Lec12_DNA Repair.ppt
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Transcript of Lec12_DNA Repair.ppt
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DNA Repair & Recombination
All 3 genomes in plants constantly being
damaged by UV and other forms of
radiation, chemicals, and otherstresses (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 UVlight (UV-C (
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Types of DNA Damage (cont.)
5. Oxidative damage: guanine oxidizes to 8-oxo-guanine, also cause SS and DS breaks, veryimportant for organelles
6. Replication errors: wrong nucleotide (or modified nt)inserted
7. Double-strand breaks (DSB): induced by ionizingradiation, transposons, topoisomerases, homingendonucleases, and mechanical stress onchromosomes
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Repair of UV-induced dimers in the light
Photoreactivation1. Light-dependent, UV-A blue light (360-420 nm)
2. Catalyzed by Photolyases:
Enzymes that convert the dimers to monomers
Use FAD as chromophore and electron donor
also have another chromophore that acts asantenna
3 classes: CPD I and II for T-T dimers, and a 6-4
photolyase for T-C dimers3. Arabidopsis has CPD II and 6-4 photolyases
4. Arabidopsis also has a photolyase in the chloroplastand possibly one in the mitochondria.
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Fig. 6.12 in Buchanan et al.
Photolyase
gene
expression
also induced
or increasedby light.
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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 inArabidopsis genome
ERCC1 and RAD25
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Nucleotide Excision Repair
(in E. coliof a T-T dimer)
Endonuclease cuts on
either side of damage
(~20 nt altogether).
Strands unwound by
helicase.
Fig. 6.14 in Buchanan et al.
1. UvrA,B
2. UvrC
3. UvrD
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Base Excision Repair (BER)
Not much known about this pathway in
plants
Probably important though, based onthe existence of 16 genes
homologous to DNA glycosylases,
and 3 homologous toAPendonucleases in theArabidopsis
genome.
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Base Excision Repair (BER)
Variety ofDNA glycosylases,
for different types of damagedbases.
AP endonuclease recognizes
sites with a missing base;
cleaves sugar-phosphate
backbone.
Deoxyribose
phosphodiesterase removes
the sugar-phosphate lacking
the base.
Deaminated C
Fig. 6.15
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Mismatch Repair
Problem: how do cells know which is the righttemplate strand?
In E. coli, new DNA not methylated right away
Mismatch recognized by mutS, then mutLbinds and attracts mutH(endonuclease thatcleaves mismatch and nearest CTAG that isnot methylated)
Eucaryotes (includingArabidopsis) have mutSand mutL homologues, but no mutH
Also have the requisite exonucleases, butnot clear how the strand specificity isdetermined
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In E.coli, A of each
GATC is methylated.
Mismatch Repair
mutHis endonuclease
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Repair of Double-strand breaks (DSBs)
2 general ways to repair DSBs:
1. Homologous recombination (HR) - repair of brokenDNA using the intact homologue. Very accurate.
2. Non-homologous end joining (NHEJ) - ligating non-homologous ends. Prone to errors, ends can be
damaged before ligation (genetic material lost), orget translocations.
Usage: NHEJ >> HR in plants and animals (in
the cells nucleus)
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RecA/Rad51
Resolvase (recG)
DSBR by HR
Modified from Fig. 6.18 in
Buchanan et al.
3 SS extensions
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RecA binds preferentially
to SS DNA and will
catalyze invasion of a DSDNA 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., Ku70and Ku80).
Processing of DSBs very important they
can block cell cycle progression and triggerapoptosis (programmed cell death).