Regulation of DNA repair by histone ADP-ribosylation

DNA is continually being exposed to a variety of agents that induce DNA damage. As such, a set of pathways known as the DNA damage response (DDR) detect DNA damage when it occurs and activate mechanisms for its repair. These pathways are critical for our health and their dysfunction leads to a variety of pathologies including increased cancer risk, neurodegeneration, congenital abnormalities and premature ageing. Therefore, understanding how cells repair DNA damage will provide information about the underlying causes of these conditions and, importantly, how they can be treated.

This strategy is exemplified by inhibition Poly(ADP-ribose)-polymerases (PARPs), a class of enzymes that promote DNA repair by attaching ADP-ribose units onto proteins through a process known as ADP-ribosylation. Inhibitors of PARPs are being used to treat breast and ovarian cancers and have the potential to treat other pathologies associated with DDR defects. However, despite their potential as therapeutic targets, our knowledge of how PARPs regulate DNA repair is limited. For example, the proteins ADP-ribosylated in response to DNA damage and how this regulates repair are ill-defined. This situation is epitomized by histones, the proteins that package DNA into the nucleus of the cell. Histones are major targets for PARPs, particularly at serine amino acids that also have the potential to be modified by phosphorylation. Given phosphorylation regulates a variety of processes, including cell growth and division, this raises the possibility that interplay between ADP-ribosylation and phosphorylation may coordinate DNA repair with a variety of pathways. However, the functional significance of these relationships remains to be tested due to difficulties in manipulating histone genes in human cells. There is therefore a need for an experimental system where histone genes can be easily manipulated to test how histone ADP-ribosylation regulates DNA repair.

Our current MRC-funded work provided key advances to these questions by developing a robust experimental pipeline in the amoeba Dictyostelium that allowed us to manipulate histone genes to assess how histone ADP-ribosylation regulates DNA repair. Our previous work pioneered the use of this system to study human DNA repair mechanisms lost in other genetic model organisms, including ADP-ribosylation. By exploiting the unique ability to manipulate histone ADP-ribosylation sites in this organism, we identified that interplay between histone ADP-ribosylation and phosphorylation is critical to maintain genome integrity by coordinating DNA repair with cell division. This provides a paradigm shift for how ADP-ribosylation integrates with other post-translational modifications to regulate the DDR and the ability to identify novel regulatory mechanisms that can be extended to human cells. The proposed work will build on these key technical and conceptual advances in Dictyostelium to identify how histone ADP-ribosylation couples DNA repair with cell cycle progression and extend these findings to human cells. In addition to providing an increased understanding of how cells promote DNA repair to prevent mutagenesis, these studies will provide information to facilitate the design of therapeutic agents that target DNA repair pathways to treat pathologies associated with a defective DDR.

We will address three critically important question regarding how ADP-ribosylation couples DNA repair and cell cycle progression: a) Which histone H3 variants regulate DNA double strand break repair? b) How does H3 ADP-ribosylation regulate mitotic entry following DNA damage? c) What factors interact with ADP-ribosylated histones and how do they regulate DNA repair?

A bottle-neck for the ADP-ribosylation field is the lack of an experimental model in which ADP-ribosylation sites can be mutated in histone genes to decipher how these modifications regulate DNA repair. The ability to manipulate histone genes in Dictyostelium, in addition to our mutation strategy that separates the function of serine ADP-ribosylation and phosphorylation, will address this deficiency. Dictyostelium H3 variants (H3a; H3b) are ADP-ribosylated in response to DSBs. Gene replacement technology will knock-in mutations at these sites, alone or in combinations, to generate histone ADP-ribosylation defective strains. DSB repair efficiency will be assessed using plasmid integration (NHEJ/HR efficiency), DNA damage sensitivity, and enrichment of repair factors at damage sites. Mitotic entry/progression in Dictyostelium and human cells will be assessed using cell synchronisation and live cell imaging, whilst immunofluorescence will assess markers of mitotic progression in different genetic backgrounds. Histone ADP-ribosylation defective strains offer a rigorously controlled platform to identify factors that interact with ADP-ribosylated histones. Immunoprecipitation of nucleosomes from wild-type and histone ADP-ribosylation defective strains will identify factors that interact with ADP-ribosylated histones by mass spectrometry. The orthologues of proteins identified in Dictyostelium will be disrupted by CRISPR/Cas9 using genome editing in human cells and the consequences on DSB repair assessed using standard assays, including repair efficiencies, enrichment of repair factors at DSBs etc.