Segregation of Nucleoid into Domains

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					Segregation of Nucleoid into Domains
                                                     A                                                                      B

                                                      Photobinding of trimethylpsoralen

                                                                                                                                Photobinding of trimethylpsoralen
• If the chromosome were simply supercoiled to
  give a condensed nucleoid, then 1 nick would relax
  the entire chromosome nucleoid structure.
• Sinden and Pettijohn used the amount of binding
  of trimethylpsoralen to the DNA to measure
  supercoiling (torsional strain) and introduced
  double stranded breaks in the DNA using x-rays
  (A at right).
• They found that, on average, 50 breaks per
  chromosome were required to relieve all of the       0                                                                                     0
                                                         0                                Supercoiling (torsional strain)                                           0 Breaks per genome
  torsional strain in the E. coli chromosome (B
  at right).
• The chromosome of E. coli consists of 50           C
  domains, each containing about 100 kb of DNA
  (C at right).                                                                                           Folded
• Nucleoid structural proteins are thought to
  mediate the domain architecture, but the roles
  of individual proteins are less than clear.
• Proteins such as HU, which restrains negative
  supercoils in vitro, and HNS bind non-specifically
  to DNA, whereas IHF binds to a specific sequence                                            Nick
  of DNA.
• Cells that lack 1 or 2 of these proteins survive,                                                       reaction                                                             Domain of
  but loss of the ability to make all three proteins                                                                                                                           100 kbp
  is lethal.
• Proteins called condensins, such as MukB in
  E. coli, condense the nucleoid by binding DNA at
  both ends of 1 protein molecule.                                                                 Nick                                                                      David Pettijohn
Semiconservative replication
• Watson and Crick had proposed that DNA replicates in a semiconservative way,
  meaning that the two old strands are conserved and serve as templates for
  the two new strands.
• Meselson and Stahl tested this hypothesis in an experiment
  outlined at right. They used heavy isotopes to label
  the old DNA and showed that after one generation
  time without the heavy isotopes, the DNA had a
  density between the light and heavy DNA.
Synthesis of DNA by DNA polymerase:
• proceeds in the 5’ to 3’ direction only (B).
• requires a DNA or RNA primer to start (A)
• requires a template strand.
• requires the energy of the triphosphate on the
  incoming dNTP.
• DNA pol III is the major polymerase in the cell.
• DNA pol I replaces RNA primers with DNA
Bidirectional replication fork
• In 1963 Cairns showed that two replication forks work simultaneously on one
  chromosome during replication. An initial heavy pulse of radiolabelled
  nucleotides was used to label the origin of replication, followed by light
  labeling to allow imaging of spread chromosomes on photographic
  film. The depiction of a spread chromosome at right as seen by Cairns
  (without the arrows) shows two replication forks traveling away from
  the origin.
• Two possibilities existed for replication at each fork: either both or just one
  strand could be replicated at each fork.
• Prescott and Kuempel showed that both strands of DNA are replicated
  simultaneously at each replication fork. They labeled replicating
  chromosomes lightly with 3H-thymidine with occasional heavy pulses.
  Spread chromosomes were then imaged using photographic film.
• The two most likely outcomes of their experiment are shown
• Heavy labeling of both parts of the replication fork indicated that replication is occurring
  on both of the the separated DNA strands of the replication fork.
• This implies that replication of one of the two strands occurs in the 3’ to 5’ direction (!!!?).

              X    One strand replicated                                    4 Both strands replicated
Model of replication must account for:

  1   Initiation of replication (Timing, location)
  2   Opposite polarity polymerization (5’ to 3’ and 3’ to 5’ - Prescott and Kuempel)
  3   Unwinding of the helix (To expose template)
  4   Protection of single-stranded DNA and prevention of secondary structure
  5   High processivity (750-800 bp per second)
  6   High fidelity (1 mistake per 1010 bp replicated)
  7   Termination of replication
Early genetic approaches to study replication

  1. Isolate Temperature-sensitive (Ts) lethal mutations
       Subgroup will be replication deficient

  2. Isolate Ts mutants that survive lytic phage
       Subgroup will be replication deficient

  3. Isolate Ts mutants that do not replicate their DNA
       Mutagenize bacteria at 30°C
       Shift to 42°C for 50 min
       Add 5-bromodeoxyuridine (5-BrdU)
       Irradiate with UV
       Cells that incorporated 5-BrdU die due to Br• free radical formation
       Ts mutants that cannot replicate DNA at 42°C survive

  Two general types of mutants isolated:
     Quick stop mutants - stop replication immediately at 42 °C
     Slow stop mutants - finish round of replication at 42 °C, then stop
Mutant phenotype
Slow stop

Quick stop
Slow or quick stop
Quick stop
Quick stop
DNA repair defective

Quick stop

Quick stop
Quick stop

Quick stop
Quick stop
Quick stop

Accumulation of nascent fragments

Slow stop
Slow stop