Journal:

Article Title: The silencing complex SAS-I links histone acetylation to the assembly of repressed chromatin by CAF-I and Asf1 in Saccharomyces cerevisiae

doi: 10.1101/gad.929001

Figure Lengend Snippet: (A) cac1Δ and sas2Δ were epistatic in HML silencing in sir1Δ strains. Stains used in a patch mating assay were MATa HMLα and sir1Δ cac1Δ (AEY2204), sir1Δ sas2Δ (AEY346), sir1Δ hir1Δ (AEY2236), sir1Δ sas2Δ cac1Δ (AEY2205), sir1Δ sas2Δ hir1Δ (AEY2228), sir1Δ cac1Δ hir1Δ (AEY2234), sir1Δ sas2Δ cac1Δ hir1Δ (AEY2230), and sas2Δ cac1Δ (AEY2206). MATα his4 as a mating tester. (B) cac2Δ and cac3Δ did not cause additional derepression in sir1Δ sas2Δ strains. The a mating ability of MATa strains AEY1290 (sir1Δ sas2Δ), AEY2205 (sir1Δ sas2Δ cac1Δ), AEY2508 (sir1Δ sas2Δ cac2Δ), and AEY2479 (sir1Δ sas2Δ cac3Δ) is shown. (C) sas2Δ and cac1Δ had additive effects on HMR repression. A colony color assay was used to assay repression of the ADE2 gene inserted at HMR. Yeast cells were incubated on YM plates supplemented with 10% of the normal adenine concentration, WT (AEY1676), sas2Δ (AEY2481), cac1Δ (AEY2483), and sas2Δ cac1Δ (AEY2486). (D) The α-factor response of MATa HMLα strains was measured by spreading them on complete medium plates containing 40 μg/mL α-factor and incubating them at 23°C. Approximately 100 cells per strain were scored after 17 h. Schmoo indicates individual cells that formed mating projections and remained arrested; schmoo cluster, individual cells that formed multiple mating projections and eventually divided at least once; and colony, cells that formed colony of round cells and did not appear to respond to α-factor. WT (AEY2), sas2Δ (AEY266), sir1Δ (AEY345), cac1Δ (AEY1403), sas2Δ cac1Δ (AEY2206), asf1Δ (AEY2430), sas2Δ asf1Δ (AEY2429), asf1Δ cac1Δ (2452), and sas2Δ asf1Δ cac1Δ (2491). (E) CAF-I activity was not altered in sas2Δ strains. (Left) CAF-I activity was immunoprecipitated with myc–Cac2. Immunoprecipitates from AEY1558 or AEY1808 transformed with pAE716 (myc–Cac2) were used in SV40 in vitro replication assays to determine nucleosome assembly activity. The autoradiogram (top) of the ethidium bromide stained gel (EtBr, bottom) shows increased negative supercoiling of replicated plasmid DNA on addition of myc–Cac2 immunoprecipitate from a wild-type strain (WT). In the absence of Cac1 (cac1Δ), immunoprecipitated myc–Cac2 was unable to increase supercoiling. (right) CAF-I activity was unaffected by the absence of Sas2. Increasing amounts of myc–Cac2 immunoprecipitates from wild-type (WT) or sas2Δ∷TRP1 strains (sas2Δ) were added to in vitro SV40 replication reactions and assayed as above.

Article Snippet: SV40 DNA replication coupled chromatin assembly assay SV40 based DNA replication reactions with HeLa extract, SV40 large T antigen, pUC-HSO plasmid DNA, and [ 32 P]dCTP were purchased from Chimerx and performed according to the manual instructions, essentially as described ( Stillman 1986 ).

Techniques: Incubation, Concentration Assay, Activity Assay, Immunoprecipitation, Transformation Assay, In Vitro, Staining, Plasmid Preparation