Structural basis of the Sir1–origin recognition complex interaction in transcriptional silencing

Abstract

The Sir1 protein plays a key role in establishing a silent chromatin structure at the cryptic mating-type loci HMR and HML in Saccharomyces cerevisiae by interacting with the bromo-adjacent homology (BAH) domain of the Orc1p subunit of the origin recognition complex (ORC). Here, we present the high-resolution crystal structures of the ORC interaction region (OIR) of Sir1p and that of the complex formed between the OIR and BAH domains. Amino acids within the OIR previously shown to be required for a Sir1p͞ORC interaction are presented on a conserved, convex surface that forms a complementary interface with a concave region of the Orc1 BAH domain that is critical for transcriptional silencing. The OIR͞ BAH interaction surface comprises a network of hydrophobic and polar͞ionic interactions between discrete structural modules in each protein and involves several residues that were not implicated in previous studies. These data provide important structural insights into a protein-protein interaction critical for the formation of a specialized chromatin domain within eukaryotic chromosomes.chromatin ͉ silencers ͉ yeast ͉ chromosomes ͉ replication

Transcriptional silencing functions of the yeast protein Orc1/Sir3 subfunctionalized after gene duplication

“…To explore this hypothesis, we generated two point mutations in KlORC1. The P185L mutation is analogous to a mutation in ScSir3 that disrupts nucleosome binding and the ability of Sir proteins to spread (46), and the E124K mutation occurs in the H domain, which interacts with Sir1 in S. cerevisiae (26) (Fig. S4).…”

“…We focused on the nucleosome-binding BAH domain, which is essential for the silencing functions of both ScSir3 and ScOrc1 but is dispensable for DNA replication in S. cerevisiae (26,31,43). We truncated the genomic copy of KlORC1, removing the sequence encoding the first 217 amino , and KlSir2-HA (LRY2566) with the replication origin KlARS503 (previously known as KARS12) were assessed by chromatin IP followed by quantitative PCR.…”

“…The deacetylation of nucleosomes by Sir2 is thought to create high-affinity binding sites for Sir3 and Sir4, which in turn recruit additional Sir2, enabling the propagation of Sir-silenced chromatin (21)(22)(23). A fourth Sir protein, Sir1, stabilizes the other Sir proteins at silencers by interacting with Orc1 but is not thought to spread (24)(25)(26)(27).…”

“…Orc1, as part of ORC, binds silencers (16), and the BAH domain of Orc1 interacts with Sir1 (26,27) to stabilize other Sir proteins at silencers. This recruitment of Sir1 is thought to be the only function of ORC in silencing because ORC can be bypassed by tethering Sir1 to the silencer (27,35).…”

Structural basis for allosteric stimulation of Sir2 activity by Sir4 binding

“…Interestingly, an eso mutant of Sir2, which has no obvious connection to interaction with Sir4, is also located in the Sir4-interacting region of Sir2 (Garcia and Pillus 2002). It is known that establishment of a silenced chromatin domain in the mating type loci involves a cascade of protein-protein interactions, including the binding of Sir1 to the silencerbound ORC1 and the recruitment of the Sir2-Sir4 complex by the interaction between Sir1 and Sir4 (Triolo and Sternglanz 1996;Bose et al 2004;Hou et al 2005;Hsu et al 2005). Thus, a weakened or disrupted Sir2-Sir4 interaction can undermine the Sir1 function of recruiting a histone deacetylase to the targeted chromatin region, and our discovery that a Sir2 mutant affecting Sir1 function is located in the Sir4 interaction region is testimony to the network of protein-protein interaction required for transcriptional silencing in yeast.…”