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DNA Replication
Biochem and medical genetics
| Question | Answer |
|---|---|
| When does DNA replication occur | During synthesis of interphase (cell cycle) |
| Structure of a chromosome | Telomere on each end - separate ends of chromosomes and prevent fusion Replication origin - where replication starts Centromere - chromosome attachment site, spindle fibres attach here |
| Why is DNA replication semi-conservative | The two strands of DNA can serve as templates for the synthesis of its partner strand During replication the two anti-parallel strands are unwound and the daughter strands are synthesised on then using dNTPs and polymerases |
| Origins of replication | Unlike bacterial chromosomes which only have one origin of replication, eukaryotic chromosomes have multiple origins of replication (in humans there are 40-80K These sequences are conserved, very between chromosomes and are located between active genes |
| Activation of replication | Pre-replication complexes are assembled at replication origins during G1. These are activated in S phase. The initiator (Orc1-6) are bound to origins. Inactive replication helicase complex is loaded (Mcm2-7) Activated by forming a holo helicase |
| What prevents the strands re-joining | The single stranded DNA is bound by proteins (RPA) to prevent re-joining of the strands Single strand binding proteins prevent re-joining Each helicase wraps around one strand to separate them |
| The primase activity | Short (30 nucleotide long) primers are synthesised by the primase Pol alpha These are RNA/DNA hybrids. First a short RNA primer is made then extended by the addition of around 20 dNTPs This occurs in the 5' to 3' direction |
| DNA polymerases | Many different protein domains DNA polymerases are highly conserved enzymes and eukaryotes have at least 25 different DNA polymerases. They operate under a division of labour principle. Main bulk of replication done by epsilon and delta |
| Why do we need a primer | DNA polymerases can only bind to and extend from existing nucleic acid strands |
| Auxiliary factors - PNCA | Proliferating cell nuclear antigen Auxiliary factors are critical for polymerase activity. PNCA is a DNA clamp essential for replication It functions as a processivity and assembly factor for DNA polymerase Loaded onto DNA by clamp loading factor RFC |
| Core replisome assembly | DNA helicase DNA polymerase A pol alpha primer PNCA On leading strand DNA pol moves in 5' to 3' direction On lagging strand it creates Okazaki fragments (backstretch mechanism) |
| Lagging strand synthesis | As DNA polymerase can only move 5' to 3', the lagging strand is replicated by a backstitch mechanism Each time more DNA opens up a new primer is placed down, creating chunks of new DNA called Okazaki fragments |
| Completing lagging strand synthesis | FEN1 recognised the fragments and cuts at the branch point to remove the RNA/DNA primer DNA polymerase and helicase push this aside and continue synthesis The missing phosphodiester bond between the fragments is added by DNA ligase |
| Replication fork movement | Replication is bidirectional, creating two forks Both strands are synthesised on each fork The helicases from the original replisome move in both directions |
| Replication machinery | Helicases - unwind DNA RPA - prevent refolding Topoisomerases - reduce strain on DNA DNA pol a - primase DNA pol - replicates DNA PNCA - tethers pol to DNA RFC - Loads clamp FEN1 - removes RNA flap Ligase - fuses fragments |
| Telomeres | Specific structures at the end of linear chromosomes that prevent shortening of chromosomes during proliferation and also clearly differentiate the ends of chromosomes from DNA with double strand breaks Associated with Shelterin protein complex TTAGGG |
| Replication of telomeres | On the leading strand telomeres can be replicated continuously On the lagging strand there is no template for primase to synthesis the next primer, so continued shortening of the telomere occurs with each replication |
| Telomerase | An enzyme which has an RNA component - Ribo-Nucleo-Protein (RNP) A reverse transcriptase and can use an RNA template to synthesise the complementary DNA strand Its RNA is complementary to telomeres, so can add nucleotides to extend the telomere strand |
| Problems with telomeres | Removal of the RNA primer leads to the shortening of the chromosome after each replication. This eventually leads to cell death RNA in telomerase acts as a template to extend telomeres in the 3' direction restoring length of chromosomal DNA. |
| Role of telomerase in cancer | Loss of telomeres prevents cells replicating indefinitely as in somatic cells telomerase is not active - telomeres shorten and cell stops dividing In cancer the telomerase is activated which maintains the length and helps prevent senescence |
| How telomerase causes cancer | Most normal somatic cells have no telomerase activity, so telomeres shorten over time and cell enters senescence. Continued shortening beyond this leads to apoptosis to avoid cancer. Telomerase activity in cancer avoids this leading to malignancy |
| Steps resulting in high fidelity DNA synthesis | 5'-3' polymerisation - 1 in 10^5 mistakes 3'-5' exonucleolytic proofreading - 1 in 10^2 mistakes Strand-directed mismatch repair - 1 in 10^2 mistakes Overall only 1 in 10^9 errors |
| Correcting mistakes - exonuclease activity | Polymerases with a 3'to5' exonuclease activity can correct mistakes. If a wrong nucleotide is inserted, base pairing is disrupted causing a shift of the pol to exo-activity This allows the pol to remove the wrong nucleotide and resume replication |
| What can exonucleases do | Dissociate the pol and remove incorrect base Proofread and chop the nucleotide strand further back before continuing Can extend beyond the mistake and mismatch repair later Can leave the mutation and propagate it |
| Recreating chromatin | Histones are removed when the replication fork moves forward. In the daughter strands one half of the histones are recycled from the parental, the other is newly synthesised. Additional proteins are dismantled and rebuilt to reform nucleosomes |
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