**8. Conclusion**

We have demonstrated an in vitro inactivation of *BRCA1*/BRCA1 by the anticancer platinum drug cisplatin. The transcriptional activation of cisplatin-modified *BRCA1*, when tested in a "one-hybrid GAL4 transcriptional assay", was inversely proportional to cisplatin doses and was dramatically diminished in the presence of a second expression vector containing multiple cisplatin-damaged sites. This indicates a repair-mediated transcriptional transactivation of cisplatin-damaged *BRCA1* as well as the lack or unavailability of cellular transcription factors at cisplatin-*BRCA1* lesions. The BRCA1 protein contained a preformed structure in the apo-form with structural changes and more resistance to limited proteolysis after Zn2+ binding. Cisplatin-bound protein exhibited an enhanced thermostability, resulting from the favourable intermolecular crosslinks driven by the free energy. Only the apo-form, not the holo-form, of BRCA1 underwent a more folded structural rearrangement with the retention of protein structure

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upon cisplatin binding with the preferential His117 site of the BRCA1 peptide 111Glu-Asn-Asn-Ser-Pro-Glu-His-Leu-Lys119. BRCA1 E3 ubiquitin ligase activity was also inactivated by the drug. These data could raise the possibility of selectively targeting the BRCA1 DNA repair for cisplatin in cancer chemotherapy.

As mentioned earlier, the BRCA1-BARD1 RING complex has an E3 ubiquitin ligase function that plays essential roles in response to DNA damage and DNA repair. Evidence from several preclinical and clinical studies have provided data showing that many cancerpredisposing mutations within the BRCA1 RING domain demonstrated a loss of ubiquitin ligase and repair of DNA double-strand break activities (Atipairin, et al., 2011a, 2011b; Morris et al., 2006, 2009; Ransburgh et al., 2010). Furthermore, the BRCA1-associated cancers conferred a hypersensitivity to ionizing radiations and chemotherapeutic agents. Therefore, it would be of great interest to identify a relationship between BRCA1-mediated ubiquitination and chemosensitivity by approaching the BRCA1 RING domain as a potentially molecular target or predictor with cisplatin.

#### **9. Acknowledgments**

The author would like to thank all the members of BRCA1 lab for discussion and comments. This work was financial supported by grants of the Synchrotron Light Research Institute (Public organization) (1-2548/LS01), the National Research Council of Thailand (02011420- 0003, PHA530097S), Prince of Songkla University (PHA530188S), and Dr. Brian Hodgson for the proof reading and assistance with the English.

#### **10. References**


upon cisplatin binding with the preferential His117 site of the BRCA1 peptide 111Glu-Asn-Asn-Ser-Pro-Glu-His-Leu-Lys119. BRCA1 E3 ubiquitin ligase activity was also inactivated by the drug. These data could raise the possibility of selectively targeting the BRCA1

As mentioned earlier, the BRCA1-BARD1 RING complex has an E3 ubiquitin ligase function that plays essential roles in response to DNA damage and DNA repair. Evidence from several preclinical and clinical studies have provided data showing that many cancerpredisposing mutations within the BRCA1 RING domain demonstrated a loss of ubiquitin ligase and repair of DNA double-strand break activities (Atipairin, et al., 2011a, 2011b; Morris et al., 2006, 2009; Ransburgh et al., 2010). Furthermore, the BRCA1-associated cancers conferred a hypersensitivity to ionizing radiations and chemotherapeutic agents. Therefore, it would be of great interest to identify a relationship between BRCA1-mediated ubiquitination and chemosensitivity by approaching the BRCA1 RING domain as a

The author would like to thank all the members of BRCA1 lab for discussion and comments. This work was financial supported by grants of the Synchrotron Light Research Institute (Public organization) (1-2548/LS01), the National Research Council of Thailand (02011420- 0003, PHA530097S), Prince of Songkla University (PHA530188S), and Dr. Brian Hodgson

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**13** 

*USA* 

*Saccharomyces cerevisiae* **as a Model** 

*Department of Biochemistry and Molecular Biology, University of Miami* 

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

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

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

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

**1. Introduction** 

initiating NER in chromatin.

CPDs, as reviewed by Smerdon (Smerdon, 1991).

damage (Zamble et al., 1996; Setlow, 2001; Wang, 2008).

**System to Study the Role of Human** 

**DDB2 in Chromatin Repair** 

Kristi L. Jones, Ling Zhang and Feng Gong

