biostudies-literatureAderounmu AMNational Cancer InstituteNCI NIH HHSSchool of MedicineNational Institute of General Medical SciencesNIGMS NIH HHSe85120https://www.ebi.ac.uk/biostudies/studies/S-EPMC10159624biostudies-literatureUnknown12Antiviral defense in ecdysozoan invertebrates requires Dicer with a helicase domain capable of ATP hydrolysis. But despite well-conserved ATPase motifs, human Dicer is incapable of ATP hydrolysis, consistent with a muted role in antiviral defense. To investigate this enigma, we used ancestral protein reconstruction to resurrect Dicer's helicase in animals and trace the evolutionary trajectory of ATP hydrolysis. Biochemical assays indicated ancient Dicer possessed ATPase function, that like extant invertebrate Dicers, is stimulated by dsRNA. Analyses revealed that dsRNA stimulates ATPase activity by increasing ATP affinity, reflected in Michaelis constants. Deuterostome Dicer-1 ancestor, while exhibiting lower dsRNA affinity, retained some ATPase activity; importantly, ATPase activity was ueLifeAncestral protein reconstruction reveals evolutionary events governing variation in Dicer helicase function.PMC10159624R35GM141262R01CA260414R01 CA2604143I Graduate Student FellowshipR35 GM141262Bass BLAderounmu AMAruscavage PJKolaczkowski BfalseAncestral protein reconstruction reveals evolutionary events governing variation in Dicer helicase function.Antiviral defense in ecdysozoan invertebrates requires Dicer with a helicase domain capable of ATP hydrolysis. But despite well-conserved ATPase motifs, human Dicer is incapable of ATP hydrolysis, consistent with a muted role in antiviral defense. To investigate this enigma, we used ancestral protein reconstruction to resurrect Dicer's helicase in animals and trace the evolutionary trajectory of ATP hydrolysis. Biochemical assays indicated ancient Dicer possessed ATPase function, that like extant invertebrate Dicers, is stimulated by dsRNA. Analyses revealed that dsRNA stimulates ATPase activity by increasing ATP affinity, reflected in Michaelis constants. Deuterostome Dicer-1 ancestor, while exhibiting lower dsRNA affinity, retained some ATPase activity; importantly, ATPase activity was u2023-01-01T00:00:00Z2023 Apr2026-05-28T11:58:11.449Z2025-02-19T04:29:22.05ZS-EPMC101596243706801110.7554/eLife.85120