DNA Repair

1
DNA Repair Recombination

• All 3 genomes in plants constantly being damaged
  by UV and other forms of radiation, chemicals,
  and other stresses (e.g., oxidative, heat).
• Some proteins involved in repair also function
  in recombination
• e.g., recombination can be used to repair
  double-strand breaks.

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Types of DNA Damage

1. Deamination (C ? U and A? hypoxanthine)
2. Depurination purine base (A or G) lost
3. T-T and T-C dimers bases become cross- linked,
   T-T more prominent, caused by UV light (UV-C
   (lt280 nm) and UV-B (280-320 nm)
4. Alkylation an alkyl group (e.g., CH3) gets added
   to bases chemical induced some harmless, some
   cause mutations by mispairing during replication
   or stop polymerase altogether

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Types of DNA Damage (cont.)

• 5. Oxidative damage guanine oxidizes to
  8-oxo-guanine, also cause SS and DS breaks, very
  important for organelles
• 6. Replication errors wrong nucleotide (or
  modified nt) inserted
• 7. Double-strand breaks (DSB) induced by
  ionizing radiation, transposons, topoisomerases,
  homing endonucleases, and mechanical stress on
  chromosomes

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Repair of UV-induced dimers in the light

• Photoreactivation
• Light-dependent, UV-A ? blue light (360-420 nm)
• Catalyzed by Photolyases
• Enzymes that convert the dimers to monomers
• Use FAD as chromophore and electron donor
• also have another chromophore that acts as
  antenna
• 3 classes CPD I and II for T-T dimers, and a 6-4
  photolyase for T-C dimers
• Arabidopsis has CPD II and 6-4 photolyases
• Arabidopsis also has a photolyase in the
  chloroplast and possibly one in the mitochondria.

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Photolyase gene expression also induced or
increased by light.
Fig. 6.12 in Buchanan et al.
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Plants also repair pyrimidine dimers in the dark

• Probably by a general Nucleotide Excision Repair
  Pathway (NER).
• Arabidopsis mutants deficient in dark repair have
  been isolated, but few genes characterized.
• rad1.
• Not much biochemistry in plants, but homologues
  of NER genes also occur in Arabidopsis genome
• ERCC1 and RAD25

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Nucleotide Excision Repair (in E. coli of a T-T
dimer)
1. UvrA,B 2. UvrC
Endonuclease cuts on either side of damage (20
nt altogether). Strands unwound by helicase.
3. UvrD
Fig. 6.14 in Buchanan et al.
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Base Excision Repair (BER)

• Not much known about this pathway in plants
• Probably important though, based on the
  existence of 16 genes homologous to DNA
  glycosylases, and 3 homologous to AP
  endonucleases in the Arabidopsis genome.

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Deaminated C
Base Excision Repair (BER)
Variety of DNA glycosylases, for different types
of damaged bases. AP endonuclease recognizes
sites with a missing base cleaves
sugar-phosphate backbone. Deoxyribose
phosphodiesterase removes the sugar-phosphate
lacking the base.
Fig. 6.15
11
Mismatch Repair

• Problem how do cells know which is the right
  template strand?
• In E. coli, new DNA not methylated right away
• Mismatch recognized by mutS, then mutL binds and
  attracts mutH (endonuclease that cleaves mismatch
  and nearest CTAG that is not methylated)
• Eucaryotes (including Arabidopsis) have mutS and
  mutL homologues, but no mutH
• Also have the requisite exonucleases, but not
  clear how the strand specificity is determined

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Mismatch Repair
In E.coli, A of each GATC is methylated.
mutH is endonuclease
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Repair of Double-strand breaks (DSBs)

• 2 general ways to repair DSBs
• Homologous recombination (HR) - repair of broken
  DNA using the intact homologue. Very accurate.
• Non-homologous end joining (NHEJ) - ligating
  non-homologous ends. Prone to errors, ends can be
  damaged before ligation (genetic material lost),
  or get translocations.
• Usage NHEJ gtgt HR in plants and animals (in the
  cells nucleus)

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DSBR by HR
3 SS extensions
RecA/Rad51
Resolvase (recG)
Modified from Fig. 6.18 in Buchanan et al.
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RecA binds preferentially to SS DNA and will
catalyze invasion of a DS DNA molecule by a SS
homologue. Important for many types of
homologous recombination, such as during meoisis
(in yeast).
Fig. 6.19
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Genes for Repair of DSBs in Arabidopsis

• Arabidopsis has rad51, resolvase (recG), and
  repA (SS DNA binding in animals) homologues,
  all needed for HR.
• Also has homologues of key genes required for
  NHEJ (e.g., Ku70 and Ku80).
• Processing of DSBs very important they can
  block cell cycle progression and trigger
  apoptosis (programmed cell death).