Molecular lec 15

• Initiator protein

  Protein that could be synthesized continuously throughout the cell cycle; accumulation of a critical amount would trigger initiation
• Inhibitor protein

  Protein that might be synthesized or activated at a fixed point and then diluted below an effective level by the increase in cell volume
• Bacterial chromosome

  • Compacted into a dense protein–DNA structure called the nucleoid, which takes up most of the space inside the cell
• Bacterial cell shape

  • Established by a rigid layer of peptidoglycan in the cell wall, which surrounds the inner membrane
• Proteins required to maintain rod-like shape of bacteria


  • MreB, PBP2, and RodA
• Eukaryotic chromosomes

  • Reside in the nucleus
  • Each chromosome consists of many units of replication called replicons
  • Replication requires coordination of these replicons to reproduce DNA during a discrete period of the cell cycle
  • The decision about whether to replicate is determined by a complex pathway that regulates the cell cycle
  • Duplicated chromosomes are segregated to daughter cells during mitosis by means of a special apparatus
• Conservative model

  Both strands of one copy would be entirely old DNA, while the other copy would have both strands of new DNA
• Dispersive model

  DNA might fragment, replicate dsDNA, and then reassemble, creating a mosaic of old and new dsDNA regions in each new chromosome
• Semiconservative model

  DNA strands separate, and a complementary strand is synthesized for each, so that sibling chromatids have one old and one new strand
• Replication initiation in prokaryotes

  1. Replication initiates at the bacterial origin when a cell passes a critical threshold of size

  2. Completion of replication produces daughter chromosomes

  3. They are separated and moved to opposite sides of the septum before the bacterium is divided into two

  
• Replicon

  A unit of the genome in which DNA is replicated. Each contains an origin for initiation of replication
• Replication bubble

  A region in which DNA has been replicated within a longer, un-replicated region
• Replication fork

  The point at which strands of parental duplex DNA are separated so that replication can proceed. A complex of proteins including DNA polymerase is found there
• Unidirectional replication

  The movement of a single replication fork from a given origin
• Bidirectional replication

  origin generates two replication forks that proceed away from the origin in opposite directions
• Initiation of replication in E. coli

  1. Replication starts when DNA at the origin of replication denatures to expose the bases, creating a replication fork

  2. Replication is usually bidirectional from the origin

  3. E. coli has one origin, oriC, which has: a minimal sequence of about 245 bp required for initiation, three copies of a 13-bp AT-rich sequence, and four copies of a 9-bp sequence

  4. Initiator proteins (DnaA) attach

  5. DNA helicase (DnaB) binds initiator proteins on the DNA, and denatures the AT-rich region using ATP

  6. DNA primase (DnaG) binds helicase to form a primosome, which synthesizes a short (5–10nt) RNA primer
• Replisome

  A large protein complex that carries out DNA replication, starting at the replication origin.
• Replication of circular DNA and the supercoiling problem

  1. As the strands separate on one side of the circle, positive supercoils form elsewhere in the molecule

  2. Topoisomerases relieve the supercoils, allowing the DNA strands to continue separating as the replication forks advance
• Rolling circle replication

  A model for replication used by several bacteriophages, where a nick (single-stranded break) at the origin of replication allows the 3' end to act as a primer for DNA polymerase III, which synthesizes a continuous strand using the intact DNA molecule as a template
• Parts of replisome machinery

  1. Leading strand
  2. Lagging strand
  3. Okazaki fragments
  4. RNA primer
  5. DNA primase
  6. DNA helicase
  7. DNA polymerase III
  8. SSBs
  9. DNA ligase
  10. Gyrase ( topoisomerase)
• Leading strand direction?

  5'-3'
• Lagging strand direction?

  3'-5' , Okazaki fragments 5'-3'