"Studies to examine Centrin's role in DNA repair"

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DESCRIPTION (provided by applicant): Rad23 and Rad4 are DNA repair proteins that function in nucleotide excision-repair (NER). A complex consisting of Rad23 and Rad4 (termed NEF2), contains a third protein called Centrin (Cdc31), whose function in NER is unknown. The characterization of Rad4 and Centrin is the focus of this investigation. Rad23 regulates Rad4 stability by preventing its degradation by the proteasome. Similarly, Centrin increases the abundance of human Xpc (Rad4 ortholog). In yeast Centrin levels were dramatically altered by growth conditions, and a coincident change in Rad4 abundance was observed. By binding and regulating the stability of Rad4, Centrin could influence the availability of the NEF2 complex for DNA repair. Furthermore, Rad4 might itself alter Centrin's role in cell-cycle control. Centrin controls mitotic growth by promoting spindle-pole body duplication (SPB), which initiates cell cycle entry. Our hypothesis is that Centrin prevents SPB duplication following DNA damage to initiate growth arrest and promote DNA repair. We determined that Centrin binds mitochondrial proteins and influences ATP levels. A Centrin mutant with increased binding to mitochondrial proteins showed higher ATP levels. One such mutant that failed to bind Rad4 was sensitive to UV light, revealing a potential link between mitochondrial function and NER. The following genetic and biochemical studies are proposed. We will determine if i) yeast Centrin stabilizes Rad4 ii), and if this interaction is affected by DNA damage. iii) Rad4 stability is also regulated by Rad23, and studies are proposed to examine Rad23/Rad4 interaction. iv) The interaction of Centrin mutant proteins with Rad4, assembly of NEF2, and binding to mitochondrial proteins will be examined. v) The subcellular distribution of Centrin and duplication of the spindle pole-body will be characterized after DNA damage, and in the presence of mutant Centrin and Rad4 proteins. Defects in the nucleotide excision repair pathway can cause xeroderma pigmentosum, a cancer- prone condition that can lead to early death. Defining the role of Centrin will allow us to determine how growth control mechanisms contribute to efficient DNA repair. Defective DNA repair and failure to arrest cell growth is a major cause of genome instability, and is observed in many forms of malignant growth. Skin cancer is an important health concern, and shows one of the fastest incidences of increase in the US population. Nucleotide excision repair plays a central role in the removal of sunlight-light induced DNA damage, and an important protein in this cellular defense mechanism is Rad4. However, the function of Rad4 is not well understood, and its characterization is the focus of this application.