DNA replication

• DNA is reproduced by semiconservative replication. One half is synthesized completely, while the other half is synthesized in fragments (discontinuously)
• The complementarity of DNA strands allows each strand to serve as a template for the synthesis of the other
• 3 modes of DNA replication are possible

  1. Conservative
  2. Semiconservative
  3. dispersive
• Conservative replication - original helix is conserved and 2 newly synthesized strands come together
• Semiconservative - each replicated DNA molecule consists of one “old” strand and one new strand
• Dispersive - parental strands are dispersed into 2 new double helices
• Meselson and Stalh (1958) using N15-labeled E. coli grown in a medium that had N14, demonstrated that DNA replication is semiconservative in prokaryotes
• Using broad bean Vicia faba, Taylor-Woods-Hughes (1957) demonstrated that DNA replication is semiconservative in eukaryotes.
• DNA replication begins at the origin of replication. Where replication is occurring, the strands of the helix are unwound, creating a replication fork.
• Replication is bidirectional; therefore, there are 2 replication forks
• Replicon - the length of DNA that is replicated following one initiation events at a single origin
• Bacteria have single circular DNA and DNA synthesis originates at a single point, the origin of replication, OriC
• The entire bacterial chromosomes constitutes one replicon (6.6 mil base pairs)
• DNA synthesis in bacteria involves 5 polymerases, as well as other enzymes
• DNA polymerase III catalyzes DNA synthesis and requires a DNA template and all 4 dNTPs
• Chain elongation occurs in the 5’ to 3’ direction by addition of one nucleotide at a time to the 3’ end
• As the nucleotide is added, the 2 terminal phosphates are cleaved off, providing a newly exposed 3’-OH group that can participate in the addition of another nucleotide as DNA synthesis proceeds
• DNA polymerases I, II, and III can elongate an existing DNA strand but can’t initiates DNA synthesis
• All 3 polymerases possess 3’ to 5’ exonuclease activity, allowing them to proofread newly synthesized DNA and remove and replace incorrect nucleotides
• Only DNA polymerase I demonstrates 5’ to 3’ exonuclease activity, excising primers and filling in the gaps left behind
• DNA polymerase III is the enzyme responsible for the 5’ to 3’ polymerization essential in vivo. Its 3’5 to 5’ exonuclease activity allows proofreading
• DNA polymerases I, II, Iv, and V are involved in various aspects of repair of DNA damaged by external forces such as UV light
• DNA polymerase III is a complex enzyme (holoenzyme) made up of 10 subunits. This enzyme and some other proteins at the replication fork form a complex called the replisome.
• There are 7 key issues to be resolved during DNA replication

  1. Unwinding of the helix
  2. Reducing increased coiling generated during unwinding
  3. Synthesis of a primer for initiation
  4. Discontinuous synthesis of the 2nd strand
  5. Removal of RNA primers
  6. Joining of the gap-filling DNA to the adjective  strand
  7. Proofreading
• DNA replication

  • Unwinding of DNA - gyrase and helicases
  • In circular DNA - proteins DnaA DnaB and DnaC are helicases
  • OriC (origin recognition complex C) region - 245 base pairs
  • 9 mers and 13 mers (9 and 13 bases repeated)
  • DnaA binds in these areas and starts initial unwinding of DNA
• Subsequent binding of DnaB and DnaC further opens and destabilizes the helix
• Proteins (DnaB DnaC) require the energy normally supplied by the hydrolysis of ATP to break hydrogen bonds and denature the double helix are called helicases
• Single-stranded binding proteins (SSBPs) stabilize the open conformation
• Unwinding produces supercoiling that is relieved by DNA gyrase, a member of a larger group of enzymes referred to as DNA topoisomerases
• Gyrase makes single or double-stranded cuts to undo the twists and knows creating during supercoiling, which are then resealed
• To elongate a polynucleotide chain, DNA polymerase III requires a primer with a free 3-hydroxyl group
• Primase synthesizes an RNA primer that provides the free 3’-hydroxyl required by DNA polymerase III.
• DNA polymerase I removes the primer and replaces it with DNA
• Priming is a universal phenomenon during initiation of DNA synthesis
• Only when the primer is removed can DNA begin adding nucleotides
• As the replication fork moves, only one strand can serve as a template for continuous DNA synthesis - the leading strand
• The opposite lagging strand undergoes discontinuous DNA synthesis
• The lagging strand is synthesized as Okazaki fragments, each with an RNA primer
• DNA polymerase I removes the primers on the lagging strand, and the fragments are joined by DNA ligase
• Both DNA strands are synthesized concurrently by looping the lagging strand to invert the physical but not biological direction of synthesis