**3.1 Photoreactivation by photolyases**

In plants the main repair pathway for direct DNA damage caused by UV-light that leads to the generation of CPDs and (6–4) photoproducts is based on the activity of photolyases (Jiang et al., 1997). Two types of photolyases have evolved that specifically recognize and repair either type of photodamage. Based on sequence homology, CPD photolyases are grouped into two different classes: while class I CPD photolyases are present in microorganisms, class II enzymes can be found in archaea, eubacteria, some animals (excluding placental mammals), and plants (Kanai et al., 1997). In comparison, (6-4) photolyases have been found in metazoans and plants, and they share sequence similarities with class I CPD photolyases (Kanai et al., 1997).

The structure and reaction mechanisms of photolyases have been intensively studied in the last decade, providing us with plentiful data on their function. Photolyases have two types of chromophoric co-factors that are used for photoreactivation (Huang et al., 2006; Ozturk et al., 2008; Hitomi et al., 2009). One chromophore is FADH-, the two electron reduced form of FAD, while the second one can be either methenyltetrahydrofolate (MTHF) or 7,8 didemethyl-8-hydroxy-5-deazariboflavin (8-HDF). MTHF or 8-HDF function as the light harvesting chromophores that absorb blue light (300-600 nm), and transfer the energy to FADH- (Moldt et al., 2009; Li et al., 2010; Okafuji et al., 2010). Photolyases bind directly to CPD and (6-4) photoproducts, where an electron is transferred from the excited FADH- to

Recognition and Repair Pathways of Damaged DNA in Higher Plants 205

Photolyases have been widely described in plants and may often comprise small gene families like, for example, in *Arabidopsis*, which encodes for five members (http://www.arabidopsis.org/). While loss of single members can lead to increased UVsensitivity (Jiang et al., 1997; Landry et al., 1997; Nakajima et al., 1998; Teranishi et al., 2004), the constitutive expression of CPD photolyases has been demonstrated to markedly improve UV tolerance in higher plants (Hidema et al., 2007; Kaiser et al., 2009). Although not much is known about their regulatory aspects, it has been demonstrated in rice that phosphorylation may play a role in regulating photolyase activities (Teranishi et al., 2008) and a few reports show that light increases photolyase expression (Chen et al., 1994; Waterworth et al., 2002). It was recently indicated that in darkness basal transcription of the photolyase genes *UVR3* and *PHR1* is sustained by the light signaling transcription factors HY5 and HYH and is limited by the actions of COP1 and DET1 dependent E3 ligases (Castells et al., 2010). Upon light exposure and during photomorphogenesis, COP1 leaves the nucleus and expression of *PHR1* is greatly induced by HY5 and HYH while the repression through DET1 remains in place. These observations suggest that photoreactivation is controlled by the photomorphogenisis pathway, and the activation of

A mechanism that can substitute for photolyase activities in plants, and which is required for photodamage repair in mammals, is the nucleotide excision repair (NER) pathway. NER is light-independent and, hence, sometimes referred to as dark repair. In contrast to photoreactivation, which reduces CPDs and 6-4 photoproducts back to pyrimidine monomers, NER is based on a complex recognition and repair machinery that excises and *de novo* synthesizes single DNA strands between 24-32 bp around the lesions. NER is highly conserved among eukaryotes and has two sub-pathways: transcription coupled NER (TCR) and global genome NER (GGR). NER has been intensively studied in animals, but the findings are a model for what is being found in plants, and will be briefly summarized in

GGR and TCR recruit the same repair proteins; however, they mainly differ in their initial steps of damaged DNA recognition. GGR is genome-wide active, and its initial steps include the xeroderma pigmentosum group C factor (XPC), which is able to sense thermodynamic destabilizations of the Watson-Crick duplex caused by a flipping-out of the affected bases from the strands (Min and Pavletich, 2007). XPC in itself is capable of detecting most bulky DNA lesions, but for the recognition of CPDs it is supported by WD-40 protein Damaged DNA Binding 2 (DDB2) (Aboussekhra et al., 1995; Mu et al., 1995; Mu et al., 1996; Moser et al., 2005; Min and Pavletich, 2007; Scrima et al., 2008). DDB2 binds with high affinity to photoproducts, induces a bending of the DNA to approximately 40° and facilitates the flipping of the affected bases that are recognized and bound by the XPC/hHR23B complex, which further introduces structural changes into the DNA (Min and Pavletich, 2007; Scrima et al., 2008). DDB2 is part of a DDB1-CUL4-RBX1 (DCX) E3 ligase that mediates the polyubiquitination of histones, XPC and DDB2 itself (Rapic-Otrin et al., 2002; Fitch et al., 2003; Sugasawa et al., 2005; Chen et al., 2006; Kapetanaki et al., 2006; Wang et al., 2006). As a consequence, DDB2 is degraded via the 26S proteasome clearing the way for later repair stages (Rapic-Otrin et al., 2002; Fitch et al., 2003; Chen et al., 2006). Interestingly ubiquitination has the opposite effect on XPC leading to its stabilization and activation (Sugasawa et al., 2005). The DDB2-dependent ubiquitination of histones H2A, H3, and H4

the PHR1 is dependent on photomorphogenetic regulators.

**3.2 Nucleotide excision repair** 

the following paragraph.

the dimers generating pyrimidine monomers, upon which the enzyme is released (Li et al., 2010; Okafuji et al., 2010) (Fig. 2).

Fig. 2. Photodamage and potential repair pathways in plants.

**(A)** Direct DNA damage caused by UV can be recognized and repaired by **(B)** photolyases in a light-dependent reaction. Alternatively, repair can follow **(C)** the global genome nucleotide excision repair (GGR) or **(D)** the transcription coupled NER (TCR).

Photolyases have been widely described in plants and may often comprise small gene families like, for example, in *Arabidopsis*, which encodes for five members (http://www.arabidopsis.org/). While loss of single members can lead to increased UVsensitivity (Jiang et al., 1997; Landry et al., 1997; Nakajima et al., 1998; Teranishi et al., 2004), the constitutive expression of CPD photolyases has been demonstrated to markedly improve UV tolerance in higher plants (Hidema et al., 2007; Kaiser et al., 2009). Although not much is known about their regulatory aspects, it has been demonstrated in rice that phosphorylation may play a role in regulating photolyase activities (Teranishi et al., 2008) and a few reports show that light increases photolyase expression (Chen et al., 1994; Waterworth et al., 2002). It was recently indicated that in darkness basal transcription of the photolyase genes *UVR3* and *PHR1* is sustained by the light signaling transcription factors HY5 and HYH and is limited by the actions of COP1 and DET1 dependent E3 ligases (Castells et al., 2010). Upon light exposure and during photomorphogenesis, COP1 leaves the nucleus and expression of *PHR1* is greatly induced by HY5 and HYH while the repression through DET1 remains in place. These observations suggest that photoreactivation is controlled by the photomorphogenisis pathway, and the activation of the PHR1 is dependent on photomorphogenetic regulators.

#### **3.2 Nucleotide excision repair**

204 Selected Topics in DNA Repair

the dimers generating pyrimidine monomers, upon which the enzyme is released (Li et al.,

2010; Okafuji et al., 2010) (Fig. 2).

Fig. 2. Photodamage and potential repair pathways in plants.

**(A)** Direct DNA damage caused by UV can be recognized and repaired by **(B)** photolyases in a light-dependent reaction. Alternatively, repair can follow **(C)** the global genome nucleotide excision repair (GGR) or **(D)** the transcription coupled NER (TCR).

A mechanism that can substitute for photolyase activities in plants, and which is required for photodamage repair in mammals, is the nucleotide excision repair (NER) pathway. NER is light-independent and, hence, sometimes referred to as dark repair. In contrast to photoreactivation, which reduces CPDs and 6-4 photoproducts back to pyrimidine monomers, NER is based on a complex recognition and repair machinery that excises and *de novo* synthesizes single DNA strands between 24-32 bp around the lesions. NER is highly conserved among eukaryotes and has two sub-pathways: transcription coupled NER (TCR) and global genome NER (GGR). NER has been intensively studied in animals, but the findings are a model for what is being found in plants, and will be briefly summarized in the following paragraph.

GGR and TCR recruit the same repair proteins; however, they mainly differ in their initial steps of damaged DNA recognition. GGR is genome-wide active, and its initial steps include the xeroderma pigmentosum group C factor (XPC), which is able to sense thermodynamic destabilizations of the Watson-Crick duplex caused by a flipping-out of the affected bases from the strands (Min and Pavletich, 2007). XPC in itself is capable of detecting most bulky DNA lesions, but for the recognition of CPDs it is supported by WD-40 protein Damaged DNA Binding 2 (DDB2) (Aboussekhra et al., 1995; Mu et al., 1995; Mu et al., 1996; Moser et al., 2005; Min and Pavletich, 2007; Scrima et al., 2008). DDB2 binds with high affinity to photoproducts, induces a bending of the DNA to approximately 40° and facilitates the flipping of the affected bases that are recognized and bound by the XPC/hHR23B complex, which further introduces structural changes into the DNA (Min and Pavletich, 2007; Scrima et al., 2008). DDB2 is part of a DDB1-CUL4-RBX1 (DCX) E3 ligase that mediates the polyubiquitination of histones, XPC and DDB2 itself (Rapic-Otrin et al., 2002; Fitch et al., 2003; Sugasawa et al., 2005; Chen et al., 2006; Kapetanaki et al., 2006; Wang et al., 2006). As a consequence, DDB2 is degraded via the 26S proteasome clearing the way for later repair stages (Rapic-Otrin et al., 2002; Fitch et al., 2003; Chen et al., 2006). Interestingly ubiquitination has the opposite effect on XPC leading to its stabilization and activation (Sugasawa et al., 2005). The DDB2-dependent ubiquitination of histones H2A, H3, and H4

Recognition and Repair Pathways of Damaged DNA in Higher Plants 207

(Kimura et al., 2004; Shaked et al., 2006), and although they are not biochemically characterized, studies in Arabidopsis demonstrate their critical role for UV tolerance (Shaked et al., 2006). Other mutants directly affected in NER factors such as Arabidopsis mutants *atcen2* and *uvh3–1/xpg,* also show decreased repair activities *in vitro* and behave hypersensitive towards UV-C exposure, respectively (Liu et al., 2000; Molinier et al., 2004b). Loss of the TFIIH transcription factor complex subunits XPB/UVH6 and XPD is lethal; however, *uvh6-1* plants expressing a mutated but potentially partially functional XPB protein already show decreased repair rates of UV-induced 6–4 photoproducts (Liu et al., 2003). Overall the current findings strongly indicate that the basic mechanisms of UVinduced damaged DNA recognition and NER based repair are comparable and highly

Not all nucleotide modifications can be repaired by NER, and many DNA lesions generated by reactive oxygen species (ROS) are not recognized by the NER proteins. Thus as an additional mechanism to ensure genomic integrity, cells utilize other repair mechanisms like base excision repair (BER). Because ROS are continuously produced as metabolic byproducts or by ionizing radiation, they represent a considerable source of the daily DNA damage. ROS-induced DNA lesions include for example 8-hydroxyguanine (8-oxoG), formamidopyrimidines, and 5-hydroxyuracil, which can potentially lead to miscoding

As a general rule BER requires the activities of DNA glycosylases, which cleave the Nglycosyl bond between the base and the sugar at the lesion site. This releases the base and leaves an abasic or apurinic/apyrimidinic (AP) site. In bacteria, fungi, plants and animals, several DNA glycosylases have been described that either specifically or broadly recognize certain lesions. For example, the mammalian DNA glycosylase OGG1 has a high affinity to 8-oxoG and some formamidopyrimidines, while another mammalian DNA glycosylase, NEIL1, efficiently repairs formamidopyrimidines but only poorly 8-oxoG (Morland et al., 2002; Parsons et al., 2005). DNA glycosylases are classified as either being mono- or bifunctional. Monofunctionally they only perform the cleavage reaction of the glycosylic bond between the deoxyribose and the target base to generate an AP site. Bifunctional DNA glycosylases/lyases, to which OGG1 and NEIL1 belong, are able to catalyze the release of the oxidized base and the cleavage of the DNA backbone at the AP site (Hazra et al., 2001). Although there is currently no evidence that plants have NEIL1 orthologs, which are common in bacteria and animals and required in part for excision of oxidized purines and pyrimidines, most other DNA glycosylases have been found. For example, plants encode for orthologs of OGG1 (Roldan-Arjona and Ariza, 2009), and their activity in excising oxidized purines has been demonstrated for the *Arabidopsis* AtOGG1 (Dany and Tissier, 2001; Garcia-Ortiz et al., 2001; Morales-Ruiz et al., 2003). In addition to OGG1, plants also encode for proteins related to the bifunctional Endonuclease III/Nth from *E. coli*, yeast, and animals, which remove a broad range of damaged pyrimidines (Breimer and Lindahl, 1980; Boorstein et al., 1989; Hatahet et al., 1994; Phadnis et al., 2006; Guay et al., 2008). Like their bacterial counterparts, *Arabidopsis* AtNTH1 also shows a broad substrate specificity and DNA glycosylase activity for DNA lesions containing modified pyrimidines (Krokan et al., 1997; Roldan-Arjona et al., 2000). Furthermore, plants encode for proteins related to MutM/Fpg, an original model DNA glycosylase/lyase from *E. coli* that excises 8-oxo-guanine and other oxidized purines from damaged DNA (Tchou et al., 1991; Tchou et al., 1993; Bhagwat and

conserved among plants and animals.

during replication and transcription.

**3.3 Base excision repair** 

may be necessary for the loosening of the DNA structure to allow the binding of other repair proteins (Kapetanaki et al., 2006; Wang et al., 2006). In a similar way the recently observed ability of DDB2 to recruit histone modifying proteins to specific DNA sequences could contribute to accessibility of the DNA for XPC and other factors (Minig et al., 2009; Roy et al., 2010). XPC is then needed for the recruitment of the core NER repair factors XPA, TFIIH, and RPA (Evans et al., 1997; Araujo et al., 2001; Thoma and Vasquez, 2003). XPA and the basal transcription factor complex TFIIH bind to the damaged site and unwind the DNA around the lesion (Reardon and Sancar, 2003; Maltseva et al., 2006; Yang et al., 2006; Kesseler et al., 2007; Krasikova et al., 2008). Unwinding is specifically performed by two subunits of TFIIH, the helicases XPB (ERCC3) and XPD (ERCC2). RPA is a heterotrimeric DNA binding protein, and while it prevents incision of the non-damaged DNA strand, together with XPA, it stabilizes the opened double helix (Blackwell et al., 1996; Camenisch et al., 2006; Maltseva et al., 2006; Yang et al., 2006). Incisions are performed by the endonucleases XPF (ERCC1) and XPG which nick the damaged DNA strand 5' and 3' around the lesion. After the damaged strand is excised, the gap is filled and ligated by the concerted activities of replication factors Proliferating Cell Nuclear Antigen (PCNA), Replication Factor C (RFC), Replication Protein A (RPA), DNA polymerases and , and DNA ligase 1 (LIG1) (Nichols and Sancar, 1992; Shivji et al., 1992; Green and Almouzni, 2003; Ogi et al., 2010). In contrast to GGR, TCR is specifically connected to DNA lesions in transcriptionally active regions. Here, RNA polymerase 2 (RP2) becomes stalled at CPD or (6-4) photoproduct containing sites (Selby and Sancar, 1997; Tornaletti and Hanawalt, 1999). Recognition of stalled RP2 has not been fully resolved. However, a critical role has been shown for Cockayne Syndrome factor B (CSB), a member of the SWI/SNF family of helicases (Selby and Sancar, 1997; van Gool et al., 1997; Citterio et al., 2000; Kamiuchi et al., 2002; Fousteri et al., 2006; Cazzalini et al., 2008). CSB binds to the stalled RP2, and this binding is a necessary trigger for recruitment of the same core repair proteins as described for GGR. Comparable to DDB2, CSB becomes a target of the DCX E3 ligase, which is mediated by another WD-40 protein, CSA. This interaction ultimately results in degradation of CSA, CSB and possibly also RP2 (Groisman et al., 2006).

Most of the proteins that play a role in GGR or TCR can be found in animals and plants, while only a few members, like XPA and TF2H3, a subunit of TFIIH, appear to be absent in plants (Kimura and Sakaguchi, 2006). It is currently open whether plants encode for functional analogs of XPA and TF23H that would perform tasks similar to these proteins. For most of the other NER proteins that are conserved among animals and plants, a role in DNA repair has been demonstrated, frequently by reverse genetic studies in *Arabidopsis thaliana*. Here, proteins shown to be involved in damaged DNA recognition in animals, such as DCX-E3 ligases, DDB2 and CSA, have also been recently described by several groups in plants (Bernhardt et al., 2006; Molinier et al., 2008; Al Khateeb and Schroeder, 2009; Bernhardt et al., 2010; Biedermann and Hellmann, 2010; Zhang et al., 2010; Zhang and Schroeder, 2010; Castells et al., 2011). While plants affected in ATCSA-1, the Arabidopsis CSA ortholog, do not display an abnormal development (Biedermann and Hellmann, 2010), loss of CUL4 or DDB2 cause a dwarf-like phenotype (Bernhardt et al., 2006; Koga et al., 2006). Interestingly, Arabidopsis *ddb2* or *atcsa-1* mutants are UV-hypersensitive but only when brought into the dark right after UV treatment, demonstrating that plants primarily rely on photoreactivation rather than NER (Biedermann and Hellmann, 2010). However, when kept in the dark both mutants have reduced repair activities when compared to wild type (Biedermann and Hellmann, 2010). CSB-like helicases are also present in plants (Kimura et al., 2004; Shaked et al., 2006), and although they are not biochemically characterized, studies in Arabidopsis demonstrate their critical role for UV tolerance (Shaked et al., 2006). Other mutants directly affected in NER factors such as Arabidopsis mutants *atcen2* and *uvh3–1/xpg,* also show decreased repair activities *in vitro* and behave hypersensitive towards UV-C exposure, respectively (Liu et al., 2000; Molinier et al., 2004b). Loss of the TFIIH transcription factor complex subunits XPB/UVH6 and XPD is lethal; however, *uvh6-1* plants expressing a mutated but potentially partially functional XPB protein already show decreased repair rates of UV-induced 6–4 photoproducts (Liu et al., 2003). Overall the current findings strongly indicate that the basic mechanisms of UVinduced damaged DNA recognition and NER based repair are comparable and highly conserved among plants and animals.

#### **3.3 Base excision repair**

206 Selected Topics in DNA Repair

may be necessary for the loosening of the DNA structure to allow the binding of other repair proteins (Kapetanaki et al., 2006; Wang et al., 2006). In a similar way the recently observed ability of DDB2 to recruit histone modifying proteins to specific DNA sequences could contribute to accessibility of the DNA for XPC and other factors (Minig et al., 2009; Roy et al., 2010). XPC is then needed for the recruitment of the core NER repair factors XPA, TFIIH, and RPA (Evans et al., 1997; Araujo et al., 2001; Thoma and Vasquez, 2003). XPA and the basal transcription factor complex TFIIH bind to the damaged site and unwind the DNA around the lesion (Reardon and Sancar, 2003; Maltseva et al., 2006; Yang et al., 2006; Kesseler et al., 2007; Krasikova et al., 2008). Unwinding is specifically performed by two subunits of TFIIH, the helicases XPB (ERCC3) and XPD (ERCC2). RPA is a heterotrimeric DNA binding protein, and while it prevents incision of the non-damaged DNA strand, together with XPA, it stabilizes the opened double helix (Blackwell et al., 1996; Camenisch et al., 2006; Maltseva et al., 2006; Yang et al., 2006). Incisions are performed by the endonucleases XPF (ERCC1) and XPG which nick the damaged DNA strand 5' and 3' around the lesion. After the damaged strand is excised, the gap is filled and ligated by the concerted activities of replication factors Proliferating Cell Nuclear Antigen (PCNA), Replication Factor C (RFC), Replication Protein A (RPA), DNA polymerases and , and DNA ligase 1 (LIG1) (Nichols and Sancar, 1992; Shivji et al., 1992; Green and Almouzni, 2003; Ogi et al., 2010). In contrast to GGR, TCR is specifically connected to DNA lesions in transcriptionally active regions. Here, RNA polymerase 2 (RP2) becomes stalled at CPD or (6-4) photoproduct containing sites (Selby and Sancar, 1997; Tornaletti and Hanawalt, 1999). Recognition of stalled RP2 has not been fully resolved. However, a critical role has been shown for Cockayne Syndrome factor B (CSB), a member of the SWI/SNF family of helicases (Selby and Sancar, 1997; van Gool et al., 1997; Citterio et al., 2000; Kamiuchi et al., 2002; Fousteri et al., 2006; Cazzalini et al., 2008). CSB binds to the stalled RP2, and this binding is a necessary trigger for recruitment of the same core repair proteins as described for GGR. Comparable to DDB2, CSB becomes a target of the DCX E3 ligase, which is mediated by another WD-40 protein, CSA. This interaction ultimately results in degradation

Most of the proteins that play a role in GGR or TCR can be found in animals and plants, while only a few members, like XPA and TF2H3, a subunit of TFIIH, appear to be absent in plants (Kimura and Sakaguchi, 2006). It is currently open whether plants encode for functional analogs of XPA and TF23H that would perform tasks similar to these proteins. For most of the other NER proteins that are conserved among animals and plants, a role in DNA repair has been demonstrated, frequently by reverse genetic studies in *Arabidopsis thaliana*. Here, proteins shown to be involved in damaged DNA recognition in animals, such as DCX-E3 ligases, DDB2 and CSA, have also been recently described by several groups in plants (Bernhardt et al., 2006; Molinier et al., 2008; Al Khateeb and Schroeder, 2009; Bernhardt et al., 2010; Biedermann and Hellmann, 2010; Zhang et al., 2010; Zhang and Schroeder, 2010; Castells et al., 2011). While plants affected in ATCSA-1, the Arabidopsis CSA ortholog, do not display an abnormal development (Biedermann and Hellmann, 2010), loss of CUL4 or DDB2 cause a dwarf-like phenotype (Bernhardt et al., 2006; Koga et al., 2006). Interestingly, Arabidopsis *ddb2* or *atcsa-1* mutants are UV-hypersensitive but only when brought into the dark right after UV treatment, demonstrating that plants primarily rely on photoreactivation rather than NER (Biedermann and Hellmann, 2010). However, when kept in the dark both mutants have reduced repair activities when compared to wild type (Biedermann and Hellmann, 2010). CSB-like helicases are also present in plants

of CSA, CSB and possibly also RP2 (Groisman et al., 2006).

Not all nucleotide modifications can be repaired by NER, and many DNA lesions generated by reactive oxygen species (ROS) are not recognized by the NER proteins. Thus as an additional mechanism to ensure genomic integrity, cells utilize other repair mechanisms like base excision repair (BER). Because ROS are continuously produced as metabolic byproducts or by ionizing radiation, they represent a considerable source of the daily DNA damage. ROS-induced DNA lesions include for example 8-hydroxyguanine (8-oxoG), formamidopyrimidines, and 5-hydroxyuracil, which can potentially lead to miscoding during replication and transcription.

As a general rule BER requires the activities of DNA glycosylases, which cleave the Nglycosyl bond between the base and the sugar at the lesion site. This releases the base and leaves an abasic or apurinic/apyrimidinic (AP) site. In bacteria, fungi, plants and animals, several DNA glycosylases have been described that either specifically or broadly recognize certain lesions. For example, the mammalian DNA glycosylase OGG1 has a high affinity to 8-oxoG and some formamidopyrimidines, while another mammalian DNA glycosylase, NEIL1, efficiently repairs formamidopyrimidines but only poorly 8-oxoG (Morland et al., 2002; Parsons et al., 2005). DNA glycosylases are classified as either being mono- or bifunctional. Monofunctionally they only perform the cleavage reaction of the glycosylic bond between the deoxyribose and the target base to generate an AP site. Bifunctional DNA glycosylases/lyases, to which OGG1 and NEIL1 belong, are able to catalyze the release of the oxidized base and the cleavage of the DNA backbone at the AP site (Hazra et al., 2001). Although there is currently no evidence that plants have NEIL1 orthologs, which are

common in bacteria and animals and required in part for excision of oxidized purines and pyrimidines, most other DNA glycosylases have been found. For example, plants encode for orthologs of OGG1 (Roldan-Arjona and Ariza, 2009), and their activity in excising oxidized purines has been demonstrated for the *Arabidopsis* AtOGG1 (Dany and Tissier, 2001; Garcia-Ortiz et al., 2001; Morales-Ruiz et al., 2003). In addition to OGG1, plants also encode for proteins related to the bifunctional Endonuclease III/Nth from *E. coli*, yeast, and animals, which remove a broad range of damaged pyrimidines (Breimer and Lindahl, 1980; Boorstein et al., 1989; Hatahet et al., 1994; Phadnis et al., 2006; Guay et al., 2008). Like their bacterial counterparts, *Arabidopsis* AtNTH1 also shows a broad substrate specificity and DNA glycosylase activity for DNA lesions containing modified pyrimidines (Krokan et al., 1997; Roldan-Arjona et al., 2000). Furthermore, plants encode for proteins related to MutM/Fpg, an original model DNA glycosylase/lyase from *E. coli* that excises 8-oxo-guanine and other oxidized purines from damaged DNA (Tchou et al., 1991; Tchou et al., 1993; Bhagwat and

Recognition and Repair Pathways of Damaged DNA in Higher Plants 209

gap-filling repair mediated by a DNA polymerase (Demple and Harrison, 1994; Roldan-Arjona et al., 2000; Garcia-Ortiz et al., 2001). Removal of 3'dRP is mediated in plants and animals by AP endonucleases (APE) which also work on AP sites generated by either monofunctional DNA glycosylases or those that occurred through spontaneous degradation

Subsequently to APE, two separate BER repair pathways can become active in mammalian cells. First, the short-patch repair pathway, which relies on the concerted activities of DNA polymerase (Pol), X-ray repair cross-complementing protein 1 (XRCC1), and the DNA ligase 3 (LIG3). Pol has an intrinsic 3'dRP activity and can remove deoxyribose sugar itself if required (Caldecott, 2001). XRCC1 interacts with LIG3 and other BER proteins and may function as a repair coordinating protein (Vidal et al., 2001) (Fig. 3). Alternatively, the long patch-repair pathway can be employed in mammalian cells, which requires activities of DNA polymerases and , RFC, PCNA, and flap endonuclease 1 (FEN1) to remove and resynthesize up to 10 nucleotides 3' to the AP site, while the nick is ligated by LIG1

While most proteins are present in plants that can participate in long-patch repair (Kimura and Sakaguchi, 2006), it is currently open whether a short-patch pathway exists in plants since no obvious homologs of POL and LIG3 are identified so far (Kimura and Sakaguchi, 2006; Roldan-Arjona and Ariza, 2009). In addition, plant XRCC1-like proteins lack domains that are necessary for complex assembly with POL and LIG3, and it is therefore currently open whether the protein participates in BER (Vidal et al., 2001; Taylor et al., 2002). Although no POL proteins are described in plants so far, it is possible that their function is conducted by POL. Both polymerases belong to the X superfamily of DNA polymerases and several amino acid residues are conserved between POL and (Garcia-Diaz et al., 2000; Uchiyama et al., 2004). In addition, POL has been demonstrated in rice to possess intrinsic 3'dRP activity and its expression is mainly found in meristematic and proliferating

An important role in the recognition and repair of SSB and activation of BER involves poly(ADP-ribose) polymerases (PARP). PARP proteins belong to small protein families with, for example, 18 members in human, and they are highly conserved among eukaryotes (Ame et al., 2004); however, it is PARP1 and PARP2 that have been brought in context with damaged DNA recognition and DNA repair processes. PARP1 is a 113 kDa protein that contains a modular set of domains that enable it to fulfill multiple functions in the cell. At its N-terminal region PARP1 contains a DNA break recognition fold that is composed of a duplicated zinc finger similar to DNA ligase III. A BRCT motif is present in the center that can be found in many proteins connected with maintenance of genomic integrity and cell cycle checkpoints. The motif also functions as the main interface for protein–protein interactions. Finally, at its COOH-terminal region, PARP1 has motifs with different catalytic activities including NAD+ hydrolysis as well as initiation, elongation, branching and termination of ADP-ribose polymers (Citarelli et al., 2010). It has been shown in mammalian cells that, upon binding a DNA lesion PARP1 poly(ADP)ribosylates itself as well as nearby histones (H1 and H2B), which relaxes the chromatin structure allowing better access for XRCC1 and other repair proteins to the damaged site (Poirier et al., 1982; Masson et al., 1998; Pleschke et al., 2000). Plant PARP1 and PARP2 are nuclear localized like their animal counterparts, and they become transcriptionally activated upon genotoxic stress conditions such as ionizing radiation or oxidative stress (Puchta et al., 1995; Babiychuk et al., 1998;

of the DNA (Babiychuk et al., 1994; Demple et al., 1997; Pascucci et al., 2002) (Fig. 3).

(Matsumoto, 2001) (Fig. 3).

tissues (Uchiyama et al., 2004).

Gerlt, 1996; Ohtsubo et al., 1998; Murphy and Gao, 2001; Roldan-Arjona and Ariza, 2009). Although enzymatic function for all three types of plant DNA glycosylases is established, there is unfortunately no information available on how loss of these proteins affects development or ROS sensitivity of mutant plants.

Fig. 3. Schematic model for base excision repair (BER).

DNA lesions caused by ROS are recognized and modified by the concerted activities of a DNA glycosylase and APE, after which cells can either take the route of long-patch repair or alternatively the short-patch repair pathway. Currently evidence indicates for plants that the long-patch repair is employed for BER.

Plant OGG1 and NTH proteins generate 3' phospho ,-unsaturated aldehydes (3' dRP) at the strand breaks, and these need to be removed to generate free 3' hydroxyl ends to allow

Gerlt, 1996; Ohtsubo et al., 1998; Murphy and Gao, 2001; Roldan-Arjona and Ariza, 2009). Although enzymatic function for all three types of plant DNA glycosylases is established, there is unfortunately no information available on how loss of these proteins affects

development or ROS sensitivity of mutant plants.

Fig. 3. Schematic model for base excision repair (BER).

long-patch repair is employed for BER.

DNA lesions caused by ROS are recognized and modified by the concerted activities of a DNA glycosylase and APE, after which cells can either take the route of long-patch repair or alternatively the short-patch repair pathway. Currently evidence indicates for plants that the

Plant OGG1 and NTH proteins generate 3' phospho ,-unsaturated aldehydes (3' dRP) at the strand breaks, and these need to be removed to generate free 3' hydroxyl ends to allow gap-filling repair mediated by a DNA polymerase (Demple and Harrison, 1994; Roldan-Arjona et al., 2000; Garcia-Ortiz et al., 2001). Removal of 3'dRP is mediated in plants and animals by AP endonucleases (APE) which also work on AP sites generated by either monofunctional DNA glycosylases or those that occurred through spontaneous degradation of the DNA (Babiychuk et al., 1994; Demple et al., 1997; Pascucci et al., 2002) (Fig. 3).

Subsequently to APE, two separate BER repair pathways can become active in mammalian cells. First, the short-patch repair pathway, which relies on the concerted activities of DNA polymerase (Pol), X-ray repair cross-complementing protein 1 (XRCC1), and the DNA ligase 3 (LIG3). Pol has an intrinsic 3'dRP activity and can remove deoxyribose sugar itself if required (Caldecott, 2001). XRCC1 interacts with LIG3 and other BER proteins and may function as a repair coordinating protein (Vidal et al., 2001) (Fig. 3). Alternatively, the long patch-repair pathway can be employed in mammalian cells, which requires activities of DNA polymerases and , RFC, PCNA, and flap endonuclease 1 (FEN1) to remove and resynthesize up to 10 nucleotides 3' to the AP site, while the nick is ligated by LIG1 (Matsumoto, 2001) (Fig. 3).

While most proteins are present in plants that can participate in long-patch repair (Kimura and Sakaguchi, 2006), it is currently open whether a short-patch pathway exists in plants since no obvious homologs of POL and LIG3 are identified so far (Kimura and Sakaguchi, 2006; Roldan-Arjona and Ariza, 2009). In addition, plant XRCC1-like proteins lack domains that are necessary for complex assembly with POL and LIG3, and it is therefore currently open whether the protein participates in BER (Vidal et al., 2001; Taylor et al., 2002). Although no POL proteins are described in plants so far, it is possible that their function is conducted by POL. Both polymerases belong to the X superfamily of DNA polymerases and several amino acid residues are conserved between POL and (Garcia-Diaz et al., 2000; Uchiyama et al., 2004). In addition, POL has been demonstrated in rice to possess intrinsic 3'dRP activity and its expression is mainly found in meristematic and proliferating tissues (Uchiyama et al., 2004).

An important role in the recognition and repair of SSB and activation of BER involves poly(ADP-ribose) polymerases (PARP). PARP proteins belong to small protein families with, for example, 18 members in human, and they are highly conserved among eukaryotes (Ame et al., 2004); however, it is PARP1 and PARP2 that have been brought in context with damaged DNA recognition and DNA repair processes. PARP1 is a 113 kDa protein that contains a modular set of domains that enable it to fulfill multiple functions in the cell. At its N-terminal region PARP1 contains a DNA break recognition fold that is composed of a duplicated zinc finger similar to DNA ligase III. A BRCT motif is present in the center that can be found in many proteins connected with maintenance of genomic integrity and cell cycle checkpoints. The motif also functions as the main interface for protein–protein interactions. Finally, at its COOH-terminal region, PARP1 has motifs with different catalytic activities including NAD+ hydrolysis as well as initiation, elongation, branching and termination of ADP-ribose polymers (Citarelli et al., 2010). It has been shown in mammalian cells that, upon binding a DNA lesion PARP1 poly(ADP)ribosylates itself as well as nearby histones (H1 and H2B), which relaxes the chromatin structure allowing better access for XRCC1 and other repair proteins to the damaged site (Poirier et al., 1982; Masson et al., 1998; Pleschke et al., 2000). Plant PARP1 and PARP2 are nuclear localized like their animal counterparts, and they become transcriptionally activated upon genotoxic stress conditions such as ionizing radiation or oxidative stress (Puchta et al., 1995; Babiychuk et al., 1998;

Recognition and Repair Pathways of Damaged DNA in Higher Plants 211

complementary strands to anneal the ends of the break, after which non-homologous tails are detached and nicks can be ligated (Tissier et al., 1995; Puchta, 2005; Blanck et al., 2009; Mannuss et al., 2010). Because HR is less likely to cause changes in the genetic information than NHEJ, it is likely that the extent to which either NHEJ or HR repair pathways are

employed in DSB repair may impact genome evolution in living organisms.

Fig. 4. ROS induced repair of double-strand breaks.

**3.5 DNA repair in chloroplasts** 

Two alternative pathways for which strong evidence is present to exist in plants are shown. Double-strand breaks can either be repaired by nonhomologous DNA end joining pathway

Based on their high metabolic activities in respiration and photosynthesis, organelles are centers of ROS production. Both mitochondria and chloroplasts possess their own repair

(NHEJ; left hand side), or by homologous recombination (HR; right hand side).

Doucet-Chabeaud et al., 2001; Chen et al., 2003). However, although a similar role of plant PARP1 and PARP2 in damaged DNA recognition and initiation of DNA repair is likely, a detailed *in planta* functional description is still missing for these proteins.
