DNA Replication

• what is the function of helicase?

  • unzipping and separating 2 strands of DNA double helix
  • travels along DNA backbone, catalysing reaction to unzip
  • to provide a single-stranded DNA for replication
• what type of replication is DNA replication?

  semi-conservative - each of the two new DNA helices contain one original strand and one newly synthesised strand
• what is the function of topo-isomerase?

  • binds to double stranded DNA
  • cuts phosphate backbone of one/both strands
  • prevents knotting/supercoiling
• what is the function of DNA polymerase?

  • catalysis formation of phosphodiester bonds between nucleotides
  • create DNA molecules by assembling nucleotides
• what is the function of DNA ligase?

  • create phosphodiester bonds between 3’ hydroxyl of one nucleotide and 5’ phosphate of another
• what are the requirements of DNA ligase?

  it depends of Mg2+ ions and ATP/NAD+ cofactors
• what are the requirements for DNA polymerase?

  It depends of a template nucleotides, primers and Mg2+
• why is DNA ligase important?

  • for DNA recombination, repair and replication
• why is DNA polymerase important?

  • important for DNA replication, repair and recombination technology
• roughly how many base pairs are replicated?

  3 billion
• what are the steps of DNA replication?

  • there is a short representative portion of DNA, which is about to undergo replication
  • DNA heliase causes the leading and lagging strand to unzip
  • Free nucleotides that have been activated are attracted to complementary bases
  • activated nucleotides are joined together by DNA ligase
  • a complete polynucleotide chain is formed - two identical molecules of DNA are formed using DNA polymerase to join complementary nucleotides, each composed of one original strand and one newly formed molecule (semi-conservative replication)
• what does the anti parallel property of a DNA strand mean?

  Opposite directionality of the two DNA strands - leading strand starts with a carbon 3 and ends with a carbon 5, and the lagging strand starts with carbon 5 and ends with carbon 3
• which direction does DNA polymerase always move along the template strand?

  • it can only bind to the 3’ (OH) end, so reads in the 3’ to 5’ direction and makes the new strand in the 5’ to 3’ direction
• what is meant by continuous replication?

  Replication that occurs on the leading strand, that is unzipped from the 3’ end can be continuously replicated as the strand is unzipped
• what is meant by discontinuous replication?

  Replication that occurs on the lagging strand, that is unzipped from the 5’ end, so the DNA polymerase has to wait until a section of DNA has unzipped (sections called Okazaki fragments which then have to be joined)
• how are Okazaki fragments joined?

  DNA ligase
• how does each Okazaki fragment start?

  with an RNA primer - exonuclease removed the primer
• why does the leading and the lagging strand replicate in different ways?

  as DNA polymerase only travels in the 3’ to 5’ direction, only the leading strand of DNA can be replicated continuously as the DNA unwinds but the lagging strand has to be replicated in the opposite direction in the short sections called Okazaki fragments
• what does semi-conservative replication mean?

  DNA replication where each new double-stranded DNA molecule consists of one original strand and one newly synthesised strand, called an intermediate molecule
• what does DNA conservative replication mean?

  where one DNA molecule is an identical replication of the original DNA molecule, and the other is an entirely new synthesised DNA molecule
• what does dispersive DNA replication mean?

  where the new DNA molecules contain different proportions of original and newly DNA within each polynucleotide strand
• what is meant by the term isotope?

  different forms of the same element that have the same number of protons, but a different number of neutrons
• what are two isotopes of nitrogen?

  Nitrogen - 14 (light) and Nitrogen - 15 (heavy)
• what is a centrifuge?

  A machine that separates substances of different densities using centrifugal force

  • spins quickly
  • contains different salt concentrations
• how can DNA be separated according to its weight using caesium chloride?

  allows different densities of DNA to be separated
• what is the Messelsohn and Stahl's experiment?

  • grew bacteria on a medium only containing N-15 (all DNA contains N-15)
  • DNA will move further down density gradient of CsCl in centrifuge (is heavy)
  • Switched to growing bacteria on medium only containing N-14 (light)
  • New DNA contains one strand with N-15 and one strand with N-14 (intermediate DNA) - doesn't move down the density gradient as far
  • subsequently replicated DNA contains one old strand (with either N-14 or N-15) and one strand with N-14