Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2019 Nov 19;19(1):e13061. doi: 10.1111/acel.13061

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© 2019 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

PMC Copyright notice

Amino acids 392–394 (SHY) facilitate Rap1‐histone interactions. (a) Location of alpha‐helices within the SANT domain, redrawn from Konig et al. (1996). Triple alanine mutants were generated from amino acids 359–410. (b) Immunoblot analysis of in vitro GST pull‐down assay of histones showing representative triple alanine mutants. Pull‐down was performed with equimolar GST‐SANT (0.5 μM) and histones (0.5 μM) at 400 mM NaCl. Bottom panel is the blot stained with Ponceau S as a loading control. (c) Quantitation of triple alanine mutants binding to H3/H4, normalized to Ponceau stain signal, and with WT SANT set to 1.0. Error bars for all quantitations indicate standard error of the mean (N = 2). Only mutant 12 (amino acids 392–394, SHY) showed a significant loss of H3 signal. (d) Two views of the SANT domain bound to DNA. Amino acids SHY side chains are colored in magenta. SHY is located immediately C‐terminal to helix 2, with side chains facing away from the Rap1‐DNA interaction surface. Image generated using Pymol (PDB ID: 3UKG). (e) Representative immunoblot analysis of GST pull‐down histone binding assay with full‐length Rap1 and Rap1^SHY. Pull‐down was performed with 0.5 μM each Rap1 and H3/H4 at 400 mM NaCl. Rap1^SHY displays a ~50% loss of histone binding. Bottom panel is a loading control gel stained with Coomassie blue. (f) Quantitation of full‐length Rap1 and Rap1^SHY binding to H3/H4 (N = 3). (g) Representative immunoblot analysis of GST pull‐down histone assay using two truncated versions of Rap1 lacking the C‐terminus, Rap1^CΔ and Rap1^{643 Δ}. Pull‐down was performed with 2 μM Rap1 truncated constructs and 2 μM H3/H4 at 400 mM NaCl. Both truncated forms show a significant and similar loss of histone signal when amino acid SHY is mutated to AAA (rightmost two lanes). Bottom panel is Coomassie loading control. (h) Quantitation of g (N = 3). (i) Representative coimmunoprecipitation of histone H3 with immunoprecipitated HA‐Rap1 and HA‐Rap1^SHY. Input is 5% of the WCE, and Rap1^SHY shows a significant loss of histone binding in the extracts. (j) Quantitation of the ratio of co‐immunoprecipated H3 to input H3 signals in i (N = 3)