DNA Replication

Principles Module 47


  • Explain how the structure of DNA suggests a mechanism for its replication
  • Describe the design of the Meselson and Stahl experiment that demonstrated semiconservative replication
  • Discuss the process of DNA replication
  • Identify the key enzymes involved in replication and their functions

Key points

DNA structure

  • each nucleotide is composed of a pentose sugar with a phosphate and a nitrogenous base
  • early work showed A in equal amount as T, G equal to C
  • X-ray diffraction studies by Franklin and Wilkins gave clues to structure
  • Watson and Crick made the major conceptual breakthrough, proposing the double helix
Semiconservative Replication
  • Meselson and Stahl designed an experiment to test different models of replication using ‘heavy’ N to label the nitrogenous base part of DNA
    • grew bacteria in 15N media for many generations, then transferred to 14N
    • separated DNA by density gradient centrifugation
    • different replication models made distinct, testable predictions:
      • Semiconservative replication predicts 1 density of DNA after 1 round, 2 after 2 or more rounds of replication
      • Conservative replication predicts 2 densities of DNA after 1 or more rounds of replication
      • Dispersive replication predicts 1 density after 1 or more rounds of replication
Features of semiconservative replication
Overview of Replication
  • initiation: proteins open double helix, exposing single strands of DNA to enzymes
  • elongation: DNA polymerases add free nucleotide bases in 5′ to 3′ direction
  • termination: polymerases fall off template strands
Replication in Prokaryotes in detail
  • begins at a specific sequence of DNA known as the origin of replication (oriC), rich in AT sequences
  • DNA polymerase enzymes only add free nucleotides to the -OH on the 3′ carbon, thus replication proceeds from 5′ end to 3′ end of polynucleotide
    • DNA polymerase III is the key enzyme for replication in prokaryotes
  • primase adds a short sequence of RNA ‘primer’ to DNA template
    • primase does not require the free 3′-OH group to catalyze addition
  • DNA helicases unwind the double helix, require ATP
  • once unwound, DNA binds to single-strand binding proteins (SSBs) that prevent re-pairing
  • topoisomerases bind to double-stranded DNA and relieve torsional strain
Leading and lagging strands
  • leading strand synthesis is toward the replication fork (where DNA is unwinding), can be replicated from a single primer
  • lagging strand is away from replication fork, requires repeated priming events, which creates Okazaki fragments
  • DNA replication on the lagging strand is discontinuous
  • DNA polymerase I replaces RNA primers with DNA
  • DNA ligase joins Okazaki fragments by forming phosphodiester bond
Eukaryotic differences
  • larger genome of Eukaryotes requires multiple origins, forming distinct replicons
  • the overall process is similar but involves more components
  • Eukaryotic chromosomes are linear, not circular, presenting a problem for replication on the lagging strand at the end
  • telomerase solves this problem by having a built-in RNA template for repeating telomere sequence
  • keeps telomeres from shortening after each replication cycle

In-class activities

Questions for Practice

  • In what way did the structure of DNA proposed by Watson and Crick contain a mechanism for replication ‘built-in’?
  • Describe the experiment that showed DNA replication was semi-conservative.
  • What is the role of _________ (helicase, topoisomerase, primase, DNA polymerase III, polymerase I) in DNA replication?
  • What is an Okazaki fragment?