DNA replication

DNA replication：Initiation、Elongation、Terminations、some terms' defination、some enzymatic conventions……

DNA replication

• Initiation
  • initiation site
    • Prokaryotic
      • OriC
        • repeated nucleotides
          • bind initiator proteins
          • 4*9-mer repeats
        • A-T rich sequence
          • 3*13-mer repeats
          • open site
        • 240bp/4Mb Ecoli genome
      • single replicon
      • initiator proteins
        • DnaA
          • DnaAs bind and wrap around 9-mer repeats make A-T rich sequence opens
    • Eukaryotic
      • no specific origins
        • may depends on both sequence and chromatin structure
        • 0.5-55kbp intiation zone with no signal sequences
      • multiple replicons
        • no define number
          • devolopment dependent
          • increases with size of chromosome
      • initiator proteins
        • ORC(origin recognition complex)
          • six-subunit protein
  • Replisome
    • Helicase
      • role
        • Unwinds DNA double helix
          • produces torsional strain
            • cause supercoiling
      • Prokaryotic
        • DnaB
          • helicase loader(DnaC) loads 2 Helicase(DnaB) to the 2 replication folks of the opened A-T rich region
            • DNA replication is always bidirectional (exept some viruses)
            • loading requires 1 ATP per Helicase
            • forms prepriming complex
        • Structure
          • hexamer with 1.7nm hole
      • Eukaryotic
        • MCM
          • Mini Chromosome maintenance
      • Moves 5’ to 3’ on lagging strand template
        • with hydrolysis of ATPs
    • Topoisomerases
      • Type I
        • transiently nick one strand, pass the intact strand through the nick, then reseal the nicked strand.
      • Type II
        • break both strands and pass a double helical section through the double-strand break. They require ATP.
          • e.x.DNA gyrase
            • relieve supercoiling
    • DNA Primase
      • role
        • leading strand
          • synthesis once at initiation
        • lagging strand
          • synthesis periodically
      • Prokaryotic
        • only RNA polymerase
          • syntheses 10-20bp long RNA primers
      • Eukaryotic
        • Primase–Pol α complexes
          • contains both RNA & DNA polymerase
            • Primase synthesis first short RNA and extend with DNA by pol α
      • After each synthesis, Primase dissociates
    • DNA polymerase
      • Prokaryotic
        • DNA polymerase I
          • DNA synthesis
            • 5'-to-3' DNA polymerase activity
          • proofreading
            • 3'-to-5' exonuclease activity
              • larger fragment
                • res-324 to 928 = Klenow fragment
          • role
            • replaces primer with DNA
              • 1. erases primer
                • 5'-to-3' exonuclease activity
                  • smaller fragment
                    • res-1 to 323
              • 2. fill gaps
        • DNA polymerase II
          • DNA synthesis
            • 5'-to-3' DNA polymerase activity
          • proofreading
            • 3'-to-5' exonuclease activity
          • role
            • DNA repair
        • DNA polymerase III
          • DNA synthesis
            • 5'-to-3' DNA polymerase activity
          • proofreading
            • 3'-to-5' exonuclease activity
          • role
            • main replication enzyme
          • subunitits
            • β subunit
              • provides high processivity
              • 2 identical protein chains
                • form a circular sliding clamp
                  • requires clamp lodder to open it, load it on DNA, and close it
        • DNA polymerase IV
        • DNA polymerase V
      • Eukaryotic
        • 2 main replication enzymes
          • polymerase delta(pol δ）
            • PCNA-Rfc（Replication factor C)-Pol δ displaces primase-polα
              • increases processivity
              • increases efficiency
                • 5 times faster than pol α
                  • it's easier if you just view it as part of primer..
          • polymerase epsilon(pol ε)
    • Single strand binding proteins(SSB)
      • role
        • stabalize exposed hydrophilic base in aqueous environment
      • Eukaryotic
        • RPA proteins
    • sliding clamps
      • Prokaryotic
        • β subunit of DNA pol III
          • Ring shaped dimer
            • each monomer having three globular domains
      • Eukaryotic
        • PCNA (proliferating cell nuclear antigen)
          • discovery
            • because this protien exist in proliferating cell nucleus and induce antibody productin
          • structure
            • Ring shaped trimer
    • Clamp loading complex
      • role
        • loads clamps
          • 1. loads a β subunit on DNA single strand
            • leading strand
              • load onece
            • lagging strand
              • load periodically
          • 2. transfers DNA pol III to every loaded clamp
        • holds 2 DNA pol III together with other accessory proteins
      • structure
        • claw domain
          • catch and loads clamp
        • 2 DNA pol III main body attaching domain
          • constantly attached to the main body
        • tail domain
          • binds to helicase on lagging strand
    • conserved arrangement in all 3 domains
      • clamp loader, sliding clamp, 2 polymerases, helicase, and primases at replication folk
• Elongation
  • leading strand
    • single priming event
  • lagging strand
    • looping
      • make DNA pol IIIs on 2 strands(DNA pol dimmer) move in same direction, but DNA synthesis in two strands under different directions
        • because DNA polymerase III periodically dettaches
    • perioditity
      • Clamp
        • it detaches spontaneously when the elongating okazaki fragment reaches previous primer
          • Then this primer is removable by DNApol I
      • Clamp loader
        • it always holds 2 DNA pol III major subunit
        • it periodically catches a β clamp, encircle it on new primer site of DNA template and transfer the free DNA polIII body on it
      • DNA primase
        • periodically synthesis primers
      • DNA polymerase III
        • periodically dettaches from DNA template
          • because β clamp is periodically dettached
            • and main body dissociates with β clamp
        • and reassociates with β clamp next round
      • loop
        • grows
          • loop grows when DNApolIII elongating okazaki fragemts
        • refills
          • after the new dettach & loading event, the loop is refilled
    • Eukaryotic okazaki fragments maturation
      • No DNA polymease 1
      • so pol δ replaces the primer, then RPA covers the protruding and Dna2 enonuclease cleaves it, finally DNA ligase I stichs the nick using 1 molecule of ATP
  • Protein changes
    • primer removals
      • prokaryotic
        • DNA pol I
      • eukaryotic
        • pol δ displaces backward from 5' end
    • stich nicks/DNA ligase
      • prokaryotic
        • DNA ligase
          • 1. NAD+
      • Eukaryotic
        • DNA ligase I
          • 1 ATP
• Terminations
  • termination site
    • Prokaryotic
      • Ter
        • conserved sequence: GTGTGTTGT
        • roughly opposite to oriC
    • Eukaryotic
      • telomere
        • elongated by telomerase
  • termination proteins
    • Prokaryotic
      • Tus
        • contra-helicase
  • DNA intertwining
    • only prokaryotic issue
      • opened by DNA gyrase
• some terms' defination
  • Replicon
    • The DNA contolled by an origin
  • topological state of DNA
    • the coiling of double helix
      • torque
        • tortion strain
      • supercoiling
        • coil in space
      • intertwining
        • like two rings in chains
  • leading strand
    • continuous strand
  • lagging strand
    • discontinuous strand
  • Okazaku fragments
    • DNA fragments synthesized on the lagging strand
  • replication fork
    • the partial opening DNA helix, here replisome forms
  • processivity
    • The ability of a polymerase to remain attached to the template
  • Replisome
    • The assembly of enzymes involved in DNA replication
    • subdivision
      • primosome
        • primase complex with helicase
      • DNA pol III dimer
• some enzymatic conventions
  • all polymerase synthesize nucleotides from 5' to 3' direction
  • all DNA polymerases require primers, but RNA polymerase don't