ja308908p_si_001.pdf (1.26 MB)
Stability of Nucleosomes Containing Homogenously Ubiquitylated H2A and H2B Prepared Using Semisynthesis
journal contribution
posted on 2012-12-05, 00:00 authored by Beat Fierz, Sinan Kilic, Aaron R. Hieb, Karolin Luger, Tom W. MuirPost-translational modifications (PTMs) of histones are
an essential
feature in the dynamic regulation of chromatin. One of these modifications,
ubiquitylation, has been speculated to directly influence the stability
of the nucleosome, which represents the basic building block of chromatin.
Here we report a strategy for the semisynthesis of site-specifically
ubiquitylated histone H2A (uH2A). This branched protein was generated
through a three-piece expressed protein ligation approach including
a traceless ligation at valine. uH2A could be efficiently incorporated
into nucleosomes, thereby opening the way to detailed biochemical
and biophysical studies on the function of this PTM. Accordingly,
we used uH2A, as well as a previously generated ubiquitylated H2B,
in chaperone-coupled nucleosome stability assays to demonstrate that
the direct effect of ubiquitylated histones on nucleosomal stability
is in fact modest.