DNA replication

Semi-conservative
DNA synthesis
1. occurs in a 5’ -> 3’ direction by breakage and formation of phosphodiester bonds
  1. Strand orientation is antiparallel
    1. Leading Strand and Lagging Strand synthesis are oriented antiparallel to one another within the Replication Fork
      1. Leading Strand synthesis is continuous and occurs 5’ -> 3’
      2. Lagging Strand synthesis is discontinuous and also occurs 5’ -> 3’
2. effectively an irreversible reaction because it is coupled to breakdown of PPi to 2Pi by pyrophosphatase
  1. The free energy required for DNA synthesis is provided by the breakage of 2 high energy phosphate bonds
3. All DNA synthesis is initiated by extension of a short primer made of RNA
  1. The RNA primer is synthesised by DNA Primase and only requires a DNA template and NTPs
    1. A surprising role for RNA in the initiation of DNA Synthesis!
4. Lagging Strand synthesis requires DNA Primase, DNA Polymerase, Ribonuclease H and DNA Ligase to convert Okazaki fragments into a continuous strand of DNA
  1. DNA Primase – makes RNA primer
  2. DNA Polymerase – extends RNA primer
  3. Ribonuclease H – removes RNA primer
  4. DNA Polymerase – extends across gap
  5. DNA Ligase seals the nick
    1. DNA Ligase uses the energy of ATP hydrolysis to ligate newly synthesised, adjacent DNA fragments in a two-step catalytic reaction
      1. The ligation process is rendered energetically highly favourable by the conversion of PPi to 2Pi by pyrophosphatase
DNA Replication in cells is bidirectional: 2 Replication Forks are created that move in opposite directions
1. DNA Helicase uses ATP to separate parental DNA strands at the Replication Fork and move the Replication Fork forward
  1. A simple DNA strand separation assay reveals that DNA Helicase activity is ATP-dependent and Magnesium-dependent
  2. rBLM protein is an ATP-dependent, Mg-dependent DNA Helicase that unwinds DNA
  3. Mutations in genes encoding DNA helicases cause human diseases such as Werner Syndrome: a progeria (premature ageing)
    1. Werner Syndrome mutations are autosomal recessive, occurring in RECQ helicase gene WRN
2. Single-stranded Binding Proteins (SSBs)
  1. expose single-stranded DNA in the replication fork
    1. making it available for templating synthesis of the new DNA strand
    2. and easing replication fork progression
3. DNA topoisomerases
  1. prevent DNA from becoming tangled during DNA replication
    1. Unwinding of parental DNA strands at the Replication Fork introduces superhelical tension into the DNA Helix.
      1. Tension is relaxed by DNA Topisomerases, which nick and reseal the backbone of the parental helix
        Type I Topoisomerases nick and reseal one of the 2 DNA strands, no ATP required
        Type II Topoisomerases nick and reseal both DNA strands, ATP required (!)
The processivity of DNA Polymerases is greatly enhanced by their association with a Sliding Clamp
1. Processivity
  1. Once the first step of DNA synthesis has been accomplished, interaction of enzyme with the Primer:Template junction is maintained and addition of further nucleotides is very rapid
2. The Sliding Clamp (ATP-dependent!) is positioned close to the Primer:Template Junction by a Clamp Loader
  1. Sliding clamps encircle the DNA like a nut on a bolt and help to move DNA Polymerase forward
DNA replication requires the same set of proteins in all organisms
1. Helicase: disrupts base pairing in dsDNA, enables replication fork progress
2. Single-stranded binding proteins (SSBs): make template an “easy read”
3. Primase: synthesises RNA primer
4. DNA Polymerase: extends primers annealed to ssDNA template from 3’-OH end
5. Sliding Clamp and Clamp Loader: ensure processivity of DNA Polymerase
6. Ribonuclease H: removes RNA primer
7. DNA Ligase: ligates adjacent single-stranded DNA fragments
8. Topoisomerases: break and rejoin phosphodiester bonds in DNA backbone, relax supercoils generated by Helicase
Control of DNA Replication: Initiation at replication origin
1. Replicators - direct the initiation of DNA replication by recruiting Replication Initiator proteins
2. Initiation of DNA replication in eukaryotes is biphasic:
  1. Replicator Selection - formation of a pre-Replicative Complex - occurs in G1 phase
    1. Eukaryotic Replicator Selection occurs in G1 and leads to the formation of a Pre-Replicative Complex (pre-RC)
      1. Origin Recognition Complex Binds to Replicator sequence
        Helicase loading proteins Cdc6 and Cdt1 bind to ORC
        The Helicase Mcm2-7 binds to complete formation of pre-RC
      2. High levels of Cyclin-dependent kinase (Cdk) activity in S-phase activates existing pre-RC but prevents formation of new pre-RCs
      3. Close relationships between pre-RC function, Cdk levels and cell cycle ensures that chromosomes are replicated exactly once per cell cycle
  2. Origin Activation - unwinding of DNA and recruitment of DNA Polymerase - occurs in S phase
  3. Temporal separation of these 2 events ensures that each chromosome is only replicated exactly once per cell cycle

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DNA replication

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	Criado por Kristi Brogden aproximadamente 10 anos atrás