DNA replication

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DNA replication in man, an eukaryote, is a conserved mechanism that restricts DNA replication to once per cell cycle. The process is done by the cell organelle, the replisome, and involves multiple multiple protein sub complexes and regulatory mechanisms. While DNA replication results in the synthesis of a DNA strand complementary to the original template strand by DNA polymerases, much more has to be involved given the complexity of DNA packing and the necessity with large genomes of synthesizing multiple DNA strands in parallel. Initiation sites need to be selected, the double-stranded DNA must be unwound by DNA helicases ahead of polymerases that work on the two single-stranded templates generated by the replication fork and the DNA needs repacking well before it moves into the two daughter cells at mitosis. The process which is only reasonably well understood in eukaryote organisms like yeast can thus be split into:
  1. Initiation
  2. Elongation
    • 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)
  3. Termination
    • 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[1]. 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.