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