L9: Enzymology of DNA replication

• What is the title of Dr. Robert Spooner's lecture in 2024?

  Enzymology of DNA replication
• What are the key features of DNA replication summarized in the 1950s?

  • DNA replication is semi-conservative
  • DNA strands are anti-parallel
  • New DNA is synthesized in the 5’ → 3’ direction
  • DNA synthesis is semi-continuous with leading and lagging strands
  • DNA polymerase has proof-reading 3’ → 5’ exonuclease activity
• Who established the directionality of DNA synthesis?

  Reiji and Tsuneko Okazaki
• How is the leading strand synthesized during DNA replication?

  It is synthesized continuously in the same direction as the replication fork.
• How is the lagging strand synthesized during DNA replication?

  It is synthesized discontinuously in the opposite direction to that of the replication fork.
• What are Okazaki fragments?

  Short segments of DNA synthesized on the lagging strand.
• Why do Okazaki fragments occur during DNA replication?

  Because new DNA is built in the 5’ → 3’ direction only.
• What is the typical length of Okazaki fragments in eukaryotes?

  ~100-200 nucleotides
• What is the typical length of Okazaki fragments in E. coli?

  ~1000-2000 nucleotides
• Why is there no 3’ → 5’ synthesis of new DNA?

  Because DNA polymerase can only synthesize DNA in the 5’ → 3’ direction.
• What role does hydrolysis of the incoming nucleotide play in DNA synthesis?

  It provides the energy for polymerization.
• What type of genome does the M13 bacteriophage have?

  A single-stranded DNA genome.
• How long is the M13 phage?

  ~900 nm long
• How thick is the M13 phage?

  ~6 nm thick
• What is the approximate size of Escherichia coli?

  ~1-2 μm long
• What is the approximate width of Escherichia coli?

  ~0.5 μm wide
• What prevents the replication of M13 phage DNA in E. coli extracts?

  Rifampicin, an inhibitor of E. coli RNA polymerase.
• What did Tsuneko Okazaki discover about Okazaki fragments?

  DNase cannot completely destroy Okazaki fragments, leaving small RNA pieces.
• What is the role of DNA primase in DNA replication?

  It synthesizes an RNA primer to initiate DNA synthesis on the lagging strand.
• What are the steps involved in lagging strand synthesis?

  1. RNA primers are synthesized by DNA primase. 2. DNA Pol III extends the RNA primer to make Okazaki fragments.
• What happens to old RNA primers during lagging strand synthesis?

  They are erased by the 5’ → 3’ exonuclease activity of Pol I and replaced with new DNA.
• How is the gap between Okazaki fragments sealed?

  By DNA ligase, which joins the Okazaki fragment to the growing chain.
• What is the role of DNA ligase in joining Okazaki fragments?

  It uses ATP as an energy source to form a phosphodiester bond between fragments.
• What is processivity in the context of DNA polymerase?

  It is a measure of an enzyme's ability to catalyze consecutive reactions without releasing its substrate.
• How does clamping affect the processivity of DNA Pol III?

  Clamping converts Pol III to high processivity, allowing it to replicate long stretches of DNA.
• How does the lagging strand template behave during synthesis?

  It is forced into a loop to accommodate the synthesis of Okazaki fragments.
• What happens as helicase unwinds the DNA during lagging strand synthesis?

  Pol III replicates the leading strand continuously and extends the new primer on the lagging strand.
• What is the replication speed of DNA polymerase III?

  ~1000 bases per second
• What does the hand flexing analogy represent in the context of DNA polymerase?

  It represents how DNA polymerase grips and releases the DNA duplex during replication.
• How is leading strand synthesis characterized?

  It is straightforward and continuous.
• What is the direction of synthesis for the leading strand?

  5’ to 3’ direction
• What role does the clamp holder play in DNA replication?

  It transfers the two halves of the β clamp to DNA polymerase III.
• What does clamping do for DNA polymerase III?

  It converts Pol III to high processivity, allowing it to replicate long stretches of DNA.
• How does helicase contribute to leading strand synthesis?

  It unwinds the DNA, allowing Pol III to replicate the leading strand continuously.
• What is the primary challenge of lagging strand synthesis?

  It is more complex than leading strand synthesis.
• What does DNA primase do on the lagging strand template?

  It manufactures an RNA primer.
• What happens to the primed duplex on the lagging strand?

  It is captured by Pol III and clamped, forcing the lagging strand template into a loop.
• Why does DNA Pol III need to have low processivity during lagging strand synthesis?

  Low processivity allows it to release the new Okazaki fragment easily.
• What is the torsional problem in DNA replication?

  Unwinding of DNA causes tightening, which stimulates supercoiling.
• What type of supercoiling is most common in DNA?

  Most DNA is negatively supercoiled.