DNA Replication

• DNA (RNA) strand is synthesised in the 5' to 3' direction. Template strand synthesised in the 3' to 5' direction.
• What is needed to make a DNA copy?

  • new nucleotides
  • unwinding DNA double helix to give 2 parental templates
  • unwinding is the release of tension generated
  • prevention of unwounded DNA stranded helical
  • joining the ends of new synthesised fragments together
• Replication is semi-continuous
• Lagging strand is discontinuously synthesised 5' - 3' direction as okazaki fragments.
• Okazaki fragments are joined by ligase enzyme.
• Leading strand is continuously synthesised 5' - 3' direction.
• Primase is an enzyme that makes an RNA primer, which is the starting point for RNA polymerase.
• Dna polymerisation is the main DNA replicative. It can add thousands of nucleotides to a growing DNA strand without dissociating from the template strand. It needs an OH group onto which phosphate group of the incoming nucleotide can be attached.
• DNA polymerisation makes the DNA 5' - 3' direction
• DNA polymerisation is an enzyme that synthesises a new DNA strand by adding nucleotides complimentary to DNA strand.
• Initiating DNA replication

  A) Primase

  B) Replication Fork

  C) Helicase

  D) Single strand binding proteins

  E) Topoisomerase

  
• Making new DNA strands

  A) Leading strand

  B) Lagging strand

  C) Leading strand template

  D) Lagging strand

  
• DNA polymerase has 2 activities

  1. Removes RNA primers
  2. Fills the gap with DNA nucleotides
• When removing RNA primers, an endonuclease enzyme that recognises DNA:RNA hybrids and degrades the RNA part
• When filling the gap with DNA nucleotides, DNA polymerase synthesises DNA by adding nucleotides complementary to parental DNA template by lagging strand.
• DNA ligase joined new synthesised fragments together once RNA primers have been removed and replaced by DNA nucleotides.
• DNA ligase also joined newly synthesised fragments from the bubble including leading strands.
• During replication -> exonuclease

  After replication -> endonuclease
• Exonuclease is when they show high level of accuracy. Replication error rate of 1 -> 10^8 to 10^10 base pairs replicated, by proof reading mechanism.
• Proof reading mechanism adds new nucleotides to growing DNA strand. It checks newly inserted base against the template strand.
• Endonuclease is a variety that can cause damage:

  • incorrectly insert bases are not corrected by DNA pol (||)
  • radiation damage
  • chemical modifications of bases
• The damaged or incorrect DNA is removed by an endonuclease. Then a DNA polymerase makes new DNA. This DNA ligase joins new DNA to existing DNA.
• Importance of correcting DNA errors

  If DNA errors are not corrected, they become part of the DNA template strand and can be a mutation where the DNA error will be permanent.
• Polymerase Chain Reaction (PCR) in the vitro method making multiple DNA copies that there's enough DNA material to work with.
• PCR

  • only targets DNA that will be copied.
  • rapid exponential increase of DNA molecules
  • method utilises cycles of heating and cooling
• PCR is important because of the amplification of DNA, detection and identifications of mutations and insertions, gene cloning, manipulation and forensic analysis.
• DNA template is a DNA molecule to which complementary nucleotides can be matched to make identical copies
• Primers provide free 3OH group and defines the region of DNA molecule to be replicated
• DNA polymerase is an enzyme which adds nucleotides and joins them together forming a phosphodiester bond
• dNTPs are free nucleotides