Transcript DNA Repair & Recombination

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.
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 crosslinked, T-T more prominent, caused by UV
light (UV-C (<280 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
Types of DNA Damage (cont.)
5. Oxidative damage: guanine oxidizes to 8-oxoguanine, 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
Repair of UV-induced dimers in the light
•
Photoreactivation
1. 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 as
antenna
• 3 classes: CPD I and II for T-T dimers, and a 6-4
photolyase for T-C dimers
3. Arabidopsis has CPD II and 6-4 photolyases
4. Arabidopsis also has a photolyase in the chloroplast
and possibly one in the mitochondria.
Photolyase
gene
expression
also induced
or increased
by light.
Fig. 6.12 in Buchanan et al.
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
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).
3. UvrD
Strands unwound by
helicase.
Fig. 6.14 in Buchanan et al.
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.
Base Excision Repair (BER)
Deaminated C
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
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
Mismatch Repair
In E.coli, A of each
GATC is methylated.
mutH is endonuclease
Repair of Double-strand breaks (DSBs)
2 general ways to repair DSBs:
1.
Homologous recombination (HR) - repair of broken
DNA using the intact homologue. Very accurate.
2.
Non-homologous end joining (NHEJ) - ligating nonhomologous ends. Prone to errors, ends can be
damaged before ligation (genetic material lost), or
get translocations.
Usage: NHEJ >> HR in plants and animals (in
the cells’ nucleus)
DSBR by HR
3’ SS extensions
RecA/Rad51
Resolvase (recG)
Modified from Fig. 6.18 in
Buchanan et al.
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
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).