Lecture 1

• Prokaryotic genomes


  Typically consist of a single, circular DNA molecule located within the nucleoid
• DNA Topology

  • The dominant structure of DNA in cells is the B-form, characterized by a right-handed double helix, about 10.5 base pairs per turn, and a diameter of 2 nm
  • The helix is not static; it can coil in three dimensions, altering the number of base pairs per helix turn to accommodate torsional stress
• Supercoiling


  A mechanism that occurs when additional turns are introduced into the DNA double helix (positive supercoiling) or if turns are removed (negative supercoiling)
• Linking number (L)

  Represents the total twists in the DNA double helix
• Topoisomerases

  • Enzymes that manage DNA supercoiling by cutting and rejoining DNA strands
  • Type I topoisomerases alter the linking number by ±1, usually relieving negative supercoils
  • Type II topoisomerases, including DNA gyrase in E. coli, introduce negative supercoils using ATP
• DNA Organization in Bacteria

  • E. coli's DNA is structured into supercoiled loops anchored to a protein core, evidencing a highly organized architecture within the nucleoid
  • Nucleoid-Associated Proteins (NAPs) maintain supercoiled states and assist in compacting the DNA
• Not all prokaryotic genomes are circular; some, like Borrelia burgdorferi, possess linear genomes
• Some bacteria contain genomes divided into multiple DNA molecules, challenging the distinction between essential genomic components and plasmids
• Horizontal Gene Transfer (HGT)

  1. Transformation: The uptake of free DNA fragments from the environment by a bacterium

  2. Conjugation: Transfer of DNA material (often plasmids or transposons) between bacterial cells through direct cell-to-cell contact

  3. Transduction: The transfer of bacterial DNA from one bacterium to another via bacteriophages

  4. Gene Transfer Agents (GTAs): Virus-like particles produced by some bacteria that can package and transfer fragments of the producing cell's DNA to other cells
• Prophages and Genomic Islands

  • Prophages: Bacterial genomes often harbor prophage elements from past infections, which can contribute to bacterial virulence
  • Genomic Islands: Large DNA segments acquired through HGT, often containing clusters of genes with related functions, such as antibiotic resistance or virulence
• DNA Replication and Cell Cycle

  1. Replication initiates at a single origin (oriC) and is tightly linked to the cell cycle, ensuring each replicon is replicated once per cycle

  2. The replication process involves leading and lagging strands, with DNA Pol III performing bulk synthesis, and DNA Pol I filling in Okazaki fragments
• Replication Control

  1. Initiation: Involves DnaA binding to oriC, forming a complex that unwinds DNA for replication to commence

  2. Termination: Concludes when replication forks meet and fuse in the terminus region, facilitated by terminator proteins and sites that ensure replication concludes properly
• Homologous Recombination


  A fundamental biological process allowing the exchange of genetic material between two similar or identical DNA molecules, playing a crucial role in genetic diversity, repair of DNA damages, and chromosome segregation during meiosis in eukaryotes
• The Meselson-Radding Model

  1. Cleavage: The process begins with an endonuclease cleaving one DNA strand, creating a single-stranded (ssDNA) tail

  2. Chain Displacement and DNA Synthesis: DNA synthesis commences, displacing a strand and extending the ssDNA tail

  3. Strand Invasion: The ssDNA tail, facilitated by recombinase proteins, invades a homologous double-stranded DNA molecule, forming a displacement loop (D-loop)

  4. Chain Removal: The displaced ssDNA chain is either used as a template for repair synthesis or is digested

  5. Ligation and Branch Migration: The invading strand is ligated to the complementary strand, and branch migration, mediated by proteins, extends the heteroduplex region by moving the Holliday junction

  6. Isomerization: The Holliday junction undergoes spontaneous isomerization, changing the strands that cross

  7. Resolution: The final step involves the resolution of the Holliday junction by endonucleases, leading to the separation of the recombined DNA molecules
• RecBCD Pathway in E. coli

  1. RecBCD Binding: Binds to a blunt end of DSBs, unwinding and degrading DNA to produce a 3' ssDNA tail

  2. RecA Filament Formation: RecA coats the ssDNA tail, facilitating strand invasion into a homologous DNA sequence

  3. RecBCD and Chi Sites: RecBCD's activity changes upon encountering Chi sites, reducing degradation of the 3' tail and promoting recombination
• What are the seven parts of the meselon-radding model

  1. cleavage
  2. chain displacement
  3. strand invasion
  4. chain removal
  5. ligation and branch migration
  6. isomerisation
  7. resolution