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

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.

SUBMITTER: Kilic S

PROVIDER: S-EPMC5935033 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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

Kilic Sinan S Boichenko Iuliia I Lechner Carolin C CC Fierz Beat B

Chemical science 20180321 15

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 ...[more]

PMID: 29780501

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Project description:Global genome nucleotide excision repair removes DNA damage from transcriptionally silent regions of the genome. Relatively little is known about the molecular events that initiate and regulate this process in the context of chromatin. We've shown that, in response to UV radiation-induced DNA damage, increased histone H3 acetylation at lysine 9 and 14 correlates with changes in chromatin structure, and these alterations are associated with efficient global genome nucleotide excision repair in yeast. These changes depend on the presence of the Rad16 protein. Remarkably, constitutive hyperacetylation of histone H3 can suppress the requirement for Rad7 and Rad16, two components of a global genome repair complex, during repair. This reveals the connection between histone H3 acetylation and DNA repair. Here, we investigate how chromatin structure is modified following UV irradiation to facilitate DNA repair in yeast. Using a combination of chromatin immunoprecipitation to measure histone acetylation levels, histone acetylase occupancy in chromatin, MNase digestion, or restriction enzyme endonuclease accessibility assays to analyse chromatin structure, and finally nucleotide excision repair assays to examine DNA repair, we demonstrate that global genome nucleotide excision repair drives UV-induced chromatin remodelling by controlling histone H3 acetylation levels in chromatin. The concerted action of the ATPase and C3HC4 RING domains of Rad16 combine to regulate the occupancy of the histone acetyl transferase Gcn5 on chromatin in response to UV damage. We conclude that the global genome repair complex in yeast regulates UV-induced histone H3 acetylation by controlling the accessibility of the histone acetyl transferase Gcn5 in chromatin. The resultant changes in histone H3 acetylation promote chromatin remodelling necessary for efficient repair of DNA damage. Recent evidence suggests that GCN5 plays a role in NER in human cells. Our work provides important insight into how GG-NER operates in chromatin.

Dataset Information

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

Publications

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

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