6.8:

The Replisome

JoVE Core
Molecular Biology
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JoVE Core Molecular Biology
The Replisome

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03:01 min

November 23, 2020

DNA replication is carried out by a large complex of proteins that act in a coordinated matter to achieve high-fidelity DNA replication. Together this complex is known as the DNA replication machinery or the replisome.

The synthesis of the leading and lagging strands is a highly coordinated process. To explain this, the “Trombone model” was proposed by Bruce Alberts in 1980. The DNA loop formation starts when a primer is synthesized on the parent lagging strand. The loop grows with the synthesis of an Okazaki fragment and finally dissolves when synthesis of the fragment ends. The DNA polymerase then attaches to another primer and the whole cycle, from loop formation to collapse, is repeated. The replisome allows all of the activities to be coordinated as a replication machinery complex.

Replisome components

Helicases unwind and separate double-stranded DNA.  These enzymes are present as a single hexamer ring in prokaryotes and as a double hexamer ring in eukaryotes. Eukaryotic helicases depend on additional proteins, Cdc45 and GINS, to function.  Single-stranded DNA binding (SSB) proteins keep the DNA strands from reannealing.  In prokaryotes, SSBs consist of a single subunit, whereas in eukaryotes, they are a heterotrimeric protein known as replication protein A (RPA).

Primase adds an RNA primer to where DNA synthesis will originate.  In prokaryotes, primase is present as a single subunit enzyme called DnaG, which synthesizes an RNA primer of around 12 nucleotides.  In eukaryotes, a multisubunit enzyme, DNA polymerase-α primase, synthesizes an RNA-DNA hybrid primer of around 25 nucleotides. In addition to polymerase-α, replicative polymerase will extend the newly synthesized DNA. While a single type of replicative polymerase, DNA polymerase III, is present in prokaryotes, two different types of replicative polymerases, Pol ε and Pol δ, are present in eukaryotes, for leading strand and lagging strand synthesis, respectively. 

Sliding Clamp proteins keep the polymerases attached to the DNA template.  β-clamp, a homodimeric protein, acts as the clamp in prokaryotes. In eukaryotes, the same task is performed by proliferating cell nuclear antigen (PCNA), a homotrimeric protein. The central pore of the sliding clamp is positively charged, which enables it to have electrostatic interactions with the negatively charged phosphate backbone of the DNA. The clamp is attached to the DNA by a Clamp Loader. These pentameric proteins belong to the AAA+ class of ATPases. The eukaryotic clamp loader is known as replication factor C, while the prokaryotic E. coli clamp loader is known as the γ complex; however, the clamp proteins and clamp loaders are thought to be evolutionary homologs unlike many other components of the replisome.