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Circular Oligonucleotide Synthesis
Immediately after infection by the ssDNA genome (the plus strand), synthesis of the complementary strand (the minus strand) begins using host enzymes. As all the phage mRNA is synthesized using the minus strand as template strand, no phage messenger RNA is synthesized at this stage. Synthesis of the minus strand is primed using an RNA primer made at a specific site on the plus strand, called minus ori or single-strand ori (sso), (step 1 in Fig. 1). Then the host elongation proteins synthesize the minus strand from the primer terminus. Three different modes of priming are known: by RNA polymerase (2), by DnaG primase (3), and by a seven-componentprimosome (4). A general feature of minus ori is an extensive region of secondary structure, which probably stabilizes the recognition sequences for the primer synthesis against melting by single-stranded DNA binding protein (SSB). After completion of the minus-strand synthesis, the initial product is a duplex circular DNA (RFII) consisting of an intact viral DNA and a nearly full-length linear complementary DNA plus the RNA primer (step 2 in Fig. 1). Then DNA polymerase removes the RNA primer and fills the resultant gap. Subsequently, the nick is sealed by DNA Ligase, and the duplex DNA is converted to the superhelical form (RFI) by the action of DNA gyrase (step 3 in Fig. 1) (see DNA Topology). The RFI DNA serves as a template for RNA transcription, and the production of phage proteins begins.
The molecular mechanism of action of the initiator nuclease of ssDNA phage has been best characterized for that of fX174, gpA (product of gene A) (5). The gpA nuclease recognizes and binds to a specific sequence in the plus ori and introduces a nick in a nearby sequence. Then the gpA protein covalently attaches to the 5 ‘ -P end at the nick by a phospho bond to a tyrosine residue, and the energy liberated by the cleavage of the DNA strand is stored in the protein-DNA complex (see step 4 in Fig. 1). GpA has affinity for the host Rep DNA helicase and recruits it to the nicked site. Furthermore, the interaction locks the gpA protein to the template throughout the replication (see steps 5 and 6 in Fig. 1). When synthesis of the new plus strand reaches the regenerated plus ori, the gpA nuclease introduces a second cleavage in the plus strand, and the 5′-P group of the old strand attached to the gpA protein is transferred to the newly created 3 -OH end, to produce circular ssDNA. At the same time, the new 5′ -P end is transferred to the Tyr residue of the gpA protein, and the next cycle of the rolling circle replication begins (see step 7 in Fig. 1). The fX174 circle is synthesized in about 10 sec, and 20 or more ssDNA circles are released from a single rolling circle intermediate. It should be noted that supercoiling of the template is required for the first cleavage, whereas the second cleavage occurs on the relaxed template. An explanation for this difference may be that the supercoiling is required for specific binding of the gpA protein to the template and that, since the gpA is already bound to the DNA, recognition of the nicking site is sufficient for the second cleavage.
Rolling circle replication - Wikipedia
Certain small bacteriophages contain a circular, single-stranded DNA (ssDNA) as genome. Replication of the single-stranded DNA has been characterized extensively for two groups of Escherichia coli phage, filamentous phages represented by M13 and spherical phages represented by fX174 (1). Their replication can be divided into three steps (Fig.1);(1) conversion of the ssDNA genome to a double-stranded form, called the replicative form (RF); (2) multiplication of RF DNA by rolling circle replication; and (3) generation of ssDNA genome for packaging into phage from the RF DNA. Thus, replication of the ssDNA genome depends on the synthesis by DNA polymerase of a complementary strand in a sophisticated manner.
Multiplication of RF DNA is initiated by introducing a nick by an initiator nuclease encoded in the phage genome at a particular site in the plus strand (plus ori or double-strand ori, dso) (step 4 in Fig. 1). The free 3′-OH end at the nick site serves as a primer and is elongated by DNA polymerase III holoenzyme, as the host Rep DNA helicase peels off the existing plus strand (step 5 in Fig. 1). When synthesis of the new plus strand reaches the plus ori (step 6 in Fig. 1), the nick is introduced again at the junction of the old and newly synthesized plus strand, to form linear ssDNA of one unit length (step 7 in Fig.1). The linear form is then converted into the circular form, a further reaction catalyzed by the initiator nuclease. Thus, one round of the rolling circular replication produces one new plus strand, and it is used as a template to form another RF DNA.
How do I generate circular ssDNA from linear dsDNA?
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