Rolling Circles Are Used to Replicate Phage Genomes

KEY CONCEPT
- The ФX174 A protein is a cis-acting relaxase that generates single-stranded circles from the tail produced by rolling circle replication.
Replication by rolling circles is common among bacteriophages. Unit genomes can be cleaved from the displaced tail, generating monomers that can be packaged into phage particles or used for further replication cycles. FIGURE 1 provides a more detailed view of a phage replication cycle that is centered on the rolling circle.

FIGURE 1. ФX174 RF DNA is a template for synthesizing singlestranded viral circles. The A protein remains attached to the same genome through indefinite revolutions, each time nicking the origin on the viral (+) strand and transferring to the new 5′ end. At the same time, the released viral strand is circularized.
Phage ФX174 consists of a single-stranded circular DNA known as the plus (+) strand. A complementary strand, called the minus (−) strand, is synthesized. This action generates the duplex circle shown at the top of Figure 1, which is then replicated by a rolling circle mechanism.
The duplex circle is converted to a covalently closed form, which becomes supercoiled. A protein encoded by the phage genome, the A protein, nicks the (+) strand of the duplex DNA at a specific site that defines the origin for replication. After nicking the origin, the A protein remains connected to the 5′ end that it generates, while the 3′ end is extended by DNA polymerase.
The structure of the DNA plays an important role in this reaction, for the DNA can be nicked only when it is negatively supercoiled (i.e., wound around its axis in space in the opposite sense from the handedness of the double helix; supercoiling is discussed in the chapter titled Genes Are DNA and Encode RNAs and Polypeptides). The A protein is able to bind to a single-stranded decamer fragment of DNA that surrounds the site of the nick. This suggests that the supercoiling is needed to assist the formation of a single-stranded region that provides the A protein with its binding site. (An enzymatic activity in which a protein cleaves duplex DNA and binds to a released 5′ end is sometimes called a relaxase.)
The nick generates a 3′–OH end and a 5′–phosphate end (covalently attached to the A protein), both of which have roles to play in ФX174 replication.
Using the rolling circle, the 3′–OH end of the nick is extended into a new chain. The chain is elongated around the circular (−) strand template until it reaches the starting point and displaces the origin.
Now the A protein functions again. It remains connected with the rolling circle as well as to the 5′ end of the displaced tail, and is therefore in the vicinity as the growing point returns past the origin. Thus, the same A protein is available again to recognize the origin and nick it, now attaching to the end generated by the new nick. The cycle can be repeated indefinitely.
Following this nicking event, the displaced single (+) strand is freed as a circle. The A protein is involved in the circularization. In fact, the joining of the 3′ and 5′ ends of the (+) strand product is accomplished by the A protein as part of the reaction by which it is released at the end of one cycle of replication, and starts another cycle.
The A protein has an unusual property that may be connected with these activities. It is cis-acting in vivo. (This behavior is not reproduced in vitro, as can be seen from its activity on any DNA template in a cell-free system.) The implication is that in vivo the A protein synthesized by a particular genome can attach only to the DNA of that genome. Researchers do not know how this is accomplished. Its activity in vitro, however, shows how it remains associated with the same parental (−) strand template. The A protein has two active sites; this might allow it to cleave the “new” origin while still retaining the “old” origin. It then ligates the displaced strand into a circle.
The displaced (+) strand can follow either of two fates after circularization. During the replication phase of viral infection, it might be used as a template to synthesize the complementary (−) strand.
The duplex circle can then be used as a rolling circle to generate more progeny. During phage morphogenesis, the displaced (+) strand is packaged into the phage virion.

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