ESR4: “Molecular role of mitochondrial ADP-ribosylation in DNA damage and replication”

LMU (A. Ladurner group): Molecular role of mitochondrial ADP-ribosylation in DNA damage and replication

i. Objective of research: We will establish how the reversible ADP-ribosylation of the mitochondrial DNA replication machinery contributes to mitochondrial DNA damage and repair.

ii. Current state of the art: Mutations in the mitochondrial genome contribute to disease. Further, mitochondria play an essential role in cellular homeostasis. While mitochondrial metabolism and signaling are integrated with nuclear gene expression and genome maintenance, molecular mechanisms that link mitochondrial metabolism to DNA damage are not well understood. Dynamic post-translational modifications such as ADP-ribosylation regulate enzyme activities, including the oncogene and chromatin remodeler ALC1/CHD1L, as well as vital mitochondrial proteins. Cellular ADP-ribosylation depends on the mitochondrial metabolite NAD+ and often involves the modification of glutamates. Our knowledge of the presence and relevance of ADP-ribosylation in mitochondria is limited. Taking advantage of our discovery of ADP-ribose-binding macrodomains, we identified the mitochondrial ADP-ribosylome (unpublished) and interfered with MacroD1, a mitochondrial enzyme that reverses ADP-ribosylation. Interestingly, the main targets of mitochondrial ADP-ribosylation include DNA polymerase γ, Twinkle and Topoisomerase1, enzymes responsible for mitochondrial DNA replication and repair. Knocking out MACROD1 accumulates mitochondrial DNA damage and alters metabolism, indicating a loss of mitochondrial homeostasis.

iii. Research methodology and approach: We will characterize how deletion of the MACROD1 gene affects the integrity of mitochondrial DNA (mtDNA), testing for mtDNA deletions and depletion in cultured human cells, and test how oxidative DNA damage agents enhance the deletion phenotype. Since DNA Pol-gamma and Twinkle are direct targets of mitochondrial ADP-ribosylation (unpublished observations), we will identify the peptide(-s) and residue(-s) within these proteins that carry the ADP-ribosylation mark, synthesize ADP-ribosylated peptides, test how the MacroD1 macrodomain recognizes these substrates and determine the crystal structure of the macrodomain–ADP-ribosyl-peptide complex. Since fundamental enzymatic activities of mitochondria are conserved in yeast, there is a MACROD1 orthologue, and large-scale surveys indicate abnormal mtDNA maintenance and decreased anaerobic growth, we will measure quantitatively how POA1 alters mtDNA maintenance and metabolism, test whether mitochondrial DNA replication components are ADP-ribosylated, map ADP-ribosylation sites and introduce site-directed mutations in the target residues.

iv. Originality and innovative aspects of the ESR project: Mitochondrial genome maintenance and mitochondrial metabolism are vital for survival. Since nuclear and mitochondrial DNA metabolism and NAD+ signaling are interdependent, our project sheds light on the fundamental role of ADP-ribosylation in human health.

v. Integration of the ESR project to the overall research programme: Our ESR will collaborate with the Polo, Legube and Mailand groups on the role of chromatin remodeling enzymes, histone and histone variants, including linker histones in nuclear DNA damage and with the Schumacher group and Norgenotech on the organismal responses to DNA damage.