Lec12_DNA Repair.ppt

7/30/2019 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).

Lec12_DNA Repair.ppt

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