Quantification and characterization of the 5' exonuclease activity of the lysosomal nuclease PLD3 by a novel cell-based assay.

ABSTRACT: 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 ac

SUBMITTER: Cappel C 

PROVIDER: S-EPMC7857491 | biostudies-literature | 2021 Jan-Jun

REPOSITORIES: biostudies-literature