might be sufficient at a
replication fork, whereas a greater quantity would be n e e d e d to coat the
nascent viral strand completely. Possibly a low level of suppression in the
nonpermissive host might provide sufficient gene-V protein to allow some
asymmetric synthesis but not e n o u g h to allow SS D N A accumulation. R e s olution of this question will be accomplished most easily in an in vitro
system with purified proteins.
The intermediates in SS D N A synthesis contain a closed, circular com-
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D. S. Ray
plementary strand and an elongated viral strand (Ray 1 9 6 9 ; Forsheit et al.
1 9 7 1 ; S t a u d e n b a u e r and Hofschneider 1972a; Kluge 1974). Electron microscopic analysis of these R I shows open, circular, duplex rings with singlestranded tails (Allison et al. 1 9 7 7 ; Ray 1977) of the type proposed by
Gilbert and Dressier ( 1 9 6 9 ) . T h e viral strand initiates within the intergenic
region between genes II and IV and is synthesized unidirectionally in a
counterclockwise direction on the standard physical and genetic maps
(Horiuchi and Z i n d e r 1976; Suggs and Ray 1 9 7 7 ; Allison et al. 1977). The
origin-terminus of the viral strand has b e e n determined by three independ e n t methods: (1) location of the site of the discontinuity in the late life cycle
of R F I I molecules, (2) determination of the temporal o r d e r of synthesis of
each segment of the viral strand of the product R F I , and (3) electron
microscopic mapping of the 5' e n d of R I . O n e of the products of a round of
viral-strand synthesis is an R F I I molecule containing a single discontinuity.
T h e site of this discontinuity has b e e n located by carrying out limited repair
synthesis with polymerase I and labeled triphosphates and determining the
site of the repair label by restriction analysis. T h e discontinuity has been
located n e a r the center of the intergenic space. In the second method,
supercoils formed during SS D N A synthesis have been analyzed in a Dintzis
type of terminal-labeling experiment. These results have established the
direction of synthesis of the viral strand and place the origin-terminus of the
viral strand in the same region as the discontinuity in the product R F I I .
Partial denaturation mapping of rolling-circle intermediates likewise places
the viral-strand origin at this same site on the g e n o m e (Allison et al. 1977).
Entry of an R F I molecule into the replication process requires the introduction of the gene-II-specific discontinuity into the viral strand of the R F
(Fidanian and Ray 1972). A s discussed above, the role of the discontinuity
in R F I I molecules is uncertain. A l t h o u g h it has generally been assumed to
serve as a primer for viral-strand synthesis, it could also serve some other
function, such as to permit unwinding of the circular D N A and propagation
of a viral strand initiated at another site. T h e appealing simplicity of the
rolling-circle m o d e l (Gilbert and Dressier 1969) and the discovery that
single-stranded phages specify functions required for the accumulation of
specifically nicked R F molecules (Francke and Ray 1 9 7 1 ; Fidanian and Ray
1972) have p e r h a p s inhibited consideration of de n o v o initiation of the viral
strand. H o w e v e r , there are now several observations that we think require
such consideration.
First of all, t h e r e is the unexplained sensitivity of viral-strand synthesis to
rifampicin ( S t a u d e n b a u e r and Hofschneider 1 9 7 2 a , b ; Mitra 1 9 7 2 ; Fidanian
and Ray 1974). This observation alone is not entirely compelling, since
viral-strand synthesis is much less sensitive to rifampicin than is complementary-strand synthesis and since at least two rounds of rifampicin-resistant
viral-strand synthesis can occur u n d e r conditions that allow accumulation
of excess gene-V protein prior to addition of the drug (Fidanian and Ray
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Filamentous Phage DNA Replication
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1974). Yet, rifampicin inhibits viral-strand synthesis much m o r e rapidly
than does chloramphenicol, which suggests that R N A synthesis plays some
role o t h e r than just for the continued expression of gene V ( S t a u d e n b a u e r
a n d Hofschneider 1 9 7 2 a , b ) . Second, a m u t a t i o n affecting only the 5' —» 3 '
exonuclease activity of D N A polymerase I ( K o n r a d and L e h m a n 1974)
inhibits completely the sealing of the discontinuity in R F I I molecules
formed during SS D N A synthesis (Chen and R a y 1976; T.-C. Chen and D .
S. Ray, in p r e p . ) . T h e lack of closure of the viral strands of these molecules in
the absence of a functional D N A polymerase 1 5 ' —*• 3 ' exonuclease suggests
that a primer that would normally be r e m o v e d by this activity still remains at
the 5' terminus and inhibits closure of the R F I I . This interpretation is
supported by the observation of greatly increased a m o u n t s of R N A - p r i m e d
Okazaki fragments in this m u t a n t (Kurosawa et al. 1 9 7 5 ) . Finally, there is
the observed r e q u i r e m e n t in vivo for b o t h dnaB and dnaG functions for R F
- » R F replication b u t not for RF—* SS synthesis (Olsen et al. 1 9 7 2 ; Ray et al.
1975). T h e very high sensitivity of complementary-strand synthesis to
rifampicin suggests that the c o m p l e m e n t a r y strand may be primed by R N A
polymerase just as in the SS —» R F reaction in vitro. In view of the evidence
implicating dnaB and dnaG functions in D N A chain initiation (cf. M c M a c ken et al.; Wickner; b o t h this v o l u m e ) , the r e q u i r e m e n t s for dnaB
mddnaG
functions during R F D N A replication may reflect an involvement of these
functions in initiating viral-strand synthesis at this stage of the replication
cycle.
T h e possibility of a positive role for gene-V protein a n d the rifampicin
sensitivity of SS D N A synthesis have led to some speculation that gene-V
protein and R N A polymerase might play a role in the initiation of the viral
strand as gene-V protein accumulates. H o w e v e r , there is n o direct evidence
in support of this hypothesis.
SUMMARY
Replication of filamentous phage D N A involves duplex R I in which the two
strands are replicated by different mechanisms. A s y m m e t r y is introduced
into the replication process by the phage gene-II protein, which is required
for the formation of R F I I molecules specifically nicked in the viral strand.
Synthesis of viral single strands also requires the function of an additional
phage protein, the gene-V protein, a D N A - b i n d i n g protein that prevents
synthesis of the c o m p l e m e n t a r y strand late in infection. Initiation of the viral
strand occurs in the intergenic space b e t w e e n genes II and I V at a site close
to the complementary-strand origin.
During b o t h phage - * R F synthesis and R F - » R F replication, synthesis
of the complementary strand is initiated by a rifampicin-sensitive mechanism at a single, specific site within the intergenic space. However,
complementary-strand synthesis during the phage - > R F reaction is cata-
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336
D. S. Ray
lyzed by a nalidixic acid-resistant mechanism, whereas that occurring during
R F —» R F replication is sensitive to this drug.
Gene-II-specific nicking of the viral strand is required for viral-strand
synthesis during both R F - » R F replication and R F - * SS synthesis, but the
role of the discontinuity remains obscure. T h e simplest hypothesis is that the
3' terminus created by nicking of the viral strand serves as a primer for direct
elongation of the viral strand. Several lines of evidence now suggest that
consideration must also be given to the possibility of de novo initiation of the
viral strand. Purification of the proteins required for R F D N A replication
and for SS D N A synthesis and the reconstitution in vitro of these stages of
the filamentous phage replication cycle should provide us with considerably
greater insight into the precise biochemical mechanisms regulating the
initiation a n d elongation of both strands of the viral genome.
ACKNOWLEDGMENTS
Research in the a u t h o r ' s laboratory was supported by grants from the
National Institutes of Health ( A l 10752) and the National Science Foundation ( P C M 7 6 - 0 2 7 0 9 ) .
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