In this article we will discuss about the replication of ssDNA phage.
The cell wall surface of E.coli contains specific receptor sites. For example, the receptor site of ØX114 present on outer membrane of cell wall of Salmonella typhimurium is a lipopolysaccharide.
The phage gets adsorbed on bacterial cell surface through any one of 12 icosahedral vertices. The spikes differ each other in specificity for attachment. For the attachment to host cell wall the phage needs Ca++ or Mg++ ions. Therefore, to find out the specific receptor site, the adsorption may be reversible.
There starts eclipse period. Viral DNA is injected into the bacterial cell. Phage gene H encodes proteins which act as pilot protein, conveys the DNA into the bacterial cell.
After the introduction of phage DNA into the bacterial cell, there starts synthesis of viral DNA and protein. However, these processes are rather very complex in ØX174. Mitra (1980) has reviewed the DNA replication in viruses. The phage genome is in circular form consisting of an infectious hairpin duplex. Phage genome acts as template (+ strand).
It replicates immediately after infection which is accomplished in three different stages:
(a) Synthesis of a replicative from (RF) of viral DNA,
(b) Replication of parental RF to progeny RF DNA, and
(c) Conversion of RF molecules into rolling circle molecules.
(a) Formation of replicative form (RF) of DNA:
Denhard and Hours (1978) have described the DNA replication of ØX174. Soon after penetration, the ssDNA of phage synthesizes a complementary RF DNA and results in formation of the double stranded DNA. The newly synthesized RF strand is known as (-) strand (Fig. 18.10A-F).
Synthesis of RF of DNA is completed in certain stages. Soon after entry of ssDNA, the DNA unwinding proteins extend ssDNA leaving the hairpin duplex which is a promoter region. This step requires for the presence of ssDNA, dnaB protein, dnaC protein, unwinding protein and two protein factors (the X, Y, and Z, and ATP). These proteins form an intermediate substrate for the synthesis of dnaG protein (molecular weight 60,000 Dalton).
The unwinding proteins of which about 800 molecules are present, binds to ssDNA but not dsDNA. This protein establishes the ssDNA in a state which can act as a template for synthesis of its complementary strand. DNA polymerase is used since this process occurs before transcription of mRNA. It allows the initiation of DNA synthesis at the point of origin of replication present in dsDNA.
The presence of RNA primer in ssDNA is essential for the synthesis of DNA. However, after pre-priming with dnaB and dnaC proteins, dnaG protein catalyses the RNA priming in ØX174. The RNA polymerase is not required for the synthesis of RNA primer. On RNA primer a complementary (-) strand is extended by using the (+) ssDNA as the template.
Chain elongation of primed DNA takes place by DNA polymerase III holoenzyme (i.e. DNA polymerase III plus DNA co-polymerase III) in Okazaki fragments (the newly synthesized DNA in the form of short segments which later on are covalendy joined to yield a continuous (-) strand).
This structure is known as RF II which is converted to RF when gap is sealed. DNA polymerase through 5′ → 3′ exonuclease activity removes the RNA primer from (-) DNA strand. This gap is filled up by the activity of DNA polymerase I.
The two ends of the DNA molecule are ligated by the enzyme DNA ligase which results in a circular DNA molecule. Thereafter, the cellular polymerase forms a pool of progeny RF molecules (RF RF). The ssDNA strands are also synthesized.
(b) Replication of RF (RF → RF):
The RF molecules produced in this way replicate to form a pool of progeny RF molecules (Fig. 18.10 G-M). For the replication of parental RF molecule, the A gene protein is needed that makes a nick on the viral strand of RF molecule. Possibly the protein acts as hairpin in (-) DNA super-helix existing at palindromic sequences.
For the replication of RF molecule the host cell machinery is required that includes proteins encoded by rep gene and dnaB, dnaC, dnoE, dnaG, dnoH and dnaZ genes. Probably the origin of replication is present in gene A. Two models, reciprocatory strand model and rolling circle model, have been suggested for the replication of RF DNA duplex.
i. Reciprocatory Strand Model:
After the formation of RNA primers of a new strand begins at the origin of replication present on gene A (Fig. 18.10 G-J). In ØX174 the RNA priming is catalyzed by dnaG protein when a short sequence of DNA is formed.
Positive super-helical twists accumulate which impose the strain. The viral strand is synthesized around the genome continuously in unidirectional and in clockwise directions. The growing point moves for its origin to terminus around the circular genome.
Moreover, the complementary strand primed by RNA is synthesized in discontinuous manner. Synthesis of complementary strand is lagged behind the viral strand. After synthesis of the nascent complementary strand, there occurs the repeated exchange reaction between the nascent strand and bacterial strand.
The two parental strands are completely unwound. A strand exchange reaction releases two separate, circular but incomplete duplexes. One duplex has a single gap in the nascent viral strand (V-strand gap), and the other duplex has several gaps that separate nascent complementary strand of DNA segments (many C-strand gaps).
ii. Rolling circle model:
Dressier and Wolfson (1970) suggested that the rolling circle mechanism results in replication of RF → RF as well as RF → ss. Replication of RF → RF takes place by semi-conservative method (Fig. 18.10 KM.).
A nick is made on the outer strand, and the 5′ end tail serves as template for synthesis of a small DNA segment. The growing point moves from its joint by the enzyme DNA ligase to produce a dsDNA molecule. In turn, each strand of dsDNA molecules acts as a template for the synthesis of complementary DNA molecule.
(c) Synthesis of ssDNA (RF → ss DNA):
For the first time Gilbert and Dressier (1968) suggested the rolling circle mechanism for ssDNA synthesis of 0X174 (Fig. 18.10 N-R). They demonstrated that during the first round of replication RF molecules are produced. The rolling circle mechanism operates during the first stage of replication and produces ssDNA molecules.
However, no complementary strand is synthesized on the tail during RF → ss DNA stage of synthesis.
To begin the replication a virus coded enzyme makes a specific single stranded nick in the plus strand of the RF. This generates the 3′-OH and 5′-phosphate ends on the strand. The deoxynucleotides (dATP, dCTP, dGTP, dTTP) are added to free 3′-OH end. The open single strand rolls off the circle as a free tail with the progress of synthesis. By using the minus strand as a template and deoxynucleotide precursor a new plus strand is synthesized.
With the rolling of the strand, structural proteins of phage bind to the elongating tail. Nuclease acts within the hairpin loops present on DNA and release the plus strand which becomes circularized by binding the cut ends and forming the complete hairpin loop. The enzyme ligase fills up the gap.
IV. Assembly and Release:
The phage proteins are synthesized in the bacterial cell. Soon after synthesis, the progeny ssDNA molecules are packed into phage particles (Fig. 18.10R). In 1967, success has been achieved in artificial synthesis of ØX174 by A. Romberg and coworkers at Stanford University and by R.L. Sinsheimer at California Institute of Technology.