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.

2. 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 (<280 nm) and UV-B (280-320 nm) 4.Alkylation: an alkyl group (e.g., CH 3 ) gets added to bases; chemical induced; some harmless, some cause mutations by mispairing during replication or stop polymerase altogether

4. 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

5. 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.

6. Fig. 6.12 in Buchanan et al. Photolyase gene expression also induced or increased by light.

7. 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

8. Nucleotide Excision Repair (in E. coli of 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

9. 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.

10. 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. Deaminated C 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

12. In E.coli, A of each GATC is methylated. Mismatch Repair mutH is endonuclease

13. 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 non- homologous 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)

14. RecA/Rad51 Resolvase (recG) DSBR by HR Modified from Fig. 6.18 in Buchanan et al. 3’ SS extensions

15. 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

16. 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).