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DNA Replication

Meselson-Stahl experiment - supported Watson/Crick’s theories on DNA replication 

  • DNA replication theories
    • semiconservative replication - each strand of DNA duplex used when forming new DNA
    • conservative replication - original DNA duplex remains intact, new DNA has only new molecules
    • dispersive replication - original DNA gets scattered in new DNA, which contains new/old molecules on each strand
  • bacteria w/ heavier nitrogen (15N) isotope in DNA grown and then transferred to bacteria w/ lighter nitrogen isotope (14N)
  • centrifuge used to determine density of DNA after replication
  • results showed that DNA replicates in a semiconservative way

replication process - must be fast/accurate 

  • starts at origin, goes bidirectionally towards the terminus
  • replicon - functional unit containing chromosome and origin
  • polymerase - enzyme that synthesizes nucleic acids
    • 3 main ones: pol I, pol II, pol III
    • DNA pol II used mainly for DNA repair
    • DNA pol III - made up of alpha subunit (main catalytic part) and beta subunit (forms ring around template, acting as sliding clamp)
    • primer - short stretch of DNA/RNA nucleotides hydrogen-bonded to the complementary strand
    • cannot start synthesis of DNA w/o primer
  • endonucleases - cuts DNA internally
  • exonucleases - chews away at end of DNA; helps proofread
    • used by DNA pol I to remove the primers after replication
  • leading strand - can be replicated as 1 continuous strand, uses 1 primer
  • lagging strand - replicated only in short stretches (Okazaki fragments), uses multiple primers
  • DNA primase - synthesizes short RNA primer
    • RNA polymerases don’t need primers to start
  • DNA helicase - enzyme that unwinds/opens the DNA strands
  • DNA gyrase - form of topoisomerase that takes away the torsional strain (coiling up of strands)
  • single-strand binding protein (ssb) - covers the hydrophobic single, unwound DNA strands
  • DNA ligase - creates phosphodiester bond to join the Okazaki fragments
  • replication fork - site where DNA strands open and replication occurs
  • replisome - replication organelle, assembly of proteins
    • primosome - made up of primase/helicase and other proteins
    • 2 DNA pol III, 1 for each strand
  • both pol III move in the same direction, but 1 of the strands looped

stages of replication -

  • initiation - occurs at the origin (OriC)
    • initiator protein recognizes specific sites within the OriC
    • opens up helix at A-T rich region (very few triple bonds)
    • primosome assembled onto strands
    • 2 replication forks form as replication goes bidirectionally
  • elongation - takes up most of the time during replication
    • pol III add new nucleotides to the template strand
    • more complicated process on lagging strand than on leading strand
  • termination - termination site located opposite the origin on circular chromosome
    • DNA gyrase keeps new DNA molecules from intertwining

eukaryotic DNA replication - main difference in amount of DNA reproduced  

  • uses multiple origins for replication
  • more origins/replicons formed when divisions need to be rapid
  • PCNA (proliferating cell nuclear antigen) - replaces beta subunit in eukaryotes
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