Replisome

For prokaryotes, each dividing nucleoid (region containing genetic material which is not a nucleus) requires two replisomes for bidirectional replication.

The replisome remains at a fixed, midcell location in the cell, attached to the membrane, and the template DNA threads through it.

The replisome is a system in which various factors work together to solve the structural and chemical challenges of DNA replication.

As a result, the replication factors that solve these problems are highly conserved in terms of structure, chemistry, functionality, or sequence.

Many of the structural and chemical problems associated with DNA replication are managed by molecular machinery that is highly conserved across organisms.

In prokaryotic organisms, the helicases are better identified and include dnaB, which moves 5' to 3' on the strand opposite the DNA polymerase.

An expanded schematic reveals the underlying chemistry of the problem: the potential for hydrogen bond formation between unrelated base pairs.

This is because the chemical reactions catalysed by replicative polymerases require a free 3' OH in order to initiate nucleotide chain elongation.

In prokaryotes, the primase creates an RNA primer at the beginning of the newly separated leading and lagging strands.

In eukaryotes, DNA polymerase alpha creates an RNA primer at the beginning of the newly separated leading and lagging strands, and, unlike primase, DNA polymerase alpha also synthesizes a short chain of deoxynucleotides after creating the primer.

High processivity is in part ensured by ring-shaped proteins referred to as 'clamps' that help replicative polymerases stay associated with the leading and lagging strands.

There are other variables as well: from a chemical perspective, strand binding proteins stimulate polymerisation and provide extra thermodynamic energy for the reaction.

Clamp loader is a generic term that refers to replication factors called gamma (bacteria) or RFC (eukaryotes).

[3][4] Sliding clamp is a generic term that refers to ring-shaped replication factors called beta (bacteria) or PCNA (eukaryotes and archaea).

At this point, the lagging strand replicative polymerase associates with the clamp and primer in order to start polymerisation.

Many replicative polymerases contain an "error correction" mechanism in the form of a 3' to 5' exonuclease domain that is capable of removing base pairs from the exposed 3' end of the growing chain.

[8] Subsequently, the chemical reaction in the exonuclease unit takes over and removes nucleotides from the exposed 3' end of the growing chain.

[9] Once an error is removed, the structure and chemistry of the polymerisation unit returns to normal and DNA replication continues.

Working collectively in this fashion, the polymerisation active site can be thought of as the "proof-reader", since it senses mismatches, and the exonuclease is the "editor", since it corrects the errors.

In general, DNA repair enzymes complete the Okazaki fragments through a variety of means, including: base pair excision and 5' to 3' exonuclease activity that removes the chemically unstable ribonucleotides from the lagging duplex and replaces them with stable deoxynucleotides.

Primer removal and nick ligation can be thought of as DNA repair processes that produce a chemically-stable, error-free duplex.

After DNA repair factors replace the ribonucleotides of the primer with deoxynucleotides, a single gap remains in the sugar-phosphate backbone between each Okazaki fragment in the lagging duplex.

In vertebrate cells, replication of an ICL-containing chromatin template triggers recruitment of more than 90 DNA repair and genome maintenance factors.

If the replisomes moved like a train on a track, the polymerase-GFP protein would be found at different positions in each cell.

Cellular DNA stained with a blue fluorescent dye (DAPI) clearly occupied most of the cytoplasmic space.