Targeting of SIR1 protein establishes transcriptional silencing at HM loci and telomeres in yeast

Chien, Ching‐Te; Buck, Stephen W.; Sternglanz, Rolf; Shore, David

doi:10.1016/0092-8674(93)90387-6

Cited by 191 publications

(225 citation statements)

References 56 publications

Supporting

Mentioning

213

Contrasting

Unclassified

Order By: Relevance

“…E. coli strain BL21 (DE3) transformed with pET28b-(His) 6 -Sir1p-OIR* was grown in LB medium supplemented with 100 g͞ml kanamycin at 37°C. At an OD 600 of Ϸ0.5, (His) 6 -OIR* expression was induced with 1 mM isopropyl ␤-D-thiogalactopyranoside for 3 h. Cells were harvested by centrifugation, frozen at Ϫ80°C, resuspended at 4°C in lysis buffer (50 mM Hepes, pH 6.5͞500 mM NaCl͞10% glycerol͞20 mM imidazole), supplemented with protease inhibitors and DNaseI, and lysed by…”

Section: Methodsmentioning

confidence: 99%

“…The sequence of S. cerevisiae SIR1 that encodes the Sir1p-OIR (amino acids 480-611 of Sir1p) was amplified by PCR and introduced into the NheI site of pET28b (Novagen), creating pET28b-(His) 6 -Sir1p-OIR for expression of (His) 6 -OIR. A mutation that replaces the Cys-593 codon with an Ala codon was made in pET28b-(His) 6 -Sir1p-OIR by using QuikChange mutagenesis (Stratagene), creating pET28b-(His) 6 -Sir1p-OIR*.…”

Section: Methodsmentioning

confidence: 99%

“…The extract, after clearing by centrifugation (38,000 ϫ g for 15 min), was incubated with Ni-NTA beads (Novagen) for 1 h at 4°C and packed into a column (settled bed volume of 2 ml). The resin was washed with 10 column volumes of lysis buffer, and (His) 6 -OIR* was eluted with elution buffer (lysis buffer with 500 mM imidazole). Eluted (His) 6 -OIR* was buffer-exchanged into lysis buffer with 50 units of thrombin to remove the (His) 6 tag.…”

Section: Methodsmentioning

confidence: 99%

“…The resin was washed with 10 column volumes of lysis buffer, and (His) 6 -OIR* was eluted with elution buffer (lysis buffer with 500 mM imidazole). Eluted (His) 6 -OIR* was buffer-exchanged into lysis buffer with 50 units of thrombin to remove the (His) 6 tag. The cleaved protein was then passed over a Ni-NTA column a second time, and unbound protein was dialyzed into 50 mM Hepes, pH 6.5͞50 mM NaCl͞10% glycerol and loaded onto a SP-Sepharose column.…”

Section: Methodsmentioning

confidence: 99%

“…Analyses of HMR-E, a silencer that is necessary and sufficient to target silent chromatin to HMR, has established that the primary role for ORC in silencing is to recruit Sir1p to the silencer through direct protein-protein interactions between the Orc1p subunit of ORC and Sir1p (6)(7)(8)(9)(10)(11). ORC-bound Sir1p nucleates and͞or stabilizes a silent chromatin domain through direct protein-protein interactions with other Sir proteins, in particular Sir4p (7,11).…”

mentioning

confidence: 99%

See 4 more Smart Citations

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

Hou

Bernstein

Fox

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

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

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

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

Hou

Bernstein

Fox

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

Silent chromatin in yeast: an orchestrated medley featuring Sir3p

Stone

Pillus

1998

Bioessays

View full text Add to dashboard Cite

Extensive regions of chromosomes can be transcriptionally repressed through silencing mechanisms mediated by complex chromatin structures. One of the most refined molecular portraits of silenced chromatin comes from studies of the silent mating‐type loci and telomeres of S. cerevisiae. In this budding yeast, the Sir3p silent information regulator emerges as a critically important silencing component that interacts with nucleosomes and other silencing proteins. Not only is it essential for silencing, but Sir3p is also capable of spreading silenced chromatin when its dosage is increased. Sir3p is a target of mitogen‐activated protein (MAP) kinase cascade regulation and has significant similarity to the Orc1p subunit of the DNA replication origin recognition complex. Thus, in concert with other silencing proteins, Sir3p appears poised to respond to cellular signals and reprogram silencing through replication‐associated assembly of repressive chromatin structures. BioEssays 20:30–40, 1998. © 1998 John Wiley & Sons, Inc.

show abstract