The copper chaperone as a dual regulator of effects related to oxidative stress and chromatin remodeling

The goal of this thesis was to understand the biological roles of the CCS copper chaperone. CCS is known to activate Cu/Zn superoxide dismutase (SOD1), although the precise mechanism is unclear. Guided by published three-dimensional structures of CCS, we used site directed mutagenesis in Chapter 2 to explore CCS activation of SOD1. The N-terminal Domain I of CCS contains a CXXC motif that is believed to coordinate copper together with a CXC motif at the C-terminal Domain 3. Surprisingly, however, we observed that only the CXC cysteines of yeast Ccs1p Domain 3 are required for Sod1p activation; the CXXC site of Domain 1 is not. Moreover, mutations expected to disturb a flexible linker region in Domain 1 also had no effect on yeast Sod1p activation. The Domain 1 CXXC site is clearly not as critical as once thought. CCS Domain 2 docks with SOD1, and we examined specific structural differences between yeast and human Domain 2, namely the disulfide cysteines of human CCS and the extended loop region of yeast Ccs1p. Neither was found necessary for SOD1 activation under physiological conditions but may become important stabilizing features under certain types of stress. Lastly, Chapter 2 addresses the CCS-SOD1 docked complex. The crystal structure of the yeast Ccs1p-Sod1p complex curiously revealed a heterotetramer rather than heterodimer and an unexpected intermolecular disulfide between CCS and SOD1. We probed these findings by mutagenesis and observed that residues expected to promote CCS-CCS interactions in the heterotetramer, as well as a lysine predicted to stabilize the CCS-SOD1 intermolecular disulfide, were all important for SOD1 activity. The crystal structure may in fact represent an accurate snap shot of CCS in action.;Chapter 3 addresses a novel role of yeast Ccs1p, namely in chromatin silencing. Previous microarray analysis in the Culotta laboratory noted that loss of Ccs1p in yeast is associated with de-repression of the HMR mating type and subtelomeric "silenced" loci. Here we confirm these findings by RT and real time PCR and demonstrate that the third silenced loci in yeast, the rDNA, is also de-repressed in ccs1Delta strains. Moreover, de-repression of these silenced loci is not observed in sod1Delta strains and loss of rDNA silencing in ccs1Delta strains is retained in an anaerobic environment. Hence, the effects of ccs1Delta mutations on chromatin silencing are independent of SOD1 and oxidative stress.;In Chapter 4, we explore possible mechanisms by which loss of Ccs1p affects chromatin silencing. The NAD dependent histone deacetylase Sir2p of yeast is known to mediate silencing of all three loci, however, our studies failed to demonstrate an effect of ccs1Delta mutations on Sir2p protein levels or enzymatic activity. Yet during the course of these studies, we uncovered a new effect of ccs1Delta mutations on chromatin remodeling that is dependent on oxidative stress. We demonstrate that in cells lacking either CCS1 or SOD1, the histone 3 lysine 56 (H3 K56) remains hyperacetylated at the G2/M phase of the cell cycle, similar to what is observed in cells lacking the histone deactylase HST3. Overall these studies help establish new roles for CCS in chromatin remodeling that are both independent (silenced loci) and dependent (H3 K56 aceylation) of oxidative stress. Possible mechanisms are discussed herein.