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

Abstract

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.

Temporal dynamics of base excision / single-strand break repair protein complex assembly and disassembly are modulated by the PARP1/NAD+/SIRT6 axis

“…To identify how NAD + availability affects Polβ/XRCC1 repair complex formation, the NAMPT inhibitor FK866 was used to decrease cellular NAD + levels, and dihydronicotinamide riboside (NRH) was used to enhance cellular NAD + levels. Due to factors in serum that alter NAD + catabolite stability (Wilk et al, 2020), we used heat-inactivated fetal bovine serum (HI-FBS) for these studies. We noted that the recruitment profiles of Polβ, XRCC1, or RealPAR with HI-FBS, as compared to normal fetal bovine serum, were unchanged ( Figures S6A-C ).…”

Towards resolving the enigma of the dichotomy of resveratrol: cis- and trans-resveratrol have opposite effects on TyrRS-regulated PARP1 activation

“…Intriguingly, the presence of PARP1 on the broken DNA impairs efficient DNA repair [302,303], suggesting that eviction of PARP1 from the DNA through auto-poly-ADP-ribos(PAR)ylation is required for efficient DNA repair [304,305]. Consistently, NAD + supplementation that enhances auto-PARylation of PARP1 facilitates PARP1-dependent DNA repair [306]. Interestingly, our work demonstrated that nuclear localization of TyrRS after either serum starvation, heat shock, or endoplasmic reticulum (ER) stress stimulates the auto-PARylation of PARP1 suggesting that eukaryotic TyrRS activates PARP1 in a DNA-independent manner [285] to facilitate TyrRSmediated DNA repair [295,296].…”

NAD+ metabolism: pathophysiologic mechanisms and therapeutic potential

“… 133 , 183 , 184 As expected, decreased NAD + levels induce the accumulation of DNA damage, whereas replenishing intracellular NAD + stimulates the DNA repair. 185 – 187 In contrast to the positive effect of NAD + on DNA repair, NADP + suppresses the ADP-ribosylation-mediated DNA damage repair via functioning as an endogenous inhibitor of PARPs. The structure of NADP + similar to that of NAD + renders its binding to PARPs.…”