Common Name:DNA replication
Important Issues in Man
Relevant Clinical Literature
Wikipedia on DNA replication (Less technical, ? quality control)
- DNA replication is initiated from specific DNA sequences called origins of replication, and eukaryotic cells have multiple replication origins.
- The first step is the formation of a pre-replication complex (pre-RC) on one of these origins of replication.
- MCM factors bind to the origin recognition complex and separate the duplex DNA stands, forming a replication bubble.
- Formation of pre-initiation complex. Replication protein A (RPA, which has three subunits) binds to the single stranded DNA and then replication factor C (clamp loading factor, 5 subunits) binds to RPA.
- Formation of the initiation complex. RFC deposits the sliding clamp (proliferating cell nuclear antigen complex) and attracts DNA polymerases such as alpha (α), delta (δ), epsilon (ε).
- The Mcm2-7 complex acts as a helicase (an enzyme which breaks hydrogen bonds between the base pairs in the middle of the DNA duplex so separating the strands).
- Gyrases (a form of topoisomerase) relaxes and undoes the supercoiling caused by helicase ahead of the replication fork
- Replication protein A binds to the exposed bases of each fork to prevent improper ligation.
- Priming DNA polymerase alpha creates an RNA primer at the beginning of the newly separated leading and lagging strands and also synthesizes a short chain of deoxynucleotides after creating the RNA primer.
- Replication factor C acts as a clamp loading factor and sliding clamp ring-shaped replication factors (proliferating cell nuclear antigen complex) stabilise the synthesis
- The replicative polymerases DNA polymerase epsilon and DNA polymerase delta form an asymmetric dimer at the replication fork by binding to sub-units of replication factor C. Collectively, leading and lagging strand synthesis is referred to as being 'semidiscontinuous' as while leading strand synthesis with DNA polymerase epsilon proceeds first and one way, lagging stand synthesis with DNA polymerase delta is done (backwards) in about 150 base bits (Okazaki fragments)
- Proof-reading and error correction is built into the machinery of the replicative polymerases through say 3' to 5' exonuclease domains
- A number of DNA repair enzymes then act to complete primer removal and nick ligation of the lagging strand (there is a nick between each okazaki fragment that needs a DNA ligase to make whole)
- This is not yet fully understood in eukaryotes. The vertebrate replisome (CMG complex) has to be unloaded from chromatin. By analogy in man an E3 ubiquitin-protein ligase is key. The termination of DNA replication forks takes place when two replication forks coming from neighbouring origins meet each other usually in the midpoint of the replicon. At this stage, the remaining fragments of DNA have to be unwound, all remaining DNA replicated and newly synthesised strands ligated to produce continuous sister chromatids. Finally, the replication machinery has to be taken off, chromatin re-assembled, and the entwisted sister chromatids resolved topologically.