**3.1.1.2 Cullin based E3 ligases**

The 127 kDa DDB1 homologs function as substrate adaptors for CULLIN4 based E3 ubiquitin (Ub) ligases (Groisman et al., 2003). E3 Ub ligases are multimeric complexes that add ubiquitin moieties to target proteins and contain CULLIN proteins as scaffolding

Nucleotide excision repair (NER) is a light independent repair process involving a series of reactions: initial damaged DNA recognition, DNA unwinding, dual incision bracketing the lesion, repair synthesis and final ligation to seal the repaired site. NER initiates with specific sub-pathways for transcriptionally active (Transcription Coupled Repair (TC-NER)) or silent (Global Genomic Repair (GG-NER)) DNA. TC-NER and GG-NER exhibit different damage recognition strategies followed by a common repair pathway (Gillet & Scharer, 2006) (Fig. 2). Defects in human NER genes result in the photosensitive syndromes Xeroderma pigmentosum (XP) or Cockayne syndrome (CS). Eight genetic complementation groups for XP have been identified (XPA-G, V) as well as two for CS (CSA and CSB). While XP-V mutation uniquely results in defects in translesion synthesis, XP -A, -B, -D, -F, and -G mutation results in both TC-NER and GG-NER defects, while XP –C and -E mutation results in GG-NER defects only. CSA and CSB mutation results exclusively in TC-NER defects (Hoeijmakers, 2001; Svejstrup, 2002). Bioinformatic analysis of the plant NER protein machinery suggests the molecular mechanisms are largely but not entirely conserved with that of the extensively studied yeast *S. cerevisiae* and mammalian cells (Kimura & Sakaguchi, 2006; Kunz et al., 2005, 2006). NER in plants has been studied primarily in rice and *Arabidopsis* (Singh et al., 2010). Many *Arabidopsis* NER related genes were initially isolated by analysis of UV hypersensitive (*uvh*) and UV repair defective (*uvr*) mutants which were

In mammalian systems, damage detection in the GG-NER pathway involves UV-Damaged DNA Binding protein 1 and 2 (DDB1 and DDB2) followed by the XPC-HR23B-CEN2 complex. In humans DDB2 complements the *XPE* mutation and plays a role in recognition of the UV-induced lesion, while DDB1 is required for high affinity interaction of the DDB1- DDB2 complex (Groisman et al., 2003; Luijsterburg et al., 2007; Rapic-Otrin et al., 2002). *S. pombe* Ddb1 knockouts result in chromosomal segregation defects, UV sensitivity and slow S phase progression leading to defects in meiosis (Holmberg et al., 2005). DDB1 and DDB2 homologues have been identified in rice, where they are UV-induced in proliferating tissues (Ishibashi et al., 2003). *Arabidopsis thaliana* encodes two homologs of DDB1 – DDB1A and DDB1B. These proteins are 91% identical with redundant function. Although *ddb1b* null alleles appear lethal, a viable partial loss of function allele exhibits no developmental or UV sensitive phenotypes (Bernhardt et al., 2010; Schroeder et al., 2002). Overexpression of DDB1A in *Arabidopsis* confers enhanced UV resistance and *ddb1a* knockouts exhibit mild UV sensitivity (Al Khateeb & Schroeder, 2009; Molinier et al., 2008). *AtDDB2* encodes a 48 kDa nuclear localized protein with upregulated expression upon UV-induction. *AtDDB2* loss of function results in UV sensitivity while overexpression increases UV tolerance (Biedermann

The 127 kDa DDB1 homologs function as substrate adaptors for CULLIN4 based E3 ubiquitin (Ub) ligases (Groisman et al., 2003). E3 Ub ligases are multimeric complexes that add ubiquitin moieties to target proteins and contain CULLIN proteins as scaffolding

subsequently mapped to homologues of the human XP genes (Table 1).

& Hellmann, 2010; Koga et al., 2006; Molinier et al., 2008).

**3. Nucleotide excision repair** 

**3.1 Global genomic repair 3.1.1 Damage recognition 3.1.1.1 DDB1 & DDB2** 

**3.1.1.2 Cullin based E3 ligases** 

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.

UV Damaged DNA Repair & Tolerance in Plants 79

**Human Yeast Function ATG no. Arabidopsis** 

*ND ND* 6-4 PP Photolyase At3g15620 *UVR3 ND PHR1* Class II CPD Photolyase At1g12370 *PHR1/UVR2* 

> At4g05420 At4g21100

> At1g79650 At1g16190 At3g02540 At5g38470

> At5g41360

At1g55750 At3g61420 At1g05055 At1g18340 At4g17020 At1g12400 At1g62886

At2g29570

At5g22010 At1g21690 At1g77470 At1g63160 At5g27740

At2g06510 At4g19130 At5g08020 At5g45400 At5g61000 At2g24490 At3g02920 At3g52630 At4g18590

At1g19750

*AtDDB1a AtDDB1b* 

*AtRAD23A AtRAD23B AtRAD23C AtRAD23D* 

> *AtXPB1 AtXPB2*

*AtTFB1-1 AtTFB1-2 Atp44 AtTFB4 AtTFB2 AtTFB5-1 AtTFB5-2* 

*AtPCNA1 AtPCNA2* 

*AtRFC1 AtRFC2 AtRFC3 AtRFC4 AtRFC5* 

*AtRPA70A AtRPA70B AtRPA70C AtRPA70D AtRPA70E AtRPA32A AtRPA32B AtRPA14A AtRPA14B* 

*AtCSA1A AtCSA1B* 

Interacts with DCAF proteins

*DDB2/XPE ND* Damaged DNA binding (DCAF) At5g58760 *AtDDB2 CUL4 CUL4* Scaffolding subunit of E3 Ub ligase At5g46210 *AtCUL4 XPC RAD4* GG-NER damage recognition At5g16630 *AtXPC* 

*CEN2 CEN2* Stabilizes XPC-HR23B complex At3g50360 *AtCEN2* 

*XPD RAD3* Subunit of TFIIH. 5'->3' helicase At1g03190 *AtXPD/UVH6* 

*XPB RAD25* Subunit of TFIIH. 3'->5' helicase At5g41370

*PCNA PCNA* RFC dependant sliding clamp At1g07370

DNA-dependent ATPase required for DNA replication and repair

ssDNA binding protein required for architectural role in dual lesion incision and repair synthesis

*CSA RAD28* TC-NER specific DCAF protein At1g27840

Core TFIIH subunits

*XPA RAD14* ssDNA binding ND *ND XPG RAD2* 3' endonuclease At3g28030 *AtXPG/UVH3 ERCC1 RAD10* 5' endonuclease with XPF At3g05210 *AtERCC1/UVR7 XPF RAD1* 5' endonuclease with ERCC1 At5g41150 *AtXPF/UVH1* 

**Photoreactivation:** 

*GTF2H1* 

*TFB1*

*SSL1 TFB4 TFB2 TFB5* 

*RFC1 RFC2 RFC3 RFC4 RFC5* 

*RFA1*

*RFA2* 

*RFA3* 

*GTF2H2 GTF2H3 GTF2H4 GTF2H5* 

> *RFC1 RFC2 RFC3 RFC4 RFC5*

*RPA70* 

*RPA32* 

*RPA14* 

**Nucleotide Excision Repair:** 

*DDB1 ND* Substrate adaptor for CUL4.

*HR23B RAD23* Binds to XPC

subunits (Hua & Vierstra, 2011). CUL4 based E3 ubiquitin ligases consist of three core subunits: CULLIN4 (CUL4), RING finger protein REGULATOR OF CULLINS1 (ROC1)/RING-BOX1 (RBX1), and DDB1. The CUL4 – RBX1 – DDB1 complex interacts with a large number of proteins containing WD40 motifs referred to as DCAF proteins (DDB1- CUL4 Associated Factor) or DWD proteins (DDB1 binding WD40 proteins) (Lee & Zhou, 2007). DDB2 is an example of a WD40 domain containing DCAF protein. WD40 motifs are characterized by 40 amino acid repeats initiated by a glycine-histidine dipeptide and terminated by a tryptophan-aspartate (WD) dipeptide facilitating protein-protein interactions. DDB1 is composed of three β propeller domains (BPA, BPB and BPC) and DDB2, in addition to the WD40 domain, contains a helix loop helix (HLX) segment in the N terminal. While the clam shaped BPA-BPC of DDB1 mediates interaction with the HLX motif of DDB2 and other DCAF substrates, BPB exhibits exclusive interactions with CUL4 (Scrima et al., 2008).

AtCUL4 is a 91 kDa protein with a conserved CH motif and an extended N terminal region of 65 amino acids that shares close sequence similarity to its human/mouse orthologs. *AtCUL4* loss of function results in abnormal plant development (Bernhardt et al., 2006; Chen et al., 2006) and UV sensitivity (Molinier et al., 2008). Examples of DCAF proteins interacting with the *Arabidopsis* CUL4–DDB1A/B complex include AtDDB2 (Bernhardt et al., 2006), AtCSA-1&2 (Biedermann & Hellmann, 2010; Zhang et al., 2010), as well as the negative regulator of photomorphogenesis DET1 (De-etiolated1) (Schroeder et al., 2002), and many other DWD proteins (Lee et al., 2008). Recent results have shed light on the cross talk between photomorphogenesis regulation and repair of UV damaged DNA. HY5, a positive regulator of photomorphogenesis, has been shown to regulate gene sets connected to UV tolerance, such as the *UVR3* and *PHR1* photolyases, as well as secondary metabolite transcriptional regulators (Oravecz et al., 2006; Ulm et al., 2004). DET1, initially identified as a nuclear localized negative regulator of photomorphogenesis, exhibits a constitutively light grown phenotype in addition to increased levels of flavanoids (Pepper et al., 1994). Recent papers show that *det1* mutants exhibit enhanced UV tolerance through increased levels of secondary metabolites reflecting/absorbing UV irradiation as well as by upregulation of photolyase genes. Further it appears that DET1 protein dosage influences UV sensitivity of plants as DET1 overexpressing lines exhibit increased UV sensitivity (Castells et al., 2010, 2011).

### **3.1.1.3 Histone ubiquitination facilitates NER machinery entry**

In mammals, in the absence of UV irradiation, DDB2-DDB1-CUL4-RBX1 (DDB2 complex) was found to be associated with the COP9 Signalosome complex (CSN). CSN shares significant structural homology with the 19S lid of 26S proteosome. The CSN deconjugates neddylation (Nedd8) from CULLINs, thereby regulating the activation, stability or the disassembly of CULLIN based E3 ligase activity (Parry & Estelle, 2004; Schwechheimer & Deng, 2001). The DDB2 - CSN complex show no ubiquitin ligase activity, but upon UV irradiation, these complexes disassociate in parallel with increased neddylation and activation of CUL4 (Groisman et al., 2003). Core histone proteins have been identified as potential targets for DDB2-DDB1-CUL4-RBX1 mediated proteosomal degradation. Kapetanaki et al. (2006) and Wang et al. (2006) describe the ubiquitination of H2A, H3 and H4 histone proteins. Reduction of histone H3 and H4 ubiquitination by knockdown of *cul4*  impairs recruitment of the repair protein XPC to the damaged foci and inhibits the repair process. Thus biochemical studies indicate that DDB-CUL4-RBX1-mediated histone

subunits (Hua & Vierstra, 2011). CUL4 based E3 ubiquitin ligases consist of three core subunits: CULLIN4 (CUL4), RING finger protein REGULATOR OF CULLINS1 (ROC1)/RING-BOX1 (RBX1), and DDB1. The CUL4 – RBX1 – DDB1 complex interacts with a large number of proteins containing WD40 motifs referred to as DCAF proteins (DDB1- CUL4 Associated Factor) or DWD proteins (DDB1 binding WD40 proteins) (Lee & Zhou, 2007). DDB2 is an example of a WD40 domain containing DCAF protein. WD40 motifs are characterized by 40 amino acid repeats initiated by a glycine-histidine dipeptide and terminated by a tryptophan-aspartate (WD) dipeptide facilitating protein-protein interactions. DDB1 is composed of three β propeller domains (BPA, BPB and BPC) and DDB2, in addition to the WD40 domain, contains a helix loop helix (HLX) segment in the N terminal. While the clam shaped BPA-BPC of DDB1 mediates interaction with the HLX motif of DDB2 and other DCAF substrates, BPB exhibits exclusive interactions with CUL4

AtCUL4 is a 91 kDa protein with a conserved CH motif and an extended N terminal region of 65 amino acids that shares close sequence similarity to its human/mouse orthologs. *AtCUL4* loss of function results in abnormal plant development (Bernhardt et al., 2006; Chen et al., 2006) and UV sensitivity (Molinier et al., 2008). Examples of DCAF proteins interacting with the *Arabidopsis* CUL4–DDB1A/B complex include AtDDB2 (Bernhardt et al., 2006), AtCSA-1&2 (Biedermann & Hellmann, 2010; Zhang et al., 2010), as well as the negative regulator of photomorphogenesis DET1 (De-etiolated1) (Schroeder et al., 2002), and many other DWD proteins (Lee et al., 2008). Recent results have shed light on the cross talk between photomorphogenesis regulation and repair of UV damaged DNA. HY5, a positive regulator of photomorphogenesis, has been shown to regulate gene sets connected to UV tolerance, such as the *UVR3* and *PHR1* photolyases, as well as secondary metabolite transcriptional regulators (Oravecz et al., 2006; Ulm et al., 2004). DET1, initially identified as a nuclear localized negative regulator of photomorphogenesis, exhibits a constitutively light grown phenotype in addition to increased levels of flavanoids (Pepper et al., 1994). Recent papers show that *det1* mutants exhibit enhanced UV tolerance through increased levels of secondary metabolites reflecting/absorbing UV irradiation as well as by upregulation of photolyase genes. Further it appears that DET1 protein dosage influences UV sensitivity of plants as DET1 overexpressing lines exhibit increased UV sensitivity (Castells et al., 2010,

In mammals, in the absence of UV irradiation, DDB2-DDB1-CUL4-RBX1 (DDB2 complex) was found to be associated with the COP9 Signalosome complex (CSN). CSN shares significant structural homology with the 19S lid of 26S proteosome. The CSN deconjugates neddylation (Nedd8) from CULLINs, thereby regulating the activation, stability or the disassembly of CULLIN based E3 ligase activity (Parry & Estelle, 2004; Schwechheimer & Deng, 2001). The DDB2 - CSN complex show no ubiquitin ligase activity, but upon UV irradiation, these complexes disassociate in parallel with increased neddylation and activation of CUL4 (Groisman et al., 2003). Core histone proteins have been identified as potential targets for DDB2-DDB1-CUL4-RBX1 mediated proteosomal degradation. Kapetanaki et al. (2006) and Wang et al. (2006) describe the ubiquitination of H2A, H3 and H4 histone proteins. Reduction of histone H3 and H4 ubiquitination by knockdown of *cul4*  impairs recruitment of the repair protein XPC to the damaged foci and inhibits the repair process. Thus biochemical studies indicate that DDB-CUL4-RBX1-mediated histone

**3.1.1.3 Histone ubiquitination facilitates NER machinery entry** 

(Scrima et al., 2008).

2011).


UV Damaged DNA Repair & Tolerance in Plants 81

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.,

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

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

**3.1.2 DNA unwinding complex assembly** 

**3.1.3 Endonuclease recruitment following unwinding** 

2006).

lesion (Reidl et al., 2003).


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 al., 2011; Molinier et al., 2008).
