**4. Translesion synthesis**

Despite the available repair mechanisms, there are times when UV damage persists and DNA replication proceeds past UV lesions via translesion synthesis (TLS). This synthesis proceeds via one of several DNA polymerases: Pol, Rev1, Pol, Pol or Pol. Pol (eta) is encoded by the *POLH* locus in humans and is homologous to yeast RAD30. Pol performs error-free bypass of CPDs, and loss of function results in increased mutation and xeroderma pigmentosa (XP-V) in humans. REV1 exhibits dCMP transferase activity but is required for 6-4 PP bypass independent of this activity, suggesting its role may be in recruitment of other polymerases. Pol is composed of REV3 and REV7 and exhibits error prone repair. REV3 is a B-family polymerase (unlike the other TLS polymerases which are all Y-family polymerases) and REV7 an accessory subunit that enhances REV3 activity (Waters et al., 2009). Pol activity is specific to N2-dG lesions in both animals and plants, while plants do not have a Pol homologue (Garcia-Diaz & Bebenek, 2007; Garcia-Ortiz et al., 2007).

Homologues of REV1, REV3 and REV7 as well as Pol have been identified in *Arabidopsis* (Sakamoto et al., 2003; Santiago et al., 2006; Takahashi et al., 2005). Mutant alleles of *REV3, REV1* and *REV7* all result in UV sensitivity, although *rev1* only weakly and *rev7* only to long-term UV exposure (Sakamoto et al., 2003; Takahashi et al., 2005). Interestingly, AtREV1 cannot insert nucleotides across from UV-induced DNA lesions, suggesting that its role is primarily in the recruitment of other TLS polymerases (Takahashi et al., 2007). The UV sensitivity of the *rev1 rev3* double mutant is similar to that of *rev3*, additional evidence that the role of REV1 may be in REV3 recruitment (Takahashi et al., 2005). Analysis of mutation frequency in *rev1* and *rev3* mutants shows a reduction relative to wildtype rate, evidence that these genes contribute to error-prone repair (Nakagawa et al., 2011).

Pol is encoded by the *Arabidopsis POLH* gene*.* The *POLH* transcript is ubiquitously expressed and alternatively spliced, and AtPol exhibits similar levels of activity *in vitro* as the human protein (Hoffman et al., 2008; Santiago et al., 2008a, 2009). *AtPOLH* overexpression results in increased UV tolerance, while *AtPOLH* loss of function results in weak UV sensitivity, but enhances the sensitivity of the *rev3* mutant, evidence that Pol is acting independently of Pol (Curtis & Hays, 2007; Santiago et al., 2008b). *POLH* mutants

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

Despite the available repair mechanisms, there are times when UV damage persists and DNA replication proceeds past UV lesions via translesion synthesis (TLS). This synthesis proceeds via one of several DNA polymerases: Pol, Rev1, Pol, Pol or Pol. Pol (eta) is encoded by the *POLH* locus in humans and is homologous to yeast RAD30. Pol performs error-free bypass of CPDs, and loss of function results in increased mutation and xeroderma pigmentosa (XP-V) in humans. REV1 exhibits dCMP transferase activity but is required for 6-4 PP bypass independent of this activity, suggesting its role may be in recruitment of other polymerases. Pol is composed of REV3 and REV7 and exhibits error prone repair. REV3 is a B-family polymerase (unlike the other TLS polymerases which are all Y-family polymerases) and REV7 an accessory subunit that enhances REV3 activity (Waters et al., 2009). Pol activity is specific to N2-dG lesions in both animals and plants, while plants do not have a

Homologues of REV1, REV3 and REV7 as well as Pol have been identified in *Arabidopsis* (Sakamoto et al., 2003; Santiago et al., 2006; Takahashi et al., 2005). Mutant alleles of *REV3, REV1* and *REV7* all result in UV sensitivity, although *rev1* only weakly and *rev7* only to long-term UV exposure (Sakamoto et al., 2003; Takahashi et al., 2005). Interestingly, AtREV1 cannot insert nucleotides across from UV-induced DNA lesions, suggesting that its role is primarily in the recruitment of other TLS polymerases (Takahashi et al., 2007). The UV sensitivity of the *rev1 rev3* double mutant is similar to that of *rev3*, additional evidence that the role of REV1 may be in REV3 recruitment (Takahashi et al., 2005). Analysis of mutation frequency in *rev1* and *rev3* mutants shows a reduction relative to wildtype rate, evidence

Pol is encoded by the *Arabidopsis POLH* gene*.* The *POLH* transcript is ubiquitously expressed and alternatively spliced, and AtPol exhibits similar levels of activity *in vitro* as the human protein (Hoffman et al., 2008; Santiago et al., 2008a, 2009). *AtPOLH* overexpression results in increased UV tolerance, while *AtPOLH* loss of function results in weak UV sensitivity, but enhances the sensitivity of the *rev3* mutant, evidence that Pol is acting independently of Pol (Curtis & Hays, 2007; Santiago et al., 2008b). *POLH* mutants

Pol homologue (Garcia-Diaz & Bebenek, 2007; Garcia-Ortiz et al., 2007).

that these genes contribute to error-prone repair (Nakagawa et al., 2011).

the CUL4-DDB1-CSA and CSB pathway in plants.

**4. Translesion synthesis** 

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., 2008).

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 translesion synthesis.
