6.8:
The Replisome
DNA replication is carried out by a highly coordinated multi-protein assembly known as the DNA replication machinery or the replisome, which increases the efficiency of DNA replication.
The core components of the machinery are helicase, single-strand DNA binding proteins, DNA primase, sliding clamps, a clamp loader, and multiple DNA polymerases, which are all associated with each other near the replication fork.
Replicative DNA polymerases have a processivity of around 10 nucleotides, which is the number of nucleotides it can add to the daughter strand before dissociating from the template strand.
This is too inefficient to copy entire genomes in a reasonable timeframe and this problem is solved with the help of sliding clamp proteins.
When ATP associates with the clamp loader protein, they will bind to and open the sliding clamp so that its ring-like structure can surround the primer-template DNA complex. Once bound, the clamp loader hydrolyzes the ATP to ADP, causing the clamp loader to disassociate and the clamp to close around the DNA.
Then, DNA polymerase binds to the clamp proteins and together they slide along the template DNA, tethering DNA polymerase to the strand and increasing its processivity up to 1000 nucleotides.
This increased processivity allows the DNA polymerase to carry out continuous DNA replication on the leading strand.
However, on the lagging strand template, another DNA polymerase performs discontinuous DNA replication in a manner that allows the DNA polymerase molecules to synthesize the leading and lagging strands simultaneously. This process is sometimes described as the “Trombone model.”
The lagging strand and its template strand form a loop when DNA polymerase initiates Okazaki fragment synthesis from an RNA primer.
The DNA loop grows from both directions as helicase unwinds DNA and the lagging strand is synthesized.
When the DNA polymerase encounters the next RNA primer, it detaches from the template strand. Meanwhile, primase adds another primer to the lagging strand, and the growing DNA loop is released.
The clamp and clamp loader proteins allow DNA polymerase to quickly reassociate with the primed DNA template.
The formation and subsequence collapse of the DNA loop repeats with the synthesis of each new Okazaki fragment.
6.8:
The Replisome
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.
Suggested Reading
- O’Donnell, Michael, Lance Langston, and Bruce Stillman. "Principles and concepts of DNA replication in bacteria, archaea, and eukarya." Cold Spring Harbor Perspectives in Biology 5, no. 7 (2013): a010108.
- Alberts, Bruce. "DNA replication and recombination." Nature 421, no. 6921 (2003): 431.
- Kelch, Brian A., Debora L. Makino, Mike O'Donnell, and John Kuriyan. "Clamp loader ATPases and the evolution of DNA replication machinery." BMC Biology 10, no. 1 (2012): 34.
- Hedglin, Mark, Ravindra Kumar, and Stephen J. Benkovic. "Replication clamps and clamp loaders." Cold Spring Harbor Perspectives in Biology 5, no. 4 (2013): a010165.
- Gardner, Andrew F., and Zvi Kelman. "The DNA Replication Machinery as Therapeutic Targets." Frontiers in Molecular Biosciences 6 (2019): 35.