biostudies-literatureUnknown296Cappel CHans und Ilse Breuer-StiftungAlzheimer Forschung InitiativePhospholipase D3 (PLD3) and phospholipase D4 (PLD4), the most recently described lysosomal nucleases, are associated with Alzheimer's disease, spinocerebellar ataxia, and systemic lupus erythematosus. They exhibit 5' exonuclease activity on single-stranded DNA, hydrolyzing it at the acidic pH associated with the lysosome. However, their full cellular function is inadequately understood. To examine these enzymes, we developed a robust and automatable cell-based assay based on fluorophore- and fluorescence-quencher-coupled oligonucleotides for the quantitative determination of acidic 5' exonuclease activity. We validated the assay under knockout and PLD-overexpression conditions and then applied it to characterize PLD3 and PLD4 biochemically. Our experiments revealed PLD3 as the principal acThe Journal of biological chemistry100152https://www.ebi.ac.uk/biostudies/studies/S-EPMC7857491biostudies-literatureQuantification and characterization of the 5' exonuclease activity of the lysosomal nuclease PLD3 by a novel cell-based assay.PMC7857491Gonzalez ACDamme MCappel CfalseQuantification and characterization of the 5' exonuclease activity of the lysosomal nuclease PLD3 by a novel cell-based assay.Phospholipase D3 (PLD3) and phospholipase D4 (PLD4), the most recently described lysosomal nucleases, are associated with Alzheimer's disease, spinocerebellar ataxia, and systemic lupus erythematosus. They exhibit 5' exonuclease activity on single-stranded DNA, hydrolyzing it at the acidic pH associated with the lysosome. However, their full cellular function is inadequately understood. To examine these enzymes, we developed a robust and automatable cell-based assay based on fluorophore- and fluorescence-quencher-coupled oligonucleotides for the quantitative determination of acidic 5' exonuclease activity. We validated the assay under knockout and PLD-overexpression conditions and then applied it to characterize PLD3 and PLD4 biochemically. Our experiments revealed PLD3 as the principal ac2021-01-01T00:00:00Z2021 Jan-Jun2025-04-22T08:14:09.74Z2022-02-10T21:22:50.721ZS-EPMC78574913328867410.1074/jbc.RA120.015867