L52: Recombination and Transposons

• What are the key activities of RecBCD in DNA recombination?

  • Has nuclease activity for initial DNA nick
  • Has helicase activity to unwind DNA
  • Allows binding of single stranded DNA-binding protein and RecA
• What is required for the initial nick in DNA during recombination?

  A specific DNA sequence
• What are the steps involved in homologous recombination mechanism?

  1. Cross strand exchange
  2. Second nick in DNA
  3. Branch migration
  4. Requires RuvAB helicase
  5. Extensive heteroduplexes form
• What is a Holliday junction?

  A structure formed during homologous recombination
• What happens during the isomerization of a Holliday junction?

  Strands cross and uncross
• What are the two outcomes of resolving a Holliday junction?

  RuvAB interacts with RuvC for resolution
• How can homologous recombination rescue a double stranded break?

  • Involves 5’ to 3’ exonuclease activity
  • Utilizes homologous DNA strand
  • DNA synthesis occurs
  • Strand invasion takes place
• What is the final step in resolving Holliday junctions?

  Ligation
• What is the role of Hfr strains in gene transfer?

  They can transfer their genome
• What is non-homologous recombination?

  • No sequence homology
  • Examples include insertion of λ phage
  • Involves transposon events
  • Used in double stranded break repair
• Who won the Nobel Prize in 2020 for work related to non-homologous recombination?

  Emmanuelle Charpentier and Jennifer Doudna
• What happens during non-homologous recombination after double stranded breaks?

  • Ends are rejoined
  • Sequence might be lost if degraded
  • Ends can join to the "wrong" ends
• What are insertion sequences (IS)?

  • Small DNA pieces (~1000 bp)
  • Can hop from one position to another
  • Catalyzed by transposase
  • Carries no other genes
• What is the function of transposase in insertion sequences?

  It catalyzes transposition
• What are the effects of insertion sequences on phenotype?

  • Carries no novel features
  • Can disrupt genes due to insertion
  • High degree of reversion
  • Disruption of housekeeping genes can be lethal
• What are transposons?

  • Similar to insertion sequences
  • Carry additional genes, sometimes resistance genes
  • Can knock-out genes through insertion
• What are the mechanisms of transposition?

  • Transposase binds to ends
  • Transposon is cut out
  • Chromosome is repaired
  • New target sequence is found
• What are the two types of transposition?

  • Conservative: original copy is retained
  • Replicative: original copy stays, new copy is made
• What are the key messages regarding recombination?

  • DNA repair and horizontal gene transfer depend on recombination
  • Recombination can be homologous or non-homologous
  • Transposons can convey phenotypes and knock out genes
• What is the definition of recombination?

  Breaks and joins DNA into new combinations
• Why is recombination important?

  • Creates genetic diversity
  • Allows rapid evolution
  • Necessary for horizontal gene transfer methods
• What distinguishes homologous from non-homologous recombination?

  • Homologous: requires sequence similarity
  • Non-homologous: repairs double stranded breaks by joining DNA
• What is required for homologous recombination?

  • Extensive homology
  • Can replace faulty genes
  • Involves Holliday junctions
• What is the mechanism of homologous recombination?

  1. Alignment of homologous DNA helices
  2. Breakage of one strand
  3. Invasion of free 3’ end
  4. Stabilization by SSB protein
  5. Catalyzed by RecA
• What is the role of RecA in homologous recombination?

  Essential for DNA repair
• What is a functional homolog of RecA found in all organisms?

  It binds single stranded DNA
• What is the role of RecA in branch migration?

  It catalyzes branch migration