**1. Introduction**

230 DNA Repair

Zhong, Q., Boyer, T.G., Chen, P.L. & Lee, W.H. (2002). Deficient nonhomologous end-joining

Zhu, Y., Hub, J., Hu, Y. & Liu, W. (2009). Targeting DNA repair pathways: a novel approach to reduce cancer therapeutic resistance. *Cancer Treat Rev.* 35(7): 590-596.

3970.

activity in cell-free extracts from Brca1-null fibroblasts. *Cancer Res.* 62(14): 3966-

Genetic and biochemical studies in *Saccharomyces cerevisiae* have made major contributions in elucidating the mechanism of several DNA repair pathways, including the nucleotide excision repair (NER) pathway that remove bulky DNA damage from the genome. Although NER is conserved from yeast to humans, there are differences in NER between yeast and humans. For example, no homolog of the human NER factor DNA damagebinding protein 2 (DDB2) has been identified in the budding yeast *S. cerevisiae*. Here, we present evidence suggesting that *S. cerevisiae* can be used to dissect the roles of DDB2 in initiating NER in chromatin.

Ultraviolet light (UV) is a well studied genotoxic stress that induces bulky DNA damage. These UV lesions are repaired by the NER pathway (Hanawalt, 2002; Sancar & Reardon, 2004). The particular lesions induced by UV irradiation have been characterized, namely, cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs). Both lesions result in the distortion of the DNA double helix, but 6-4PPs result in a greater distortion. Additionally, there are other minor differences between the two types of lesions. CPDs have been consistently shown to have higher incidence than 6-4PPs (Douki & Cadet, 2001). CPDs are induced both in nucleosome core and linker DNA, whereas 6-4PPs are formed with 6 fold greater frequency in linker DNA. In addition, 6-4PPs are repaired much faster than CPDs, as reviewed by Smerdon (Smerdon, 1991).

In humans, a defect in NER results in xeroderma pigmentosum (XP) and several other rare diseases (Kraemer et al., 2007). XP patients are extremely sensitive to UV light and have about 2000-fold higher incidence of sunlight induced skin cancers than the general population. NER lesion recognition is via protein interaction with the structural DNA changes that are induced. Other bulky DNA lesions repaired by NER include those induced by cigarette smoke, cisplatin treatment and a newly identified form of bulky oxidative DNA damage (Zamble et al., 1996; Setlow, 2001; Wang, 2008).

NER has been extensively studied and the basic mechanism is understood. It consists of three main steps: 1) lesion detection, 2) dual incision to remove an oligonucleotide containing the lesion and 3) repair synthesis to fill the gap. There are two sub-pathways of NER, termed transcription coupled repair (TC-NER) and global genome repair (GG-NER) (Hanawalt, 2002). TC-NER is responsible for repair of damage on the actively transcribed

*Saccharomyces cerevisiae* as a Model System to

mechanisms of NER operation in the context of chromatin.

2009; Waters et al., 2009; Zhang et al., 2009a; Jones et al., 2010).

role(s) of ubiquitination in DDB2-mediated GG-NER.

**2.1 Galactose induced expression of DDB2 in** *S. cerevisiae*

**chromatin** 

Study the Role of Human DDB2 in Chromatin Repair 233

Additionally, DDB2 is in complex with the E3 ubiquitin ligase complex consisting of DDB1, Cul4 (CULlin 4) and ROC (Ring Of Cullins) (Jackson & Xiong, 2009). E3 ubiquitin ligases transfer ubiquitin to the target protein. DDB2 is thought to be the substrate receptor targeting the E3 ubiquitin ligase complex to DNA lesion sites to facilitate GG-NER. Of note, DDB1 and Cul4 have been shown to be in complex with other proteins, including CSA, a TC-NER specific protein (Jackson & Xiong, 2009). Consistent with its classification as an E3 ubiquitin ligase, XPC, histone H2A, H3, H4, and DDB2 itself have been identified as UVdependent ubiquitination targets of the DDB1-DDB2 E3 ligase complex (Chen et al., 2001; Nag et al., 2001; Matsuda et al., 2005; Sugasawa et al., 2005; Kapetanaki et al., 2006; Wang et al., 2006). The UV-dependent mono-ubiquitination of histone H2A has been suggested to be involved in both chromatin relaxation and restoration (Kapetanaki et al., 2006; Zhu et al., 2009). Clearly, understanding the role of DDB2 in NER will yield important insights into the

Chromatin is a hierarchal structure composed of DNA and protein. The core component is the nucleosome. It is a complex of 147 base pairs of DNA wrapped around the core histone octamer. The core histone octamer consists of four subunits, H2A, H2B, H3 and H4 in a 2:2:2:2 ratio (Luger et al., 1997; Kornberg & Lorch, 1999). The innate structure of chromatin restricts DNA protein interactions. ATP-dependent chromatin reconfiguration is an important mechanism to alleviate this tight association. Several groups have demonstrated a requirement for the ATP-dependent chromatin remodeling in chromatin repair (Jiang et al., 2010; Gong et al. 2006; Zhang et al. 2009a; Zhang et al. 2009b; Zhao et al. 2009; Lans et al. 2010; Sarkar et al. 2010). How DNA repair occurs in chromatin is an emerging question and has been discussed in several recent review articles (Osley et al., 2007; Nag & Smerdon,

**2.** *S. cerevisiae* **as a model system to study DDB2-mediated GG-NER in** 

It has been demonstrated that DDB2 is the initial lesion detection factor in GG-NER (Tang et al., 2000; Wakasugi et al., 2002; Fitch et al., 2003b; Pines et al., 2009). Although it has been implicated in the recruitment of XPC to CPD sites (Fitch et al., 2003b); how DDB2 transfers these identified lesions to XPC remains controversial. It is believed that ubiquitination of DDB2 leads to its degradation at damage sites and this degradation is required for CPD repair. However, there are several lines of evidence disputing this model, including: 1) inhibition of ubiquitination-mediated DDB2 degradation in mouse via Cul4a ablation enhances CPD repair (Liu et al., 2009), 2) DDB2 degradation is not stimulated by either DNA binding or XPC association (Luijsterburg et al., 2007), and 3) crystal structures suggest that DDB2 and XPC can co-localize on the lesion (Min & Pavletich, 2007; Scrima et al., 2008). Therefore, we try to explore the budding yeast as a simplified, alternative model system to begin to dissect the

As discussed in the introduction, DDB2 has no homolog in budding yeast. However, conservation of the GG-NER pathway and interacting partners such as DDB1 are known (Zaidi et al., 2008). Therefore, we hypothesized that DDB2 would act in a physiological relevant manner in budding yeast GG-NER. We first cloned the DDB2 gene into a low copy number, galactose inducible yeast expression vector. The cloning results in a fusion protein;

strand; while GG-NER is responsible for repair in the remainder of the genome, including lesions on the non-transcribed strand of actively transcribed genes, as well as those in repressed or silent chromatin regions. Both TC-NER and GG-NER consist of all three steps, but, they differ in the lesion recognition step. In TC-NER the lesion is thought to be detected by pausing of RNA polymerase I or II (Conconi et al., 2002; Hanawalt, 2002; Fousteri & Mullenders, 2008). GG-NER, on the other hand, requires a specific lesion recognition hetero-dimeric protein complex, XPC-hRad23 (Xeroderma Pigmentosum complementation group C-human Rad23) in humans and Rad4-Rad23 (RADiation sensitive) in budding yeast (Wood 2010; Guzder et al., 1998; Jansen et al., 1998; Sugasawa, 2009). However, under certain *in vivo* circumstances, DDB2 is the pioneering damage recognition factor during GG-NER (Hwang et al., 1999; Nichols et al., 2000; Sugasawa, 2009). So far, no DDB2 homolog has been identified in the budding yeast (Fig. 1). Of note, the Rad16-Rad7 heterodimer, without a known human homolog, is required for GG-NER in the budding yeast.


Fig. 1. Conservation of NER pathway between humans and the budding yeast *S. cerevisiae*. Of note, no DDB2 counterpart has been identified in *S. cerevisiae.* Likewise, humans don't have a homolog of the Rad16-Rad7 heterodimer that is essential for GG-NER in *S. cerevisiae*.

Several lines of evidence suggest that DDB2 plays a key role in **chromatin repair** of UV damage. It has been shown that DDB2 is responsible for the lesion detection by directly interacting with the damaged DNA (Tang, et al., 2000; Scrima et al., 2008). Additionally, DDB2 binds the lesion independent of XPC (Wakasugi et al., 2002). DDB2 can co-localize with both CPDs and 6-4 PPs *in vivo*, while XPC seems to bind 6-4 PPs efficiently, but not CPDs. This suggests the necessity of DDB2 in GG-NER is specific for CPD repair (Fitch et al., 2003). Importantly, it has been suggested that the observed high affinity of DDB2 for 6-4PPs aids in the targeting of XPC to 6-4PPs when low levels of damage are present (Nishi et al., 2009).

strand; while GG-NER is responsible for repair in the remainder of the genome, including lesions on the non-transcribed strand of actively transcribed genes, as well as those in repressed or silent chromatin regions. Both TC-NER and GG-NER consist of all three steps, but, they differ in the lesion recognition step. In TC-NER the lesion is thought to be detected by pausing of RNA polymerase I or II (Conconi et al., 2002; Hanawalt, 2002; Fousteri & Mullenders, 2008). GG-NER, on the other hand, requires a specific lesion recognition hetero-dimeric protein complex, XPC-hRad23 (Xeroderma Pigmentosum complementation group C-human Rad23) in humans and Rad4-Rad23 (RADiation sensitive) in budding yeast (Wood 2010; Guzder et al., 1998; Jansen et al., 1998; Sugasawa, 2009). However, under certain *in vivo* circumstances, DDB2 is the pioneering damage recognition factor during GG-NER (Hwang et al., 1999; Nichols et al., 2000; Sugasawa, 2009). So far, no DDB2 homolog has been identified in the budding yeast (Fig. 1). Of note, the Rad16-Rad7 heterodimer, without a known human homolog, is required for GG-NER

Fig. 1. Conservation of NER pathway between humans and the budding yeast *S. cerevisiae*. Of note, no DDB2 counterpart has been identified in *S. cerevisiae.* Likewise, humans don't have a homolog of the Rad16-Rad7 heterodimer that is essential for GG-NER in *S. cerevisiae*. Several lines of evidence suggest that DDB2 plays a key role in **chromatin repair** of UV damage. It has been shown that DDB2 is responsible for the lesion detection by directly interacting with the damaged DNA (Tang, et al., 2000; Scrima et al., 2008). Additionally, DDB2 binds the lesion independent of XPC (Wakasugi et al., 2002). DDB2 can co-localize with both CPDs and 6-4 PPs *in vivo*, while XPC seems to bind 6-4 PPs efficiently, but not CPDs. This suggests the necessity of DDB2 in GG-NER is specific for CPD repair (Fitch et al., 2003). Importantly, it has been suggested that the observed high affinity of DDB2 for 6-4PPs aids in the targeting of XPC to 6-4PPs when low levels of damage are present

in the budding yeast.

(Nishi et al., 2009).

Additionally, DDB2 is in complex with the E3 ubiquitin ligase complex consisting of DDB1, Cul4 (CULlin 4) and ROC (Ring Of Cullins) (Jackson & Xiong, 2009). E3 ubiquitin ligases transfer ubiquitin to the target protein. DDB2 is thought to be the substrate receptor targeting the E3 ubiquitin ligase complex to DNA lesion sites to facilitate GG-NER. Of note, DDB1 and Cul4 have been shown to be in complex with other proteins, including CSA, a TC-NER specific protein (Jackson & Xiong, 2009). Consistent with its classification as an E3 ubiquitin ligase, XPC, histone H2A, H3, H4, and DDB2 itself have been identified as UVdependent ubiquitination targets of the DDB1-DDB2 E3 ligase complex (Chen et al., 2001; Nag et al., 2001; Matsuda et al., 2005; Sugasawa et al., 2005; Kapetanaki et al., 2006; Wang et al., 2006). The UV-dependent mono-ubiquitination of histone H2A has been suggested to be involved in both chromatin relaxation and restoration (Kapetanaki et al., 2006; Zhu et al., 2009). Clearly, understanding the role of DDB2 in NER will yield important insights into the mechanisms of NER operation in the context of chromatin.

Chromatin is a hierarchal structure composed of DNA and protein. The core component is the nucleosome. It is a complex of 147 base pairs of DNA wrapped around the core histone octamer. The core histone octamer consists of four subunits, H2A, H2B, H3 and H4 in a 2:2:2:2 ratio (Luger et al., 1997; Kornberg & Lorch, 1999). The innate structure of chromatin restricts DNA protein interactions. ATP-dependent chromatin reconfiguration is an important mechanism to alleviate this tight association. Several groups have demonstrated a requirement for the ATP-dependent chromatin remodeling in chromatin repair (Jiang et al., 2010; Gong et al. 2006; Zhang et al. 2009a; Zhang et al. 2009b; Zhao et al. 2009; Lans et al. 2010; Sarkar et al. 2010). How DNA repair occurs in chromatin is an emerging question and has been discussed in several recent review articles (Osley et al., 2007; Nag & Smerdon, 2009; Waters et al., 2009; Zhang et al., 2009a; Jones et al., 2010).
