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Issue 15, 2018
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A bi-terminal protein ligation strategy to probe chromatin structure during DNA damage

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Abstract

The cellular response to DNA damage results in a signaling cascade that primes chromatin for repair. Combinatorial post-translational modifications (PTMs) play an important role in this process by altering the physical properties of chromatin and recruiting downstream factors. One key signal integrator is the histone variant H2A.X, which is phosphorylated at a C-terminal serine (S139ph), and ubiquitylated within its N-terminal tail at lysines 13 and 15 (K13/15ub). How these PTMs directly impact chromatin structure and thereby facilitate DNA repair is not well understood. Detailed studies require synthetic access to such N- and C-terminally modified proteins. This is complicated by the requirement for protecting groups allowing multi-fragment assembly. Here, we report a semi-synthetic route to generate simultaneously N- and C-terminally modified proteins using genetically encoded orthogonal masking groups. Applied to H2A.X, expression of a central protein fragment, containing a protected N-terminal cysteine and a C-terminal thioester masked as a split intein, enables sequential C- and N-terminal protein modification and results in the convergent production of H2A.X carrying K15ub and S139ph. Using single-molecule FRET between defined nucleosomes in synthetic chromatin fibers, we then show that K15 ubiquitylation (but not S139ph) impairs nucleosome stacking in tetranucleosome units, opening chromatin during DNA repair.

Graphical abstract: A bi-terminal protein ligation strategy to probe chromatin structure during DNA damage

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Publication details

The article was received on 09 Feb 2018, accepted on 15 Mar 2018 and first published on 21 Mar 2018


Article type: Edge Article
DOI: 10.1039/C8SC00681D
Citation: Chem. Sci., 2018,9, 3704-3709
  • Open access: Creative Commons BY-NC license
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    A bi-terminal protein ligation strategy to probe chromatin structure during DNA damage

    S. Kilic, I. Boichenko, C. C. Lechner and B. Fierz, Chem. Sci., 2018, 9, 3704
    DOI: 10.1039/C8SC00681D

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