**3.1.1.4 XPC-HR23B-CEN2**

The next step in GG-NER involves the homologous heterodimers hXPC-hHR23B (in Humans) and RAD4-RAD23 (in yeast). In addition to hXPC-hHR23B, Araki et al. (2001) identified hCEN2, a Ca2+ binding protein contributing to the stability of the hXPC-hHR23B complex. Hence in mammalian systems the identified protein recognition complex is hXPChHR23B-hCEN2, however neither hHR23B nor hCEN2 bind to damaged DNA but enhance both the affinity and specificity of hXPC binding to damaged DNA (Fitch et al., 2003; Nishi et al., 2005). AtCEN2 shares 49% identity with hCEN2, *Atcen2* mutants are UV sensitive, and AtCEN2 overexpression resulted in enhanced repair. Upon UV irradiation, AtCEN2 level increases and it rapidly translocates to the nucleus. AtCEN2-AtXPC interaction in *Arabidopsis thaliana* has also been demonstrated (Liang et al., 2006; Molinier et al., 2004). Potential homologs of HR23B/RAD23 have been identified in *Arabidopsis thaliana*, *Oryza sativa* and *Daucus carota* (Schultz & Quatrano, 1997; Sturm & Leinhard, 1998). The *Arabidopsis* genome has 4 loci encoding RAD23 homologs (RAD23a, RAD23b, RAD23c, RAD23d), and although mutants exhibit multiple pleotrophic developmental defects (Farmer et al., 2010), UV sensitivity or complex interactions with the *Arabidopsis* NER machinery have not yet been reported.

**Human Yeast Function ATG no. Arabidopsis**  *CSB RAD26* SWI/SNF2 like ATPase At2g18760 *AtCSB XAB2 SYF1* Complex stabilization At5g28740 *AtXAB2 TFIIS TFIIS* TC-NER elongation factor At2g38560 *AtTFIIS HMGN1 ND* Nucleosome binding ND *ND* 

*POLH/XPV RAD30* Y-family DNA polymerase At5g44740 *AtPOLH REV1 REV1* Y-family DNA polymerase At5g44750 *AtREV1 POLZ REV3* B-family DNA polymerase At1g67500 *AtREV3 REV7 REV7* REV3 accessory subunit At1g16590 *AtREV7 POLK ND* Y-family DNA polymerase At1g49980 *AtPOLK* 

Table 1. Genes involved in UV damaged DNA repair and tolerance. ND=not detected.

ubiquitination weakens the interaction between histones and DNA to further facilitate the recruitment of repair proteins to damaged DNA (Guerrero-Santoro et al., 2008; Higa et al., 2006). The activated DDB2 complex recruits XPC via protein-protein interactions, followed by ubiquitination of XPC and DDB2. Polyubiquitinated DDB2 exhibits reduced affinity for damaged DNA and is subsequently displaced from the damaged foci, whereas polyubiquitinated XPC enhances its binding to DNA (Sugasawa et al., 2005). In *Arabidopsis,* DDB2 turnover is abrogated in *cul4, ddb1a, atr* and *det1* mutants suggesting that ATR and DET1 may co-operate with the CUL4-DDB1 E3 ligase complex in regulating NER (Castells et

The next step in GG-NER involves the homologous heterodimers hXPC-hHR23B (in Humans) and RAD4-RAD23 (in yeast). In addition to hXPC-hHR23B, Araki et al. (2001) identified hCEN2, a Ca2+ binding protein contributing to the stability of the hXPC-hHR23B complex. Hence in mammalian systems the identified protein recognition complex is hXPChHR23B-hCEN2, however neither hHR23B nor hCEN2 bind to damaged DNA but enhance both the affinity and specificity of hXPC binding to damaged DNA (Fitch et al., 2003; Nishi et al., 2005). AtCEN2 shares 49% identity with hCEN2, *Atcen2* mutants are UV sensitive, and AtCEN2 overexpression resulted in enhanced repair. Upon UV irradiation, AtCEN2 level increases and it rapidly translocates to the nucleus. AtCEN2-AtXPC interaction in *Arabidopsis thaliana* has also been demonstrated (Liang et al., 2006; Molinier et al., 2004). Potential homologs of HR23B/RAD23 have been identified in *Arabidopsis thaliana*, *Oryza sativa* and *Daucus carota* (Schultz & Quatrano, 1997; Sturm & Leinhard, 1998). The *Arabidopsis* genome has 4 loci encoding RAD23 homologs (RAD23a, RAD23b, RAD23c, RAD23d), and although mutants exhibit multiple pleotrophic developmental defects (Farmer et al., 2010), UV sensitivity or complex interactions with the *Arabidopsis* NER machinery have not yet

At1g79000 At1g67220 At1g55970 At3g12980 At1g16710

*HAC1 HAC2 HAC4 HAC5 HAC12* 

*EP300/CBP ND* Histone acetyl transferase

**Translesion Synthesis** 

al., 2011; Molinier et al., 2008). **3.1.1.4 XPC-HR23B-CEN2** 

been reported.

#### **3.1.2 DNA unwinding complex assembly**

Following recognition, the damaged region is unwound by the TFIIH transcription factor which joins the XPC-CEN2-HR23B complex. TFIIH is a complex of 10 proteins, seven of which are players in the NER pathway (helicases XPB and XPD, p62, p44, p34, p52, and p8). The last five proteins are encoded by GTF2H1, GTF2H2, GTF2H3, GTF2H4, GTF2H5 (Frit et al., 1999). TFIIH not only participates in NER of transcriptionally active and inactive DNA, but also in RNA POL II dependant transcription, cell cycle control and regulation of nuclear receptor activity (Chen & Suter, 2003). ATP dependant 5'–>3' and 3'–>5' helicase activities associated with XPD/RAD3 and XPB/RAD25 respectively unwind the DNA encompassing the lesion with the concomitant release of the recognition complex. Human XPB and the corresponding yeast RAD25 knockouts are lethal. *Arabidopsis thaliana,* unlike the sugarcane, rice or mammalian genomes, encodes two homologs of XPB – AtXPB1 and AtXPB2. These proteins are 95% identical with redundant functions and are expressed constitutively throughout plant development (Morgante et al., 2005; Ribeiro et al., 1998). Despite this redundancy, *xpb1* mutants exhibit delayed germination and flowering phenotypes but are not UV sensitive (Costa et al., 2001). Phenotypes of *Arabidopsis xpb2* or double mutants have not yet been reported. *Arabidopsis* XPD is 56% and 50% identical to human and yeast sequences. *Arabidopsis XPD/RAD3* null mutations are lethal, however viable point mutation alleles are UV sensitive and were identified as *uvh6* (*uv hypersensitive 6*) mutants. (Jenkins et al., 1997; Liu et al., 2003). Another component of the of TFIIH complex, p44, was identified in *Arabidopsis* as ATGTF2H2 and was found to interact in vivo with AtXPD (Vonarx et al., 2006).

#### **3.1.3 Endonuclease recruitment following unwinding**

TFIIH further recruits additional factors such as XPA and heterotrimeric Replication Protein A (RPA), composed of 70, 32 and 14 kDa subunits, to promote and stabilize the formation of an open intermediate essential for the dual incision by XPG and XPF-ERCC1 (Excision Repair Complementing defective repair in Chinese hamster 1) (RAD1/RAD10) endonucleases at the 3' and 5' sites respectively (Tapais et al., 2004). The RPA-XPA complex exhibits interactions with both endonucleases (He et al., 1995; Wold, 1997), however the specific function of XPA is still not evident. Initially it was thought to function as a lesion recognition complex in concert with XPC, but was later determined to be recruited after TFIIH entry and facilitate XPC complex departure (Hey et al., 2002; Volker et al., 2001). In addition, XPA homologues do not exist in plants (Kunz et al., 2005). The dual incisions catalyzed by the endonucleases excise oligonucleotides of about 20-30 bases containing the lesion (Reidl et al., 2003).

Potential homologs of ERCC1, XPF, XPG and RPA have been identified in plants. Although ERCC1 was first cloned from male germ line cells of *Lilium longiforum*, southern blots confirmed the presence of ERCC1 across diverse plant species such as *A. thaliana*, *B. napus*, *Z. mays*, *L. esculentum*, *N. tobacum*, and *O. sativa* (Xu et al., 1998). Hefner et al. (2003) mapped the *Arabidopsis uvr7* mutant to *AtERCC1*. *Atercc1* knockouts are phenotypically normal in contrast to the lethal mammalian counterparts (Weeda et al., 1997). *Atercc1* mutants are extremely sensitive to DNA damaging chemicals such as mitomycin and ionizing agents such as UV and γ – radiation (Hefner et al., 2003). More recent studies in *Arabidopsis* indicate significant roles of *AtERCC1* in concert with *AtXPF* in homologous recombination and chromosomal stability (Dubest et al., 2002, 2004; Vannier et al., 2009). Gallego et al. (2000) and Liu et al. (2000) characterized the single copy AtXPF which is 37% and 29% identical to

UV Damaged DNA Repair & Tolerance in Plants 83

The emerging picture of mammalian TC-NER is of a complex mechanism requiring two essential assembly factors (CSA and CSB), certain TC-NER specific proteins (P300, HMGN1, XAB2 and TFIIS), as well as core NER proteins. UV induced DNA damage is initially recognised by RNA POL II, which stalls when it encounters helix-distorting lesions on the template strand during transcription. Stalled RNA POL II backtracks a few nucleotides and is recognised by the CSB protein which in turn co-ordinates the recruitment of the repair

Cloning and characterization of the mammalian CSB gene revealed a nuclear protein of 168 kDa with a region of homology to the SWI2/SNF2 family of helicases. CSB has been shown to interact with RNA POL II and this interaction is enhanced and prolonged by UV exposure (van den Boom et al., 2004). Studies propose that functional CSB in the absence of UV could also serve as a component of the transcriptional machinery promoting elongation (Fousteri & Mullenders, 2008; Hanawalt & Spivak, 2008). Further, CSB facilitates the entry of the core NER factors XPA, XPG and TFIIH through complex interactions (Laine & Egly, 2006; Saxowsky & Doetsch, 2006; Svejstrup, 2002). Mammalian CSA on the other hand is a 46 kDa DWD protein containing seven WD40 repeats that associates with DDB1-CUL4 type E3 ligases and is recruited to the damaged site after CSB. CSA physically interacts with the CSB-RNA POL II complex in a UV dependant manner (Groisman et al., 2003; Fousteri et al., 2006). Interestingly, unlike the DDB2 complex, the CSA-CUL4 Ub ligase complex is active under normal conditions but is rapidly inactivated upon UV irradiation by CSN. Hence CSN plays a differential and dynamic role in regulating both pathways of Nucleotide Excision Repair. The stable CSN-CSA-CSB complex is required for the recruitment of the other NER factors. Following repair, CSN dissociates, reactivating CSA Ub ligase activity and resulting in CSB degradation. Clearance of CSB is required for the reinitiation of

Several papers over the years propose the fate of RNA POL II during the coupling process: either ubiquitinated and degraded, translocated or bypassed from the lesion site, or simply stalled during the entire repair process, is still a matter of debate (Reviewed in Tornaletti, 2009). XAB2 (XPA binding protein 2) is a RNA-binding protein with 15 tetratricopeptide repeats. In addition to interacting with XPA, XAB2 is capable of interacting with CSA, CSB and RNA POL II (Nakatsu et al., 2000). XAB2 is thought to stabilize protein assemblies by functioning as a scaffolding subunit. XAB2 knockouts in mammalian cells exhibit hypersensitivity and decreased recovery of mRNA synthesis post UV irradiation (Kuraoka et al., 2008). Increased amounts of histone acetyl transferase p300 and High Mobility Group Nucleosome binding domain containing protein 1 (HMGN1) interact with RNA POL II in a CSB-dependant manner upon UV irradiation but exhibit weak interactions under normal conditions (Fousteri et al., 2006). Both HMGN1 and p300 are nucleosome interacting proteins which remodel the chromatin structure behind the arrested polymerase and facilitating the backward translocation of RNA POL II (Hanawalt & Spivak, 2008). TFIIS, functioning as a transcription elongation factor, stimulates the arrested RNA POL II to restart elongation. This TFIIS-RNA POL II interaction is significantly increased upon UV irradiation (Fousteri et al., 2006). Hence it is proposed that TFIIS facilitates resumption of

Elucidation of the TC-NER mechanism in plants is still at its infancy. While there is no plant homologue for HMGN1, the *Arabidopsis* genome encodes homologues of XAB2,

factors required to accomplish Transcription Coupled NER (Mellon, 2005).

transcription by RNA POL II (Groisman et al., 2003, 2006).

transcription post DNA lesion removal in a CSA/B-dependent manner.

**3.2 Transcription coupled repair** 

human XPF and *S. cerevisiae* RAD1 respectively. *AtXPF* is homogenously expressed, was mapped to the *uvh1* mutant in *Arabidopsis,* and partially complements the yeast *rad1* mutant (Gallego et al., 2000). *AtXPF* point mutations result in sensitivity to ionizing radiation and mitomycin C, and impaired removal of photoproducts (Fidanstef et al., 2000; Vonarx et al., 2002). *AtXPG* was mapped to the *UVH3* locus and knockouts result in UV sensitivity as well as premature senescence and reduced seed production (Liu et al., 2001). The *XPG* rice homolog, *OsSEND-1*, exhibits upregulated mRNA levels in response to UV and DNA damaging agents (Furukawa et al., 2003a).

ssDNA binding RPA proteins in plants were first identified in rice (Ishibashi et al., 2001). Unlike most eukaryotic organisms, *Arabidopsis* and rice possess multiple copies of the RPA homologs. In addition to participating in DNA repair, RPA proteins play a role in homologous recombination and DNA replication in humans and yeast (Sakaguchi et al., 2009). The rice genome encodes three OsRPA70 (OsRPA70A, OsRPA70B, OsRPA70C), three OsRPA32 (OsRPA32-1, OsRPA32-2, OsRPA32-3) and one OsRPA14. *In vivo* interactions in rice confirms three different complex formations: OsRPA70A-OsRPA32-2-OsRPA14 (Type1); OsRPA70B-OsRPA32-1-OsRPA14 (Type2); and OsRPA70C-OsRPa32-3-OsRPA14 (Type3). Subcellular localization of all three OsRPA32 was detected in both the nucleus and chloroplasts. OsRPA70A was only in the chloroplast whereas OsRPA70B and OsRPA70C were exclusively to the nucleus. This data suggest that while the Type1 complex may participate in chloroplast DNA repair, Type2 and Type3 complexes concentrate on nuclear DNA repair (Ishibashi et al., 2006). OsRPA70A and OsRPa70B share only 33% sequence homology and exhibit differences in expression pattern (Ishibashi et al., 2001). A T-DNA insertion in *OsRPA70A* resulted in partial male sterility, complete female sterility and hypersensitivity to DNA mutagens (Chang et al., 2009). OsRPA32 protein abundance is regulated by both UV irradiation and cell cycle phase (Marwedal et al., 2002). *Arabidopsis*, on the other hand, encodes five putative RPA70 genes and two copies each of RPA32 and RPA14 (Shultz et al., 2007). *Arabidopsis* RPA70A interacts preferentially with AtRPA32A rather than AtRPA32B. Knockouts of both AtRPA70A and AtRPA70B exhibited UV sensitivity when irradiated, but exhibited wildtype characteristics under normal conditions (Ishibashi et al., 2005; Takashi et al., 2009).

#### **3.1.4 Repair synthesis and ligation**

In mammals, the gap formed by the excision is filled via PCNA (Proliferating Cell Nuclear Antigen) and RFC dependant DNA synthesis by DNA POL δ/ε. These components are likely recruited by XPG and RPA as RFC exhibits interaction with RPA (Yuzhakov et al., 1999). RFC catalyzes the ATP dependant loading of PCNA to DNA at the 3' OH. PCNA is a homotrimeric protein which forms a sliding clamp-like complex (Gulbis et al., 1996) and interacts with the DNA POL to ensure replication occurs processively (Mocquet et al., 2008). The final phase of NER is completed by phosphodiester backbone rejoining of the repaired DNA strand by DNA Ligase I.

Although PCNA and RFC homologues have been identified in plants, their specific role in nucleotide excision repair has not yet been elucidated (Furukawa et al., 2003b; Strzalka & Ziemienowicz, 2011). Recently, Roy et al. (2011) cloned and characterized a homolog of mammalian DNA POLλ in *Arabidopsis.* AtPOLλ was upregulated upon UV induction under dark conditions, and *Atpolλ* mutants exhibited UV sensitivity and decreased DNA repair. Thus, this report suggests a role for DNA POLλ in plant NER.

#### **3.2 Transcription coupled repair**

82 Selected Topics in DNA Repair

human XPF and *S. cerevisiae* RAD1 respectively. *AtXPF* is homogenously expressed, was mapped to the *uvh1* mutant in *Arabidopsis,* and partially complements the yeast *rad1* mutant (Gallego et al., 2000). *AtXPF* point mutations result in sensitivity to ionizing radiation and mitomycin C, and impaired removal of photoproducts (Fidanstef et al., 2000; Vonarx et al., 2002). *AtXPG* was mapped to the *UVH3* locus and knockouts result in UV sensitivity as well as premature senescence and reduced seed production (Liu et al., 2001). The *XPG* rice homolog, *OsSEND-1*, exhibits upregulated mRNA levels in response to UV and DNA

ssDNA binding RPA proteins in plants were first identified in rice (Ishibashi et al., 2001). Unlike most eukaryotic organisms, *Arabidopsis* and rice possess multiple copies of the RPA homologs. In addition to participating in DNA repair, RPA proteins play a role in homologous recombination and DNA replication in humans and yeast (Sakaguchi et al., 2009). The rice genome encodes three OsRPA70 (OsRPA70A, OsRPA70B, OsRPA70C), three OsRPA32 (OsRPA32-1, OsRPA32-2, OsRPA32-3) and one OsRPA14. *In vivo* interactions in rice confirms three different complex formations: OsRPA70A-OsRPA32-2-OsRPA14 (Type1); OsRPA70B-OsRPA32-1-OsRPA14 (Type2); and OsRPA70C-OsRPa32-3-OsRPA14 (Type3). Subcellular localization of all three OsRPA32 was detected in both the nucleus and chloroplasts. OsRPA70A was only in the chloroplast whereas OsRPA70B and OsRPA70C were exclusively to the nucleus. This data suggest that while the Type1 complex may participate in chloroplast DNA repair, Type2 and Type3 complexes concentrate on nuclear DNA repair (Ishibashi et al., 2006). OsRPA70A and OsRPa70B share only 33% sequence homology and exhibit differences in expression pattern (Ishibashi et al., 2001). A T-DNA insertion in *OsRPA70A* resulted in partial male sterility, complete female sterility and hypersensitivity to DNA mutagens (Chang et al., 2009). OsRPA32 protein abundance is regulated by both UV irradiation and cell cycle phase (Marwedal et al., 2002). *Arabidopsis*, on the other hand, encodes five putative RPA70 genes and two copies each of RPA32 and RPA14 (Shultz et al., 2007). *Arabidopsis* RPA70A interacts preferentially with AtRPA32A rather than AtRPA32B. Knockouts of both AtRPA70A and AtRPA70B exhibited UV sensitivity when irradiated, but exhibited wildtype characteristics under normal conditions

In mammals, the gap formed by the excision is filled via PCNA (Proliferating Cell Nuclear Antigen) and RFC dependant DNA synthesis by DNA POL δ/ε. These components are likely recruited by XPG and RPA as RFC exhibits interaction with RPA (Yuzhakov et al., 1999). RFC catalyzes the ATP dependant loading of PCNA to DNA at the 3' OH. PCNA is a homotrimeric protein which forms a sliding clamp-like complex (Gulbis et al., 1996) and interacts with the DNA POL to ensure replication occurs processively (Mocquet et al., 2008). The final phase of NER is completed by phosphodiester backbone rejoining of the repaired

Although PCNA and RFC homologues have been identified in plants, their specific role in nucleotide excision repair has not yet been elucidated (Furukawa et al., 2003b; Strzalka & Ziemienowicz, 2011). Recently, Roy et al. (2011) cloned and characterized a homolog of mammalian DNA POLλ in *Arabidopsis.* AtPOLλ was upregulated upon UV induction under dark conditions, and *Atpolλ* mutants exhibited UV sensitivity and decreased DNA repair.

Thus, this report suggests a role for DNA POLλ in plant NER.

damaging agents (Furukawa et al., 2003a).

(Ishibashi et al., 2005; Takashi et al., 2009).

**3.1.4 Repair synthesis and ligation** 

DNA strand by DNA Ligase I.

The emerging picture of mammalian TC-NER is of a complex mechanism requiring two essential assembly factors (CSA and CSB), certain TC-NER specific proteins (P300, HMGN1, XAB2 and TFIIS), as well as core NER proteins. UV induced DNA damage is initially recognised by RNA POL II, which stalls when it encounters helix-distorting lesions on the template strand during transcription. Stalled RNA POL II backtracks a few nucleotides and is recognised by the CSB protein which in turn co-ordinates the recruitment of the repair factors required to accomplish Transcription Coupled NER (Mellon, 2005).

Cloning and characterization of the mammalian CSB gene revealed a nuclear protein of 168 kDa with a region of homology to the SWI2/SNF2 family of helicases. CSB has been shown to interact with RNA POL II and this interaction is enhanced and prolonged by UV exposure (van den Boom et al., 2004). Studies propose that functional CSB in the absence of UV could also serve as a component of the transcriptional machinery promoting elongation (Fousteri & Mullenders, 2008; Hanawalt & Spivak, 2008). Further, CSB facilitates the entry of the core NER factors XPA, XPG and TFIIH through complex interactions (Laine & Egly, 2006; Saxowsky & Doetsch, 2006; Svejstrup, 2002). Mammalian CSA on the other hand is a 46 kDa DWD protein containing seven WD40 repeats that associates with DDB1-CUL4 type E3 ligases and is recruited to the damaged site after CSB. CSA physically interacts with the CSB-RNA POL II complex in a UV dependant manner (Groisman et al., 2003; Fousteri et al., 2006). Interestingly, unlike the DDB2 complex, the CSA-CUL4 Ub ligase complex is active under normal conditions but is rapidly inactivated upon UV irradiation by CSN. Hence CSN plays a differential and dynamic role in regulating both pathways of Nucleotide Excision Repair. The stable CSN-CSA-CSB complex is required for the recruitment of the other NER factors. Following repair, CSN dissociates, reactivating CSA Ub ligase activity and resulting in CSB degradation. Clearance of CSB is required for the reinitiation of transcription by RNA POL II (Groisman et al., 2003, 2006).

Several papers over the years propose the fate of RNA POL II during the coupling process: either ubiquitinated and degraded, translocated or bypassed from the lesion site, or simply stalled during the entire repair process, is still a matter of debate (Reviewed in Tornaletti, 2009). XAB2 (XPA binding protein 2) is a RNA-binding protein with 15 tetratricopeptide repeats. In addition to interacting with XPA, XAB2 is capable of interacting with CSA, CSB and RNA POL II (Nakatsu et al., 2000). XAB2 is thought to stabilize protein assemblies by functioning as a scaffolding subunit. XAB2 knockouts in mammalian cells exhibit hypersensitivity and decreased recovery of mRNA synthesis post UV irradiation (Kuraoka et al., 2008). Increased amounts of histone acetyl transferase p300 and High Mobility Group Nucleosome binding domain containing protein 1 (HMGN1) interact with RNA POL II in a CSB-dependant manner upon UV irradiation but exhibit weak interactions under normal conditions (Fousteri et al., 2006). Both HMGN1 and p300 are nucleosome interacting proteins which remodel the chromatin structure behind the arrested polymerase and facilitating the backward translocation of RNA POL II (Hanawalt & Spivak, 2008). TFIIS, functioning as a transcription elongation factor, stimulates the arrested RNA POL II to restart elongation. This TFIIS-RNA POL II interaction is significantly increased upon UV irradiation (Fousteri et al., 2006). Hence it is proposed that TFIIS facilitates resumption of transcription post DNA lesion removal in a CSA/B-dependent manner.

Elucidation of the TC-NER mechanism in plants is still at its infancy. While there is no plant homologue for HMGN1, the *Arabidopsis* genome encodes homologues of XAB2,

UV Damaged DNA Repair & Tolerance in Plants 85

exhibit an increased mutation frequency, indicating a role in error-free repair. Interestingly, this increased mutation rate is suppressed in the *rev3 polh* double mutant, indicating that REV3 is required for the increased mutations in *polh* (Nakagawa et al., 2011). AtPol interacts with the *Arabidopsis* PCNA homologues PCNA1 and PCNA2. In yeast the interaction with PCNA2 was found to be functionally important and require the PCNAinteracting protein (PIP) box and Ubiquitin-binding motif (UBM) of AtPol (Anderson et al.,

In other systems, PCNA monoubiquitination by RAD6/RAD18 is implicated in polymerase switching during translesion synthesis (Waters et al., 2009). While the *Arabidopsis* genome contains *RAD6* homologues (*AtUBC1-3*), no obvious *RAD18* homologue exists (Kraft et al., 2005). Interestingly, in mammalian cells, the CUL4-DDB1-CDT2 E3 ubiquitin ligase was recently shown to also monoubiquitinate PCNA and promote translesion synthesis (Terai et al., 2010). The CUL4-DDB1-CDT2 complex also ubiquitinates Pol (Kim & Micheal, 2008; Soria & Gottifredi, 2010), suggesting that this complex may be a key regulator of translesion synthesis (Abbas & Dutta, 2011). All components of the CUL4-DDB1-CDT2 complex exist in plants (Lee et al., 2008), so it will be interesting to examine the role of this complex in

In addition to DNA repair and TLS, UV-damaged DNA also triggers other cellular responses such as cell cycle arrest and cell death (Batista et al., 2009). In general, relatively little is known about the molecular basis of these responses in plants. As in mammalian systems, in plants the initial damage sensors include ATM (double strand breaks), ATR (ssDNA) and ATR-interacting protein ATRIP (Culligan et al., 2004; Furukawa et al., 2010; Sakamoto et al., 2009). Plants do not have a p53 homologue, in contrast damage signalling pathways converge on the SOG1 transcription factor, resulting in cell cycle arrest and cell death (Furukawa et al., 2010; Preuss & Britt, 2003; Yoshiyama et al., 2009). UV-induced cell death in plants also involves topoismerase I (Balestrazzi et al., 2010), metacaspase-8 and caspase-3-like activities (Danon et al., 2004; He et al., 2008; Zhang et al., 2009), as well as

Thus, despite the barrage of damage resulting from solar UV exposure plants face every day, they have a variety of mechanisms which allow them to survive. UV induced DNA damage is repaired by direct photoreactivation via photolyases, or by dark repair (NER) in both transcribed (TC-NER) or non-transcribed regions (GG-NER). Finally, rather than repair UV damage, plants can tolerate its presence and proceed with DNA replication via translesion synthesis, although there can be mutagenic consequences of this activity. The continued study of these pathways and the interplay between them in plants is sure to bring

Research in our laboratory is supported by a grant from the Natural Sciences &

changes in reactive oxygen species and mitochrondria (Gao et al., 2008).

2008).

translesion synthesis.

**5. Other responses** 

**6. Conclusions** 

additional insight.

**7. Acknowledgements**

Engingeering Research Council of Canada (NSERC).

TFIIS, and five p300/CBP homologues, however the role of these genes in DNA repair has not been assessed (Grasser et al., 2009; Kunz et al., 2005; Pandey et al., 2002). Only recently was the homolog of human CSA cloned and characterized in *Arabidopsis*. In contrast to mammalian systems, the *Arabidopsis* genome encodes two homologs of CSA – AtCSA1A and AtCSA1B, 92% identical DWD proteins with overlapping subcellular localization and expression patterns. These proteins exist as heterotetramers in planta and are capable of interacting with the DDB1-CUL4 E3 complex. Knockouts of either gene result in UV sensitivity and decreased photoproduct removal (Zhang et al., 2010). Concurrently, another group overexpressed AtCSA1A, which surprisingly also resulted in increased UV sensitivity. This result is hypothesised to be due to competition between CSA and with other DWD proteins to interact with the DDB1-CUL4 complex. Interestingly AtCSA1A levels remained constant upon UV induction (Biedermann & Hellmann, 2010). RNAi of a putative *Arabidopsis* CSB homolog resulted in a UV sensitive phenotype (Shaked et al., 2006). Hence, taken as a whole, these results confirm the role of the CUL4-DDB1-CSA and CSB pathway in plants.
