**1.2 Plants and UV radiation**

Plants, due to their non-motile nature, generally have a higher rate of UV tolerance than animals. Plant secondary metabolites aid in defence against both abiotic and biotic stress factors. Plants are capable of reflecting or absorbing harmful UV rays via thick layers of waxy cutin or submerin on the cell walls and intracellular accumulation of chemical substances such as flavanols or phenolics. The biological effects of UV radiation on plants include altered growth responses, reproductive deformities, epigenetic variations, plant susceptibly to biotic factors, premature senescence, damage to the photosynthetic apparatus, and altered conformation of membrane structures. A wide array of genes were found to be induced upon prolonged exposure to low doses of UV-B in the model plant *Arabidopsis thaliana* (Frohnmeyer & Staiger, 2003; Mackerness, 2000; Ries et al., 2000). Upregulated transcripts include: antioxidant/free radical scavenging enzymes, proteins involved in: DNA repair, translation, E3 ligase system, cell cycle, signal transduction, and secondary

UV Damaged DNA Repair & Tolerance in Plants 75

turn transfers electrons to the lesions, catalyzing the cleavage of the cyclobutane rings and dimer monomerization (Deisenhofer, 2000; Sancar, 2003, 2008). Multiple sequence alignment reveals that conserved homology between prokaryotic and eukaryotic CPD photolyases is limited to the C-terminal FAD binding site. It has been suggested that a common ancestor gave rise to both type I and type II photolyases but diverged at an early evolutionary stage (Yasui et al., 1994). CPD photolyases have been classified into Class I (microbial) and Class II (higher eukaryotes excluding placental mammals) groups, respectively. The 6–4 photolyases from *Drosophila* and *Arabidopsis* have strong sequence similarity to class I CPD photolyases (Nakajima et al., 1998; Todo et al., 1996). Similarly cryptochromes, the plant blue light photoreceptors, are 30% similar to the class I microbial photolyases, but demonstrate no photolyase activity (Ahmad & Cashmore, 1993). Thus, microbial Class I CPD photolyases, eukaryotic 6–4 photolyases, and blue light photoreceptors constitute the class I

Genes encoding CPD photolyases and 6-4 PP photolyases have been identified and characterized in a range of prokaryotic and eukaryotic systems (Sancar, 2003). In plants genes encoding CPD photolyases have been identified in *Arabidopsis thaliana (*Ahmed et al*.,*  1997*)*, cucumber (Takahashi et al., 2002), rice (Hirouchi et al., 2003), spinach (Yoshihara et al., 2005), and soybean (Yamamoto et al., 2008). Genes encoding 6-4 PP photolyases have been identified in *Arabidopsis* and rice (Chen et al., 1994; Singh et al., 2010). In *Arabidopsis*, the highest levels of both photolyases are associated with floral tissues, which may presumably serve to minimize lesions in germline cells. While expression of the CPD photolyase is light/UV-B regulated, 6-4 PP photolyase is constitutively expressed (Takahashi et al., 2002; Waterworth et al., 2002). The *Arabidopsis* CPD photolyase gene (*AtPHR1*) encodes a class II CPD photolyase. An *Arabidopsis* mutant (*uvr2*) lacking this gene is hypersensitive to UV. AtPHR1 is efficient in CPD photoreactivation but deficient in 6-4 photoproduct repair (Ahmed et al., 1997; Landry et al., 1997). *AtPHR1* is upregulated several fold in a UV insensitive mutant of *Arabidopsis* (*uvi1*) irrespective of light conditions, conferring constitutive protection (Tanaka et al., 2002). Overexpression of CPD photolyase in *Arabidopsis* and rice resulted in enhanced CPD removal (Hidema et al., 2007; Kaiser et al., 2009; Ueda et al., 2005). Genetic complementation of photolyase deficient *E.coli* strains with soybean, rice, spinach and *Arabidopsis* CPD photolyase genes restored photoreactivation activity (Yamamoto et al., 2007, 2008; Yoshihara et al., 2005). CPD photolyase activity in *Arabidopsis* (Pang & Hays, 1991; Waterworth et al., 2002), soybean (Yamamoto et al., 2008) and rice (Hidema et al., 2000) has been reported to be temperature sensitive. Rice CPD photolyase, encoded by a single copy gene in the nuclear genome, translocates to chloroplasts, mitochondria and nuclei to repair UV-induced CPDs in all three genomes (Takahashi et al., 2011), a phenomenon not observed in spinach chloroplasts (Hada et al., 2000) or young *Arabidopsis* seedlings (Chen et al., 1996). However, upon exposure to photoreactivating blue light, *Arabidopsis* seedlings did exhibit efficient repair of CPDs in the extracellular organelles (Draper & Hays, 2000). The *Arabidopsis* 6-4 PP photolyase, encoded by the *UVR3* gene, encodes a 62 kDa protein with 45% identity to *Drosophila* 6-4 PP photolyase and 17% identity to the Class II CPD photolyases. AtUVR3 is proficient in 6-4 photoproduct removal but deficient in CPD repair. Both *uvr2* and *uvr3* are nonsense mutations, and the double mutants are extremely sensitive to UV relative to the single mutants (Nakajima et al., 1998). Photolyases appear to be the sole repair mechanism active in non-proliferating plant tissues (Kimura et al., 2004). Hence, photolyases play an

photolyase/photoreceptor family.

important role in plant repair of UV damaged DNA.

metabolites, as well as several other genes with unknown function (Brosché et al., 2002; Jansen et al., 2008). UV-B also results in numerous changes in plant morphology. This signalling cascade is well reviewed elsewhere (Jenkins, 2009). Here we focus on plant responses to UV-induced DNA damage.

#### **1.3 UV induced DNA damage**

UV-C/B radiation is directly absorbed by DNA, inducing lesions which inhibit vital cellular functions such as transcription and DNA replication. UV-A is comparatively less efficient in lesion induction but triggers the production of reactive oxygen species (ROS) (Kunz et al., 2006). The primary UV induced DNA lesions include cyclobutane pyrimidine dimers (adjacent pyrimidines covalently linked between C-5 and C-6 carbon atoms) and secondary lesions 6-4 pyrimidine-pyrimidone photoproducts (6-4 PP) (covalent linkage between the C-4 position of a pyrimidine to the C-6 position of an adjacent pyrimidine) (Fig. 1). In order to respond to this damage, plants employ specific mechanisms (Britt, 1999). In light conditions, photoreactivation catalyses dimer monomerizations while during dark conditions, Nucleotide Excision Repair (NER) excises these helix-distorting lesions. Finally, residual lesions are bypassed via translesion synthesis (TLS).

Fig. 1. A) Normal adjacent pyrimidine residues. B) UV-induced Cyclobutane Pyrimidine Dimer (CPD) and C) 6-4 Pyrimidine-Pyrimidone photoproduct (6-4 PP).

#### **2. Photoreactivation**

Photoreactivation is a blue light dependant DNA repair mechanism catalysed by the photolyase (E.C 4.1.99.3) class of enzymes. Pyrimidine dimers are split by the action of two photoactive damage specific DNA repair enzymes – CPD photolyase and 6-4 PP photolyase. Both classes of photolyase require two co-factors, one being the two electron reduced form of Flavin Adenine Dinucleotide (FAD) and the second chromophore, a blue light harvesting photoantenna, being either 5,10- methenyltretrahydrofolate (MTHF) or 8-hydroxy-7,8 didemethyl-5-deazariboflavin (8-HDF). FAD is an essential co-factor for regulating DNA binding and repair. In contrast, the second chromophore has a higher extinction co-efficient and an absorption maximum at longer wavelengths, hence regulates the rate of repair depending on the external light intensity. MTHF or 8-HDF absorbs the photoreactivating blue light photons and transfers this excitation energy to the reduced FAD. The FADH in

metabolites, as well as several other genes with unknown function (Brosché et al., 2002; Jansen et al., 2008). UV-B also results in numerous changes in plant morphology. This signalling cascade is well reviewed elsewhere (Jenkins, 2009). Here we focus on plant

UV-C/B radiation is directly absorbed by DNA, inducing lesions which inhibit vital cellular functions such as transcription and DNA replication. UV-A is comparatively less efficient in lesion induction but triggers the production of reactive oxygen species (ROS) (Kunz et al., 2006). The primary UV induced DNA lesions include cyclobutane pyrimidine dimers (adjacent pyrimidines covalently linked between C-5 and C-6 carbon atoms) and secondary lesions 6-4 pyrimidine-pyrimidone photoproducts (6-4 PP) (covalent linkage between the C-4 position of a pyrimidine to the C-6 position of an adjacent pyrimidine) (Fig. 1). In order to respond to this damage, plants employ specific mechanisms (Britt, 1999). In light conditions, photoreactivation catalyses dimer monomerizations while during dark conditions, Nucleotide Excision Repair (NER) excises these helix-distorting lesions. Finally, residual

Fig. 1. A) Normal adjacent pyrimidine residues. B) UV-induced Cyclobutane Pyrimidine

Photoreactivation is a blue light dependant DNA repair mechanism catalysed by the photolyase (E.C 4.1.99.3) class of enzymes. Pyrimidine dimers are split by the action of two photoactive damage specific DNA repair enzymes – CPD photolyase and 6-4 PP photolyase. Both classes of photolyase require two co-factors, one being the two electron reduced form of Flavin Adenine Dinucleotide (FAD) and the second chromophore, a blue light harvesting photoantenna, being either 5,10- methenyltretrahydrofolate (MTHF) or 8-hydroxy-7,8 didemethyl-5-deazariboflavin (8-HDF). FAD is an essential co-factor for regulating DNA binding and repair. In contrast, the second chromophore has a higher extinction co-efficient and an absorption maximum at longer wavelengths, hence regulates the rate of repair depending on the external light intensity. MTHF or 8-HDF absorbs the photoreactivating blue light photons and transfers this excitation energy to the reduced FAD. The FADH-

in

Dimer (CPD) and C) 6-4 Pyrimidine-Pyrimidone photoproduct (6-4 PP).

responses to UV-induced DNA damage.

lesions are bypassed via translesion synthesis (TLS).

**1.3 UV induced DNA damage** 

**2. Photoreactivation** 

turn transfers electrons to the lesions, catalyzing the cleavage of the cyclobutane rings and dimer monomerization (Deisenhofer, 2000; Sancar, 2003, 2008). Multiple sequence alignment reveals that conserved homology between prokaryotic and eukaryotic CPD photolyases is limited to the C-terminal FAD binding site. It has been suggested that a common ancestor gave rise to both type I and type II photolyases but diverged at an early evolutionary stage (Yasui et al., 1994). CPD photolyases have been classified into Class I (microbial) and Class II (higher eukaryotes excluding placental mammals) groups, respectively. The 6–4 photolyases from *Drosophila* and *Arabidopsis* have strong sequence similarity to class I CPD photolyases (Nakajima et al., 1998; Todo et al., 1996). Similarly cryptochromes, the plant blue light photoreceptors, are 30% similar to the class I microbial photolyases, but demonstrate no photolyase activity (Ahmad & Cashmore, 1993). Thus, microbial Class I CPD photolyases, eukaryotic 6–4 photolyases, and blue light photoreceptors constitute the class I photolyase/photoreceptor family.

Genes encoding CPD photolyases and 6-4 PP photolyases have been identified and characterized in a range of prokaryotic and eukaryotic systems (Sancar, 2003). In plants genes encoding CPD photolyases have been identified in *Arabidopsis thaliana (*Ahmed et al*.,*  1997*)*, cucumber (Takahashi et al., 2002), rice (Hirouchi et al., 2003), spinach (Yoshihara et al., 2005), and soybean (Yamamoto et al., 2008). Genes encoding 6-4 PP photolyases have been identified in *Arabidopsis* and rice (Chen et al., 1994; Singh et al., 2010). In *Arabidopsis*, the highest levels of both photolyases are associated with floral tissues, which may presumably serve to minimize lesions in germline cells. While expression of the CPD photolyase is light/UV-B regulated, 6-4 PP photolyase is constitutively expressed (Takahashi et al., 2002; Waterworth et al., 2002). The *Arabidopsis* CPD photolyase gene (*AtPHR1*) encodes a class II CPD photolyase. An *Arabidopsis* mutant (*uvr2*) lacking this gene is hypersensitive to UV. AtPHR1 is efficient in CPD photoreactivation but deficient in 6-4 photoproduct repair (Ahmed et al., 1997; Landry et al., 1997). *AtPHR1* is upregulated several fold in a UV insensitive mutant of *Arabidopsis* (*uvi1*) irrespective of light conditions, conferring constitutive protection (Tanaka et al., 2002). Overexpression of CPD photolyase in *Arabidopsis* and rice resulted in enhanced CPD removal (Hidema et al., 2007; Kaiser et al., 2009; Ueda et al., 2005). Genetic complementation of photolyase deficient *E.coli* strains with soybean, rice, spinach and *Arabidopsis* CPD photolyase genes restored photoreactivation activity (Yamamoto et al., 2007, 2008; Yoshihara et al., 2005). CPD photolyase activity in *Arabidopsis* (Pang & Hays, 1991; Waterworth et al., 2002), soybean (Yamamoto et al., 2008) and rice (Hidema et al., 2000) has been reported to be temperature sensitive. Rice CPD photolyase, encoded by a single copy gene in the nuclear genome, translocates to chloroplasts, mitochondria and nuclei to repair UV-induced CPDs in all three genomes (Takahashi et al., 2011), a phenomenon not observed in spinach chloroplasts (Hada et al., 2000) or young *Arabidopsis* seedlings (Chen et al., 1996). However, upon exposure to photoreactivating blue light, *Arabidopsis* seedlings did exhibit efficient repair of CPDs in the extracellular organelles (Draper & Hays, 2000). The *Arabidopsis* 6-4 PP photolyase, encoded by the *UVR3* gene, encodes a 62 kDa protein with 45% identity to *Drosophila* 6-4 PP photolyase and 17% identity to the Class II CPD photolyases. AtUVR3 is proficient in 6-4 photoproduct removal but deficient in CPD repair. Both *uvr2* and *uvr3* are nonsense mutations, and the double mutants are extremely sensitive to UV relative to the single mutants (Nakajima et al., 1998). Photolyases appear to be the sole repair mechanism active in non-proliferating plant tissues (Kimura et al., 2004). Hence, photolyases play an important role in plant repair of UV damaged DNA.

UV Damaged DNA Repair & Tolerance in Plants 77

Fig. 2. Overview of mammalian nucleotide excision repair. In GG-NER, DDB2-CUL4 mediated histone (H) and XPC ubiquitination facilitates lesion binding. In TC-NER, stalled RNA POL II recruits CSB and the CSA-CUL4-CSN complex, followed by recruitment of other TC-NER specific factors. In both cases, NER core players follow suit: XPB and XPD helicases of the TFIIH complex, XPF-ERCC1 and XPG endonucleases, and the ssDNA binding XPA-RPA complex. The fragment encompassing the lesion is excised, followed by repair synthesis and ligation. Repair synthesis requires DNA POL δ/ε in concert with

accessory proteins PCNA, RFC and RPA. See text for details.
