Extracellular NAD + enhances PARP-dependent DNA repair capacity independently of CD73 activity

Changes in nicotinamide adenine dinucleotide (NAD + ) levels that compromise mitochondrial function trigger release of DNA damaging reactive oxygen species. NAD + levels also affect DNA repair capacity as NAD + is a substrate for PARP-enzymes (mono/poly-ADP-ribosylation) and sirtuins (deacetylation). The ecto-5′-nucleotidase CD73, an ectoenzyme highly expressed in cancer, is suggested to regulate intracellular NAD + levels by processing NAD + and its bio-precursor, nicotinamide mononucleotide (NMN), from tumor microenvironments, thereby enhancing tumor DNA repair capacity and chemotherapy resistance. We therefore investigated whether expression of CD73 impacts intracellular NAD + content and NAD + -dependent DNA repair capacity. Reduced intracellular NAD + levels suppressed recruitment of the DNA repair protein XRCC1 to sites of genomic DNA damage and impacted the amount of accumulated DNA damage. Further, decreased NAD + reduced the capacity to repair DNA damage induced by DNA alkylating agents. Overall, reversal of these outcomes through NAD + or NMN supplementation was independent of CD73. In opposition to its proposed role in extracellular NAD + bioprocessing, we found that recombinant human CD73 only poorly processes NMN but not NAD + . A positive correlation between CD73 expression and intracellular NAD + content could not be made as CD73 knockout human cells were efficient in generating intracellular NAD + when supplemented with NAD + or NMN.