DNA is very sensitive to UV damage

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G. DNA damage repair

• DNA is very sensitive to UV damage
• When there are adjacent TT bases they can be
  activated by low levels of UV radiation to react
  with each other to form dimers
• This blocks replication past that point
• One dimer therefore is lethal to a bacterium
• Initial repair of such damage is by
  PHOTORACTIVATION.
• Originally discovered in Actinomycetes (Kelner
  1949)
• Bacteria fail to recover in the dark, but do so
  in the presence of strong visible light.

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G. DNA damage repair. Cont.
Formation of DIMERS
Covalent bonds
UV Damage
Replication Blocked Lethal / no growth
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G. DNA damage repair cont...

• UV - DNA Damage - Cell Death
• UV - Bright visible light - survival !
• 3 Steps
• 1.Photolyase (encoded by phrA and phrB genes in
  E. coli) recognises distortion at dimer.
• 2.Light activates photolyase
• 3.Dimer cleaved

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G. DNA damage repair. Cont.
UV
Dimer
PHOTOREACTIVATION
Photolyase recognises distortion and binds
Energy from visible light activates lyase. Dimers
removed and photolyase released
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G. DNA damage repair cont...

• EXCISION REPAIR
• T-phage Host Cell Reactivation (Setlow 1964)
• UV resistance determinants discovered by
  screening
• uvrA,B and C mutants VERY UV sensitive
• DNA damage (dimers and other damage)
• Uvr A,B,C endonuclease cut
• DNA Pol I digestion and polymerisation
• Ligation and repair completed

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Excision repair........
UV DNA damage
Repair of gap
Endonuclease action
Ligation
Digestion and polymerisation
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H. Homologous Recombination

• Discovery of recombination functions
• Search began in 1960s using E.coli.
• Clarke (1965) and Howard-Flanders (1966)
• Hfr crosses screening for rec minus recipient
• 3 Classes of mutant found

recA(58 min) VERY UV senstitve 10-4
rec recB recC(61 min) Less UV sensitive 10-1
-10-3 rec recD discovered in 1985 Map near to
srlC and thyA genes (latter used to map by
co-transduction
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H. Homologous Recombination cont...

• Now many rec genes known to have involvement
  (over 28!)
• Other routes involving rec E,F,J,N,O,Q,R etc
  are possible (not considered here)
• Homology of over 40-50 bps needed for efficient
  rec
• Single strands or Duplexes can be involved
• Conjugation/transformation SS transferred
• Transduction / DS transferred
• Also many other DS/DS recombinations possible

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H. Homologous Recombination cont...

• Biochemical action of Rec proteins
• RecA protein is a DNA dependant ATPase and a
  specific protease.
• Binds SS DNA and promotes strand exchange
• Unwinds DNA (ATP)
• RecBCD protein complex - the RECOMBINASE
• Attaches near chi sequence (GCTGGTGG)
• ATP dependant cutting of the DNA

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H. Homologous Recombination cont...
Meselson- Radding Model for Bacterial
Recombination
1. Single strand nick
5. Ligation
2. Dissociation of ssDNA
6. Branch migration
3. Assimilation into D-Loop
7. Complete Recombination
4. D-Loop Digestion
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I. Homologous Recombination and repair.

• Repair normally at low level
• lexA gene identified as a regulator
• Recombine normally
• But NO increased UV mutagenesis (ie 30 dimers
  produces no extra mutants). Higher doses required
• LOW DOSE - Error-free repair
• HIGH DOSE - Error repair INDUCED
• Known as the SOS System in E.coli

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I. Homologous Recombination and repair.

• LexA is an autoregulated repressor
• Represses level of activity of many genes
• Collectively called DNA Inducible (din) genes
• Includes uvrA,B,C,D and sfi etc...
• RecA protease activity Cleaves LexA repressor
• Also ?CI repressor inducing lysis

In Summary DNA repair and recombination are
closely associated. They share functions and are
co-regulated. Consider the impact on evolution of
diversity for the final TUTORIAL
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SOS System in E.coli
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SOS System in E.coli