The replication fork

The replication fork

 

Many enzymes are involved in the DNA replication fork.

The replication fork is a structure which forms when DNA is being replicated. It is created through the action of helicase, which breaks the hydrogen bonds holding the two DNA strands together. The resulting structure has two branching "prongs", each one made up of a single strand of DNA.

Lagging strand synthesis

In DNA replication, the lagging strand is the DNA strand at the replication fork opposite to the leading strand. It is also oriented in the opposite direction when compared to the leading strand, with the 5' near the replication fork instead of the 3' end as is the case with the leading strand. When the enzyme helicase unwinds DNA, two single stranded regions of DNA (the "replication fork") form. DNA polymerase cannot build a strand in the 3' → 5' direction. This poses no problems for the leading strand, which can continuously synthesize DNA in a processive manner, but creates a problem for the lagging strand, which cannot be synthesized in the 3' → 5' direction. Thus, the lagging strand is synthesized in short segments known as Okazaki fragments. On the lagging strand, primase builds an RNA primer in short bursts. DNA polymerase is then able to use the free 3' hydroxyl group on the RNA primer to synthesize DNA in the 5' → 3' direction. The RNA fragments are then removed (different mechanisms are used in eukaryotes and prokaryotes) and new deoxyribonucleotides are added to fill the gaps where the RNA was present. DNA ligase is then able to join the deoxyribonucleotides together, completing the synthesis of the lagging strand. [2]

Leading strand synthesis

The leading strand is defined as the DNA strand that is read in the 3' → 5' direction but synthesized in the 5'→ 3' direction, in a continuous manner. On this strand, DNA polymerase III is able to synthesize DNA using the free 3'-OH group donated by a single RNA primer (multiple RNA primers are not used) and continuous synthesis occurs in the direction in which the replication fork is moving.

Dynamics at the replication fork

The sliding clamp in all domains of life share a similar structure, and are able to interact with the various processive and non-processive DNA polymerases found in cells. In addition, the sliding clamp serves as a processivity factor. The C-terminal end of the clamps forms loops which are able to interact with other proteins involved in DNA replication (such as DNA polymerase and the clamp loader). The inner face of the clamp allows DNA to be threaded through it. The sliding clamp forms no specific interactions with DNA. There is a large 35A hole in the middle of the clamp. This allows DNA to fit through it, and water to take up the rest of the space allowing the clamp to slide along the DNA. Once the polymerase reaches the end of the template or detects double stranded DNA (see below), the sliding clamp undergoes a conformational change which releases the DNA polymerase.

The clamp loader, a multisubunit protein, is able to bind to the sliding clamp and DNA polymerase. When ATP is hydrolyzed, it loses affinity for the sliding clamp allowing DNA polymerase to bind to it. Furthermore, the sliding clamp can only be bound to a polymerase as long as single stranded DNA is being synthesized. Once the single stranded DNA runs out, the polymerase is able to bind to the a subunit on the clamp loader and move to a new position on the lagging strand. On the leading strand, DNA polymerase III associates with the clamp loader and is bound to the sliding clamp.

Recent evidence suggests that the enzymes and proteins involved in DNA replication remain stationary at the replication forks while DNA is looped out to maintain bidirectionality observed in replication. This is a result of an interaction between DNA polymerase, the sliding clamp, and the clamp loader.

 

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