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

Research output: Contribution to journalJournal articlepeer-review

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.

Original languageEnglish
JournalChemical Science
Issue number15
Pages (from-to)3704-3709
Number of pages6
Publication statusPublished - 2018
Externally publishedYes

Bibliographical note

Funding Information:
We thank Maeva Tobler and Jade A. Chevalley for materials, and Ruud Hovius for comments on the manuscript. This work was supported by the Sandoz Family Foundation, the Swiss National Science Foundation (grant 31003A_173169), the European Research Council through the 2017 Consolidator Grant chromo-SUMMIT (724022) and EPFL (B. F.). S. K. gratefully acknowledges a Boehringer Ingelheim Fonds PhD fellowship.

Publisher Copyright:
© The Royal Society of Chemistry 2018.

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