Interaction analysis of the AAA+ domain of the heterochromatin protein Sir3

The silent information regulator 2/3/4 (SIR) complex is an archetypal heterochromatin complex required for gene silencing at the silent mating-type loci and the telomeres in the yeast Saccharomyces cerevisiae. The Sir3 component consists of a N-terminal bromo-adjacent homology (BAH) domain and a C-terminal AAA+ ATPase-like domain. In a recent study, we have determined the crystal structure of the AAA+ domain and, through an extensive mutational analysis, have identified several surface regions on this domain required for Sir3 silencing function, one of which we have shown represents a site of interaction with Sir4. Based on these results, we will determine the interaction partners of three other surface domains using a combination of genetics, molecular biology and biochemistry. Furthermore, we will define the region on Sir4 that interacts with Sir3. This detailed molecular information about protein interactions within the SIR complex and with chromatin will be complementary to structural information on the SIR complex and will for the first time provide molecular insight into a heterochromatin complex.