Rolling circle replication

Rolling circle replication

Mode of replication seen in: F plasmid, Phages (M13 phage, φX174), viroids (circular RNA genome) and in some eukaryotic viruses (HPV – 16, HHV – 6)

Direction of DNA replication: Unidirectional

  • Use host cell replicative machinery for replication (DNA polymerase, Primase, ligase, Helicase etc are usually supplied by the host cell).
  • Endonuclease enzyme responsible for creating initial nick of rolling circle replication is specific for each system.
  • Rolling circle replication is also observed in the process of conjugative transfer of bacterial plasmids.

Replication of F plasmid and ssDNA phages (eg:M13) is explained in detail. They follow almost same steps.

F plasmid: Have small circular double stranded DNA

Genetic material in M13: ssDNA (+ strand)

When these phages infect their host bacteria, viral (+) strand DNA enters host cytoplasm. Then they synthesize complementary (-) strand using bacterial enzymes and form double stranded circular DNA. This is the replicative form of the virus.

A typical DNA rolling circle replication has five steps:

Step 1: Circular dsDNA will be “nicked”.

  • Rolling circle DNA replication is initiated by an initiator protein encoded by the plasmid or bacteriophage DNA or viral DNA. (Example:Endonucleases).
  • Initiator protein nicks one strand of the double stranded circular DNA of the plasmid molecule at a site called the double-strand origin (DSO site). In case of phage the (+) outer strand is nicked. Inner (-) strand remain unbroken.
  • Break in one of the nucleotide strands creates a 3’-OH group and a 5’-phosphate group.
  • Initiator protein remains bound to the 5′ phosphate at the site of this nick. The free 3′ OH group is available as a primer for DNA synthesis by DNA polymerase III.

Step 2: Addition of nucleotides to the 3’ end of nicked strand and displacement of 5’ end of nicked strand

  • The polymerase moves along the nicked strand and new nucleotides are added to the 3’ end of the broken strand (outer strand). The inner (unbroken) strand used as a template.
  • Host encoded helicase enzyme displaces the nicked strand behind polymerase as a single-stranded DNA molecule and is stabilized by single strand binding proteins.
  • As new nucleotides are added to the 3’ end, the 5’ end of the broken strand is displaced from the template, rolling out like thread being pulled off a spool.
  • The 3’ end grows around the circle, giving rise to the name rolling-circle model.

Step 3: Circularization of displaced linear ssDNA

(Displaced DNA is a lagging strand and is made double stranded via a series of Okazaki fragments).

  • The displaced old strand is cleaved off from the circle after one round of replication by the same initiator protein (it makes another nick on outer DNA strand).

Product of this cleavage:  Double stranded circular DNA molecule + Single-stranded linear DNA molecule.

Figure 1
  • The ends of the displaced strands are ligated to make single stranded circular DNA molecule.

Step 4: Complementary strand synthesis using displaced single stranded circular DNA

  • Then RNA primase then synthesizes a primer to initiate DNA replication at the single-stranded origin site (sso) of the single-stranded DNA (ssDNA) molecule.
  • This ss DNA act as a template for the synthesis of lagging strand catalysed by DNA polymerase III.
  • Finally, DNA polymerase I remove the primer and replace it with DNA.
  • DNA ligase joins the ends to make another molecule of double-stranded circular DNA.

Step 5: Several rounds of replication of unbroken inner DNA strand (- stand) and displaced outer DNA strand (+ strand)

  • Leads to the production of multiple copies of the genetic material ( phage DNA or plasmid DNA)
Figure 2: Steps involved in rolling circle replication of F plamid and ssDNA phage

Rolling circle replication in λ phage

Genetic material: dsDNA (linear form)

  • In viruses with linear genomes, the ability to circularize once inside a cell is a crucial prerequisite for rolling circle replication. By replicating in this fashion, the virus can ensure that no genetic material is lost from its genome as a consequence of successive rounds of replication. Circularization of linear phage genomes occurs by the interaction between cos sites (cohesive sites) in the viral genome.
  • During the early phase of λ DNA replication, the phage follows the θ mode of replication to produce several copies of circular DNA. These circular DNAs are not packaged into phage particles. They serve as templates for rolling circle synthesis of linear λ DNA molecules that are packaged into the phage particles.

The overall step involved is similar to that of F plasmid replication. But slight difference is observed in some steps.

Figure 3

Step 1: Circular dsDNA will be “nicked”. (Same as that of plasmid)

Step 2: Addition of nucleotides to the 3’ end of nicked strand and displacement of 5’ end of nicked strand (Same as that of plasmid)

Step 3: Concatemer synthesis

  • The replication fork proceeds a number of revolutions around the template without the cleavage of the displaced strand. This will create numerous linear copies of DNA in a continous head to tail series called concatemer.
  • The displaced strand becomes double stranded as it is peeled off.
  • In phage λ, the displaced strand serves as the template for discontinuous, lagging strand synthesis. Primase enzyme provides RNA primer. (Generation of okazaki fragments).
  • Finally the primer is removed and okazaki fragments are ligated using host specific enzymes.

Definition of concatemer: A molecule made up of multiple copies of the same genome strung together in tandem.

Step 4:

  • The linear concatemer thus created is cleaved into one unit length and packed into the phage particles.
  • Cleavage of a unit length tail generates a copy of the original circular replicon in linear form.
Figure 4: Rolling circle replication in phage λ

 

 

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