Mechanism of DNA Replication

• DNA replication


  1. Semi-conservative replication of DNA (proven by experiments of Meselson and Stahl)

  2. The 2 DNA strands separate and provide a template for synthesis of new DNA

  3. Replication is initiated at a specific origin sequence

  4. Formation of replication fork

  
• Replicons


  • Replication of DNA molecules within cells takes place in segments known as replicons
  • A replicon is a unit of DNA in which individual acts of replication occur
  • Each replicon fires once per cell cycle
  • Each has an origin where replication initiates and a terminus where it stops
  • Any sequence not separated from an origin by a terminus is replicated as part of the replicon
  • Some sections of DNA will be copied at different times

  
• DNA replication in eukaryotes

  • This occurs in the S (synthesis) part of interphase
  • Multiple replicons may be advantageous because eukaryotes have much more DNA
  • If all replicons fired at once replication could finish within 1 hour
  • Actually, takes about 6-8 hours because entire sequence of DNA isn't being copied at once
  • Maybe 15% are active at any one time
  • Eukaryotic origin sequences are poorly understood

  
• Eukaryotic origins - yeast

  • Origins have been isolated in yeast
  • ARS = autonomously replicating sequence
  • If a piece of DNA has a sequence that is an ARS in it, it can be replicated as its giving a signal to the cell where to start copying
  • Any circular piece of DNA containing an ARS will replicate in yeast
  • Yeast origins are A+T rich – hydrogen bonding between these are weaker as they have 2 hydrogens instead of three
  • About 50bp (base pairs) long
  • There is a 14bp core region with 11bp conserved sequence
  • Mutations here disable the ARS
  • An ARS extends for 50bp and includes a consensus sequence, with some imperfect copies
  • A sequence that is highly conserved, we will always see this sequence in these regions in yeast, the act to start off DNA replication
  • Highly conserved sequence
  • If you mutate both of those bases, it wont act any longer as a signal to the cell to start replication here
  • This region lies in a stretch of 50 base pairs where you get high conservation of the sequence
• Polymerases


  • A DNA polymerase is an enzyme which can synthesize a new DNA strand from a template sequence
  • DNA polymerase has the function of creating new DNA stands on its parent template, single strand of DNA that has been exposed
  • E.coli has 5 DNA polymerases but 3 main ones: DNA polymerase I – repair of damaged DNA + replication, DNA polymerase II – implicated in repair, DNA polymerase III – multisubunit enzyme responsible for de novo synthesis of DNA
  • Eukaryotes have 5 main polymerases: Alpha, beta, gamma, delta, epsilon
  • There is much overlap in action with the bacterial polymerase
  • Job is to put together in order nucleotides to give a newly synthesised strand of DNA
  • Both eukaryotic and bacterial polymerases add nucleotides one at a time at the 3' end of a DNA strand
  • Where there is a single strand, DNA polymerase can act to do that pairing of complementary nucleotides

  
• DNA polymerase


  • DNA polymerase cannot initiate synthesis
  • They require a primer to provide a free 3' OH end from which they can extend
  • These primers are an important component of DNA replication
  • In the cell there has to be synthesis of a short region that we call a primer, which is a short region of nucleotides, a little short and single stranded section of either DNA or RNA, and then DNA polymerase can recognise this, and it can start adding nucleotide here at the three prime end and it will continue to do that until it reaches a stop sequence in the cell

  
• DNA synthesis occurs in the 5' to 3' direction


  • This refers to the newly synthesized chain
  • It is important to understand the orientation with respect to the parent and daughter strands
  • New nucleotides are added to the 3' hydroxyl (-OH) group of the extending DNA chain
  • It has to have this double stranded region for DNA polymerase, the synthesis enzyme to come in and add the next nucleotide
  • There has to be a region where there are some nucleotides already bound to it for DNA polymerase to add the next complementary base here
  • If there is an A, it will bring in a T and it will pair them together and DNA polymerase will then form that diphosphate backbone
  • It will polymerase it and add that new nucleotide so that it starts building up a new strand to copy that piece of DNA
  • So that we end up with the parent strand and a newly synthesised strand that's semi conservative
  • DNA polymerase is the enzyme that catalyses DNA synthesis

  
• Unwinding the DNA


  1. Topoisomerase relaxes supercoiled DNA

  2. Initiator protein binds DNA

  3. DNA helicase binds initiator protein, physically unwinds the DNA causing it to denature that region

  4. This requires energy (ATP)

  5. As helicase unwinds the DNA single stranded binding protein (SSB) stabilizes the DNA and prevents it from forming a 2o structure

  6. DNA is now single-stranded and can act as a template for synthesis

  7. DNA strands like to bond to each other, and hydrogen bonding occurs between the complementary bases

  8. Once the helicase enzyme has opened up those two strands, we don't want them to just bind back to each other

  9. The parts of DNA that are separated, the single strands become bound by what we cal single strand binding proteins, and this helps to keep them apart so that the other enzymes can come in and start doing DNA synthesis

  
• Priming DNA synthesis


  1. Primase binds to helicase and the denatured DNA

  2. Primase + helicase = "primosome"

  3. This activates the primary enzyme to make a short region where its double stranded, its just temporary and it makes it out of RNA nucleotides and not DNA nucleotides

  4. Primase is activated by helicase and synthesized a short RNA primer for initiation of DNA synthesis

  5. It is this RNA primer that provided the free 3'-OH group onto which DNA polymerase III can add the first nucleotide

  6. Have uracil instead of thymine

  7. Primase synthesises a short region where it becomes double stranded

  8. Now DNA polymerase has a three prime hydroxyl group that it can start adding on those new nucleotides by complementary pairing with the template to synthesise that new strand

  
• Priming DNA synthesis


  1. DNA helicase is bound, and it has opened up the DNA at that region where replication is going to start

  2. Formed a replication fork where it's opened up with the parent DNA

  3. Got two single strands

  4. Primase binds to the helicase enzymes forming the primosomes that activates and primes to then synthesis a short sequence of RNA nucleotides that are complimentary paired to the DNA

  5. DNA polymerase, which is indicated with the purple donut enzyme, DNA polymerase combined to that DNA and start the synthesis of the DNA by adding the next nucleotide by complimentary base pairing with the template

  
• New template for synthesis

  • Each replication fork has an origin and direction of travel
  • As the DNA unwinds it reveals new nucleotides to act as a template

  
• The Problem of antiparallel strands

  • But synthesis can only follow the unwinding DNA on one strand
  • This is called the leading strand
  • Nucleotides are added strictly at the 3'-OH position of the elongating strand so in any given direction only one of the two strands has a 3' end
  • The other strand is unavailable for new synthesis
  • This is called the lagging strand

  
• Okasaki fragments


  • A solution to the problem of replicating antiparallel strands is accomplished by synthesizing multiple short fragments of DNA in the normal 5'-3' direction and joining them together later
  • These pieces are called Okasaki fragments after Reiji and Tuneko Okasaki who discovered them

  
• Okasaki fragment synthesis


  1. RNA primer copied from DNA

  2. DNA polymerase III elongates RNA primer with new DNA

  3. DNA polymerase I removes 5'RNA at end of neighbouring fragment by 5'-3' exonuclease activity and replaces with DNA

  4. DNA ligase joins adjacent fragments

  
• Okasaki fragments


  • Short RNA primers are synthesized on the lagging strand (close to the replication fork)
  • DNA polymerase III lengthens the primer moving away from the replication fork and displaces SSB proteins
  • After the replication fork has moved another RNA primer is synthesized and elongated

  
• Action of DNA ligase

  Action of DNA ligase in sealing the gap between adjacent DNA fragments to form a longer, covalently continuous chain

  
• Key component of DNA replication

  • Topoisomerase
  • Helicase
  • Single stranded binding protein (SSB)
  • Primase
  • DNA polymerase III
  • DNA polymerase I
  • Ligase
  • Replisome

  
• Replisome complex


  Model for the replication machine, or replisome, the complex of key replication proteins, with the DNA at the replication fork