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Results: 7

1.
Figure 2

Figure 2. Histone suppressors of H3 A75V.. From: Compensatory Interactions between Sir3p and the Nucleosomal LRS Surface Imply Their Direct Interaction.

(A) Disc face representation of the nucleosome 1ID3 [29] rendered using PyMOL [62]. (B) Zoom view of the LRS surface of the disc face. The DNA is represented in green, the histones are represented in wheat, the LRS residues and their side chains are highlighted in magenta, and the suppressor alleles are highlighted in cyan. (C) Telomeric ADE2 silencing with wild-type, H3 A75V and H3 A75V with histone suppressors.

Anne Norris, et al. PLoS Genet. 2008 December;4(12):e1000301.
2.
Figure 3

Figure 3. Effect of histone mutations on rDNA silencing.. From: Compensatory Interactions between Sir3p and the Nucleosomal LRS Surface Imply Their Direct Interaction.

H3 D77N but not H4 H75Y suppresses the loss of rDNA silencing phenotype of H3 A75V. JPY12 yeast strain with the histone alleles on a plasmid were plated as indicated. (A) SC−Trp for growth control and SC−URA or +FOA to measure rDNA silencing. (B) MLA plates assaying for silencing of the MET15 reporter inserted into the NTS2 of the rDNA. Dark color indicates increased silencing.

Anne Norris, et al. PLoS Genet. 2008 December;4(12):e1000301.
3.
Figure 1

Figure 1. LRS residues affect the electrostatics of the nucleosome.. From: Compensatory Interactions between Sir3p and the Nucleosomal LRS Surface Imply Their Direct Interaction.

(A) Qualitative vacuum electrostatic representation of the nucleosome 1ID3 [29] rendered using PyMOL [62]. Red is electronegative and blue is electropositive. (B) A table of residues found in several studies showing that an increase in positive charge in LRS residues leads to an increase in silencing, while a decrease in charge leads to loss of telomeric silencing. * The mutation Q76 to R was found to have an increase in spreading of silent chromatin [61]. Data was compiled from the following references [1],[2],[9],[31],[32],[61].

Anne Norris, et al. PLoS Genet. 2008 December;4(12):e1000301.
4.
Figure 5

Figure 5. SIR3 D205N and L79I specifically suppress the LRS surface telomeric silencing defect.. From: Compensatory Interactions between Sir3p and the Nucleosomal LRS Surface Imply Their Direct Interaction.

SIR3 alleles L79I and D205N were assayed for their ability to suppress the loss of telomeric silencing of LRS alleles as well as other Histone alleles not on the LRS surface that lose telomeric silencing. (A) The ADE2 reporter gene was assayed on SC−Trp. A pink color indicates silencing, whereas a lighter color indicates a loss of silencing. (B) The URA3 gene was assayed for growth by serial dilution on SC−Ura. Increased growth indicates a loss of silencing, whereas decreased growth indicates an increase in silencing. (C) JPY12 yeast strain with the SIR3 alleles on a plasmid were plated on MLA plates assaying for silencing of the MET15 reporter in the rDNA.

Anne Norris, et al. PLoS Genet. 2008 December;4(12):e1000301.
5.
Figure 4

Figure 4. SIR3 suppressor mutants define a surface of Sir3p.. From: Compensatory Interactions between Sir3p and the Nucleosomal LRS Surface Imply Their Direct Interaction.

(A) Mapping SIR3 mutants onto the crystal structure of the Sir3p BAH domain 2fl7 [35]. Mutants identified in both the EMS screen and the PCR mutagenesis of the BAH domain are represented in orange. SIR3 eso mutants are represented in green [28]. (B) An alignment of Sir3p and Orc1p BAH domains with H3 A75V and eso mutants highlighted. Many of the mutants are disordered in the crystal structure, the approximate locations of missing sections, and are represented by dashed lines. Residues important for suppression of H3 A75V are highlighted in orange. H3 A75V suppressor mutants that introduce an “Orc1p like” residue are highlighted in magenta; eso mutants are highlighted in green. Highly conserved and semiconserved residues are highlighted in light blue and gray, respectively.

Anne Norris, et al. PLoS Genet. 2008 December;4(12):e1000301.
6.
Figure 6

Figure 6. SIR3 alleles restore Sir3p binding to telomeric DNA.. From: Compensatory Interactions between Sir3p and the Nucleosomal LRS Surface Imply Their Direct Interaction.

(A) ChIP of Sir3p to telomeric DNA. The histone allele is indicated, followed by the SIR3 allele on a CEN plasmid for each strain tested. Wt is wild-type, A75V is hht2-A7V5, D205N is sir3-D205N, and L79I is sir3–L79I. Wild type histones with PRS415 vector and endogenous SIR3 deleted served as a control. All values were normalized to input DNA and the PHO5 locus and wild type H3/SIR3+. Primers specific to subtelomeric sequences starting at 70-bp, and 500-bp, regions from the C1-3A repeats at the right end of Chromosome VI (Chr. VI-R). (B) Steady state Sir3p levels in strains used for ChIP analysis show that increased ChIP to telomeric DNA is not a consequence of over expression of Sir3p. Western blot of log phase cells expressing SIR3 alleles and LRS alleles probed with antibodies to Sir3p and tubulin as a loading control. Wt is wild-type, and A75V is hht2-A75V. Wild type histones with PRS415 vector and endogenous SIR3 deleted served as a control. Wt is wild-type, A75V is hht2-A7V5, D205N is sir3-D205N, and L79I is sir3–L79I.

Anne Norris, et al. PLoS Genet. 2008 December;4(12):e1000301.
7.
Figure 7

Figure 7. Model for Sir3p BAH domain binding to the LRS surface.. From: Compensatory Interactions between Sir3p and the Nucleosomal LRS Surface Imply Their Direct Interaction.

The 2Fl7 [35] crystal structure was docked to the 1ID3 nucleosome structure [29]. The helix α8 of Sir3p BAH packs against a group of Histone alpha helices consisting of H2B, H4, and H3. This juxtaposes the LRS surface with the BAH domain found to be important for suppression of LRS alleles. (A) Sighted along the SHL+/−3.5 axis, the DNA was removed to show the details of the docking. (B) A close-up of the docking structure to show the juxtaposition of the LRS surface and the Sir3p BAH domain suppressor residues. (C) An example of potential inhibitory interactions between H3 D77 and Sir3p E178. The introduction of an asparagine at H3 position 77 would ameliorate the inhibitory interactions. (D) A 90° rotation about the X axis from A, showing an overview of the docked structure. The DNA has been removed to show details of the docking. (E) A close-up of the key residues Sir3p D205 and H3 K79. H4 E74 could also be destabilizing, considering its proximity to D205. (F) Showing the interactions between the LRS residues H3 T80, D81, and H4 K79, and key residues W86 and E84. The images were made using PyMOL [62], and the docked structure is available as Datasets S1 and S2.

Anne Norris, et al. PLoS Genet. 2008 December;4(12):e1000301.

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