DNA replication

• DNA Replication


  The process by which DNA makes a copy of itself so that the information it holds can be maintained and passed to future cell generations
• As soon as Watson and Crick deciphered the structure of DNA, its mechanism for replication became obvious
• DNA Replication


  • It must be copied so that the information it holds can be maintained and passed to future cell generations
  • It is accessed to guide the manufacture of proteins
• Semiconservative DNA Replication


  1. DNA strands separate

  2. Each strand acts as a template for the synthesis of a new complementary strand

  3. Two daughter double helixes are formed, each containing one of the original DNA strands and one newly synthesized strand
• Conservative Replication


  One of the two daughter double helixes would consist entirely of original DNA strands, while the other helix would consist of two newly synthesized strands
• Dispersive Replication


  Both daughter double helixes would carry blocks of original DNA interspersed with blocks of newly synthesized material
• Meselson-Stahl Experiment


  1. Grew E. coli in 15N medium then transferred to 14N medium

  2. Isolated DNA and used density gradient centrifugation to observe DNA composition

  3. Results confirmed semiconservative replication
• DNA Polymerase


  • Requires the four deoxyribonucleoside triphosphates (dNTPs) as substrates
  • Requires a single-stranded DNA template
  • Requires a primer with a free 3' hydroxyl group
• dNTPs


  • Deoxyadenosine triphosphate (dATP), Deoxycytidine triphosphate (dCTP), Deoxyguanosine triphosphate (dGTP), Deoxythymidine triphosphate (dTTP)
  • Provide the bases for incorporation into the growing DNA strand
• DNA synthesis can only proceed by adding nucleotides to the 3' end of an existing polynucleotide
• DNA polymerase catalyzes the formation of new phosphodiester bonds, releasing pyrophosphate as a by-product
• DNA polymerase


  • Required to replicate DNA
  • Strict requirements for action: 4 dNTPs, single-stranded DNA template, primer with free 3' hydroxyl group
• Semiconservative replication


  DNA replication process where each new DNA molecule contains one original and one newly synthesized strand
• Semiconservative replication differs from conservative and dispersive replication
• The strict operating requirements for DNA polymerase action are: 4 dNTPs, single-stranded DNA template, primer with free 3' hydroxyl group
• Direction of DNA synthesis

  5' to 3'
• Basis of the direction of DNA synthesis

  DNA polymerase can only add nucleotides to the 3' end of the growing DNA chain
• Primer


  Short, single-stranded RNA molecule that provides a free 3' hydroxyl group for DNA polymerase to start synthesis
• A primer is needed during DNA replication because DNA polymerase cannot establish the first link in a new chain</b>
• Prokaryotic DNA replication


  1. Recognition of origin of replication (ori) and melting at ori

  2. Formation of replication fork

  3. Synthesis of RNA primer

  4. Leading strand synthesis

  5. Lagging strand synthesis

  6. Removal of RNA primers and joining of Okazaki fragments

  7. Termination of replication
• The primary function of DNA replication is to provide progeny with the genetic information possessed by the parent
• DNA replication must be complete and carried out in a way that maintains genetic stability
• DNA replication is complex and involves many cellular functions and verification procedures to ensure fidelity
• Initiation stage of DNA replication

  Prepares the double helix for use as a template
• Prepriming complex


  • Proteins that open up the double helix, separate the parental strands, and unwind the double helix ahead of the replication fork
  • Includes DnaA protein, DNA helicase, and single-stranded DNA-binding (SSB) proteins
• Origin of replication (ori)

  Single, unique nucleotide sequence where DNA replication begins
• DnaA protein


  Protein in prepriming complex that recognizes ori and causes melting of AT-rich regions
• Replication fork


  Small Y-shaped area where active DNA synthesis occurs as the two strands unwind and separate
• DNA helicase


  • Binds to single-stranded DNA and moves into the neighboring double-stranded region, forcing the strands apart to unwind the double helix
  • Requires energy from ATP
• Single-stranded DNA-binding (SSB) proteins

  • Bind cooperatively to single-stranded DNA generated by helicases, keeping the strands separated and providing the template for polymerases
  • Also protect DNA from nucleases
• Positive supercoils


  Accumulate in the region of DNA ahead of the replication fork as the two strands separate, interfering with further unwinding
• DNA topoisomerases


  • Enzymes that remove positive supercoils
  • Type I topoisomerases reversibly cut one DNA strand and pass the other through the break to relax supercoils
  • Type II topoisomerases bind tightly to DNA, make transient breaks in both strands, and pass another stretch of DNA through the break to relax supercoils
• DNA gyrase


  Type II topoisomerase found in bacteria and plants that can introduce negative supercoils into relaxed circular DNA, facilitating future replication and transcription
• Leading strand


  The strand being copied in the direction of the advancing replication fork, synthesized continuously
• Lagging strand


  The strand being copied in the direction away from the replication fork, synthesized discontinuously in short Okazaki fragments
• Primase


  RNA polymerase that synthesizes the short RNA primers complementary to the DNA template to initiate DNA synthesis
• DNA polymerases require an RNA primer to initiate synthesis of a complementary DNA strand
• Eukaryotic DNA replication


  1. Identification of the origins of replication (multiple ori) in linear DNA

  2. ATP hydrolysis-driven unwinding of dsDNA to provide a single-stranded DNA (ssDNA) template

  3. Prevention of reannealing of single-stranded DNA

  4. Synthesis of RNA primers

  5. Chain elongation with formation of replication bubbles

  6. Excision of RNA primers and filling of gaps

  7. Sealing of nicks

  8. Reconstitution of chromatin structure
• Differences between prokaryotic and eukaryotic DNA replication


  • Recognition of ori: Single ori; recognized by DnaA protein (prokaryotes) vs Multiple ori; recognized by origin recognition complex (ORC) (eukaryotes)
  • Unwinding: DNA helicase (prokaryotes) vs Mini-chromosome maintenance complex (MCM) (eukaryotes)
  • Prevent reannealing of separated strands: Single-stranded DNA binding protein (prokaryotes) vs Replication protein A (RPA) (eukaryotes)
  • Synthesis of RNA primer: RNA primase (DnaG) (prokaryotes) vs Pol α has primase activity which initiates synthesis on leading strand and beginning of Okazaki fragments (eukaryotes)
  • Elongation: DNA polymerase III (prokaryotes) vs Pol ε completes DNA synthesis on leading strand, Pol δ elongates Okazaki fragments; both with 3' to 5' exonuclease activity for proofreading (eukaryotes)
  • Excision of RNA primers: DNA polymerase I (prokaryotes) vs RNase H and flap endonuclease-1 (FEN1) (eukaryotes)
  • Gap filling: DNA polymerase I (prokaryotes) vs Pol β in gap filling (eukaryotes)
  • Sealing of nicks: DNA ligase (both)
  • Mitochondrial DNA replication (eukaryotes only): Pol γ replicates mitochondrial DNA
• In animal cells, including human cells, the replication of the DNA genome occurs only at a specified time during the life span of the cell, a period referred to as the synthesis or S phase