Abstract
Nucleosomes are the fundamental unit of eukaryotic chromosome packaging, comprised of 147 bp of DNA wrapped around two molecules of each of the core histone proteins H2A, H2B, H3, and H4. Nucleosomes are symmetrical, with one axis of symmetry centered on the homodimeric interaction between the C-termini of the H3 molecules. To explore the functional consequences of nucleosome symmetry, we designed an obligate pair of H3 heterodimers, termed H3X and H3Y, allowing us to compare cells with single or double H3 alterations. Our biochemical validation of the heterodimeric X-Y interaction included intra-nucleosomal H3 crosslinking using dimethyl suberimidate (DMS). Here, we provide a detailed protocol for the use of DMS to analyze yeast nucleosomes.
Highlights
Post-translational modifications of histone proteins affect every aspect of chromosome biology, including transcription, replication, repair, and recombination
To allow experimental manipulation of nucleosomal symmetry in vivo, we designed a pair of altered histone H3 proteins that have obligate heterodimeric interactions, termed H3X (L126A, L130V) and H3Y (L109I, A110W, L130I) (Ichikawa et al, 2017)
The dimethyl suberimidate (DMS) crosslinking efficiency of the X-Y heterodimeric pairs was around 10%, nearly identical to wild-type H3 homodimeric pairs; approximately 20% of the crosslinked heterodimers in the input fractions were precipitated by streptavidin-agarose (Ichikawa et al, 2017)
Summary
Post-translational modifications of histone proteins affect every aspect of chromosome biology, including transcription, replication, repair, and recombination. Examination gloves (Fisher Scientific, catalog number: 19-130-1597D) 9. Dimethyl suberimidate (DMS) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 20700). Trichloroacetic acid (TCA) (Fisher Scientific, catalog number: BP555) 12. Disodium ethylenediamine tetraacetate (EDTA) (Fisher Scientific, catalog number: S311) 15. Ammonium acetate (NH4Ac) (Fisher Scientific, catalog number: A637) 19. 2-Propanol (Fisher Scientific, catalog number: A416) 21. Agarose (Fisher Scientific, catalog number: BP160) 25. Primary antibody: anti-V5 tag (Thermo Fisher Scientific, Invitrogen, catalog number: R960-25) 31. Sodium chloride (NaCl) (Fisher Scientific, catalog number: S271) 39. Hydrochloric acid (HCl) (Fisher Scientific, catalog number: A144) 40. Glacial (Fisher Scientific, catalog number: A38) 47. Sodium bicarbonate (NaHCO3) (Fisher Scientific, catalog number: S233) 52. Sodium carbonate (Na2CO3) (Fisher Scientific, catalog number: S263) 53. Sodium hydroxide (NaOH) (Fisher Scientific, catalog number: S318) 54.
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