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.