Role of histones in DNA double-strand break repair in Dictyostelium

Thesis

Correct repair of DNA double-strand breaks is crucial for maintenance of genome integrity. Despite data showing the importance of histones variants and histone post-translational modifications in the cellular response to DNA damage, there is still a lack of knowledge concerning the role of histone H3 and its variants as well as histone ADP-ribosylation in such processes. In this work Dictyostelium discoideum was employed as genetically tractable model organism to address the role of histone H3 variants and histone ADP-ribosylation in DNA double-strand break (DSB) repair. Vegetative cells lacking two out of three histone H3 variants – H3b and H3c, were shown not to be sensitive to DNA DSB. No evidence for altered DSB repair was found as phosphorylation of histone H2AX (a marker of DSB) and one of the pathways of DSB repair, non-homologous end joining, were not altered. Altogether, this work demonstrates that H3b and c variants are not required for overall DNA DSB repair in Dictyostelium. Among the core histones histone H2B was discovered to be the major acceptor of ADP-ribosylation by major ADP-ribosyl transferase involved in DSB repair, Adprt1a, in vitro. ADP-ribosylation in vitro was shown to occur on glutamate E18 with E19 being a potential regulator of this modification. Using an epitope-tagged overexpressed H2B, in vivo H2B ADP-ribosylation in response to DSBs was observed in Dictyostelium for the first time. Decreased ADP-ribosylation of epitope-tagged H2B mutated in both E18 and E19 residues was demonstrated. Overall, this work demonstrates the presence of the ADP-ribosylation of H2B in Dictyostelium in response to DSBs and identifies the major site of this modification.

Authors: Rakhimova, A

• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Subjects: DNA repair; Biochemistry