**3. Conclusion**

236 DNA Repair

A B

Another mutation that affects DDB2's function prevents the interaction with its in vivo partner DDB1 (Nichols et al. 2000). This mutation was also constructed and is a complex mutation, consisting of both a deletion of amino acid 349 and a proline substitution for leucine at amino acid 350 (DDB2 L350P) (Fig. 4B). Like DDB2 K244E, this mutation also abrogated DDB2's ability to suppress UV sensitivity in *∆rad26* cells (Fig. 5). These data suggest DDB2-conferred UV resistance is dependent on a conserved interacting partner.

Fig. 5. DDB2 mutations and deletion of Mms1 (DDB1 homolog) abrogate suppression of UV

Although Mms1 has been identified as the budding yeast DDB1 homolog (Zaidi et al., 2008), there are no reports of it being involved in NER. However, our previous observation suggesting DDB2 function requires a conserved interacting partner prompted us to test DDB2 function in the absence of Mms1. To test this, wild type DDB2 was expressed in the *∆rad26∆mms1* double mutant and UV sensitivity was accessed by spotting assays. Indeed, this reciprocal experiment verified that Mms1 is necessary for DDB2-dependent suppression

Fig. 4. Crystal structure of DDB2 mutations modified from crystal structure solved by Scrima et al. (A) Lysine to glutamic acid substitution at aa 244 predicted to effect DDB2 DNA interaction. Red residue indicates site of mutation. Yellow indicates damaged DNA strand. (B) Deletion of aa 349 and substitution of proline for leucine at aa 350. This mutation is predicted to effect the DDB2 DDB1 interaction. Red indicates site of mutation. Mutant

DDB2 was constructed by site directed mutagenesis.

sensitivity in *∆rad26* cells.

of UV sensitivity (Fig. 5).

Studies in *Saccharomyces cerevisiae* have made major contributions to our understanding of NER. Here, we present evidence suggesting that *S. cerevisiae* can be used to dissect the roles of human DDB2 in initiating NER in chromatin. Since DDB2 functions are regulated by the ubiquitin pathway and DDB2 itself is a component of an E3 ligase, it will be interesting to explore the regulation of DDB2 functions by ubiquitination, using yeast mutants with defects in various steps of the ubiquitin pathway.

Ubiquitination is a well studied post-translational modification and recent data suggest multiple fates of ubiquitin modified proteins (Sadowski & Sarcevic, 2010). It will be important to determine if ubiquitination of DDB2 promotes its degradation or controls DDB2 association with chromatin. The budding yeast system described here will also provide an alternative system to screen the effect(s) of various DDB2 lysine mutations to determine which amino acid residue(s) is modified. Additionally, as reviewed by Kirkin et al., ubiquitin signaling is altered in many cancers (Kirkin and Dikic 2010), suggesting a potential role of ubiquitination in regulating DNA binding proteins such as transcription factors and repair proteins. Therefore, it will be interesting to determine what, if any, role ubiquitination plays in the chromatin association of other DNA binding proteins, specifically transcription factors and repair proteins. The utilization of the budding yeast model system will facilitate deciphering such questions.
