RUVB3

PF3D7_1362200 (RUVB3) is a nuclear AAA+-family DNA helicase/ATPase of Plasmodium falciparum implicated in DNA transactions and chromatin remodeling across the intraerythrocytic developmental cycle (PMID:22705314, PMID:27809838, PMID:30026605). Recombinant RUVB3 possesses ssDNA-dependent ATPase activity that depends on an intact Walker motif A, and it exhibits ATP- and divalent-cation-dependent DNA helicase activity that is stimulated by both single- and double-stranded DNA yet inactive on blunt-ended duplex; its enzymatic activity is inhibited by the helicase inhibitors doxorubicin, daunorubicin, and netropsin (PMID:22705314, PMID:27809838). RUVB3 forms a specific physical complex with PfRuvB2 (but not PfRuvB1) and acts as a modulator that stimulates PfRuvB2 helicase activity, the complex preferentially unwinding DNA in the 5'-to-3' direction (PMID:24161468). Beyond its helicase role, RUVB3 carries an insertion-domain-dependent DNA cleavage activity distinct from its ATPase and oligomerization functions, and it interacts with the chromatin-remodeling histone acetyltransferase PfMYST, co-localizing with H3K9me1, linking it to chromatin remodeling and gene transcription (PMID:30026605). RUVB3 localizes to a discrete nuclear/nucleolar compartment (co-localizing with the nucleolar marker PfNop1) during schizont stages and shows stage-varied subcellular distribution across the intraerythrocytic cycle (PMID:22705314, PMID:30026605). The physiological consequences of RUVB3 activity in vivo, and the significance of its parasitophorous-vacuole co-localization with PfMYST/H3K9me1, have not been resolved by loss-of-function studies in the available corpus.

1. 2012 High

   Whether the RUVB3 polypeptide itself is an active ATPase was unknown; assigning the catalytic activity to the protein and to a specific motif established it as a bona fide AAA+ enzyme rather than a passive complex subunit.

   Evidence In vitro ATPase assay of recombinant protein with Walker-A truncation mutagenesis and immunodepletion of endogenous protein

   PMID:22705314

   Open questions at the time

   • Helicase directionality and substrate preference of RUVB3 alone not defined here
   • No in vivo perturbation linking the ATPase activity to parasite phenotype
2. 2012 Medium

   Localizing endogenous RUVB3 placed its enzymatic activity in a defined subcellular context, showing it occupies a nuclear/nucleolar compartment that shifts upon merozoite release.

   Evidence Confocal immunofluorescence with anti-RUVB3 and nucleolar marker PfNop1

   PMID:22705314

   Open questions at the time

   • Functional meaning of the schizont-to-subnuclear relocalization not tested
   • Single-lab immunofluorescence without perturbation
3. 2013 High

   The question of RUVB3's partner within the RuvB family was resolved by demonstrating a selective RUVB3–PfRuvB2 complex, and by showing RUVB3 functionally modulates its partner's helicase activity and directional unwinding.

   Evidence Immunopurification of endogenous RUVB3 complex, in vitro interaction assay, and helicase assays with/without RUVB3

   PMID:24161468

   Open questions at the time

   • Stoichiometry and structural basis of the RUVB2/RUVB3 complex unknown
   • Why PfRuvB1 is excluded not explained mechanistically
   • In vivo requirement of the complex untested
4. 2016 High

   The cofactor and substrate requirements of RUVB3's intrinsic helicase activity were defined, characterizing it as an ATP/divalent-cation-dependent enzyme and identifying small-molecule inhibitors.

   Evidence Heterologous expression (strain K1), in vitro helicase/ATPase assays with cofactor variation and inhibitor testing, identity confirmed by mass spectrometry

   PMID:27809838

   Open questions at the time

   • Physiological DNA substrate in the parasite unknown
   • Inhibitor selectivity over host helicases not established
5. 2018 Medium

   A previously unrecognized DNA cleavage activity mapped to the insertion domain, plus a physical link to the chromatin-modifying HAT PfMYST, extended RUVB3's role beyond DNA unwinding toward chromatin remodeling and transcription.

   Evidence In vitro DNA cleavage assay with domain-deletion constructs, Co-IP with PfMYST, and IF co-localization with H3K9me1

   PMID:30026605

   Open questions at the time

   • Biological role of the DNA cleavage activity in vivo unknown
   • PfMYST interaction from single Co-IP without reciprocal validation
   • Functional consequence of H3K9me1 co-localization in the parasitophorous vacuole untested
6. 2018 Medium

   RUVB3 is expressed across all intraerythrocytic stages with varying localization, indicating persistent but stage-modulated function during the developmental cycle.

   Evidence Immunofluorescence microscopy across ring, trophozoite, and schizont stages

   PMID:30026605

   Open questions at the time

   • No functional perturbation tied to the localization changes
   • Stage-specific interaction partners not mapped

• The in vivo essentiality and physiological function of RUVB3 during schizogony, chromatin remodeling, and transcription remain unresolved.
• No knockout/knockdown phenotype
• No structural model of RUVB3 or its complexes
• Genomic targets of RUVB3 helicase/cleavage activity unidentified