Affinage · Plasmodium

RAD51

PF3D7_1107400 · DNA repair protein RAD51

Annotated
2026-09-08
23 papers in source corpus 9 papers cited in narrative 9 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

PF3D7_1107400 (PfRad51) is the RecA/Rad51-family recombinase that drives homologous recombination (HR)-mediated repair of DNA double-strand breaks (DSBs) in Plasmodium falciparum, and its transcript and protein are induced upon MMS-induced DNA damage (PMID:14527521, PMID:23631919). Biochemically, recombinant PfRad51 hydrolyzes ATP and catalyzes ATP-dependent DNA strand exchange in vitro; this activity is accelerated by PfRad54 and stimulated by the replication protein A homologue PfRPA1L, while PfRPA1S negatively modulates PfRPA1L in a dose-dependent manner (PMID:23631919). The catalytic requirement is demonstrated by the K143R active-site mutant, which acts dominant-negatively to abolish DSB repair, gene conversion, and gene targeting, establishing HR as the principal DSB repair pathway in Plasmodium (PMID:25145341). Genetic knockout confirms that PfRad51 is essential for HR-mediated repair of DSBs in the conserved core genome and produces uniform irradiation hypersensitivity across the erythrocytic cycle, whereas subtelomeric DSBs are repaired by a PfRad51-independent, homology-directed pathway (PMID:41277688). Small-molecule inhibition by B02 blocks PfRad51 ATPase and strand-exchange activities, abrogates damage-induced PfRad51 foci, causes accumulation of unrepaired DNA, and inhibits parasite growth in synergy with artemisinin and chloroquine, consistent with disruption of homomeric filament formation (PMID:30936202). Beyond these findings, PfRad51's interaction with the BRCA2 BRC-repeat interface (PMID:12967658) and with the mitochondrial exonuclease PfExomit2 (PMID:41243319) have not been experimentally validated beyond structural prediction and a single pull-down, respectively.

Mechanistic history

Synthesis pass · year-by-year structured walk · 8 steps
  1. 2003 Medium

    Whether Plasmodium encodes a bona fide Rad51 recombinase and whether it participates in damage responses was unknown; identification of a Rad51 orthologue induced by a DNA-damaging agent placed PF3D7_1107400 in recombinational repair.

    Evidence Gene identification, sequence analysis, and RT-PCR/Western blot of MMS-treated P. falciparum cultures

    PMID:14527521

    Open questions at the time
    • No in vitro recombinase activity demonstrated
    • Direct role in DSB repair not tested
  2. 2003 Low

    Whether PfRad51 could engage a BRCA2-type mediator was addressed computationally, predicting the parasite orthologue retains the BRC-repeat interaction interface unlike other family members.

    Evidence Structural fingerprint analysis from the human BRC4-RAD51 crystal structure applied to Plasmodium RAD51 sequence

    PMID:12967658

    Open questions at the time
    • Purely computational inference; no biochemical validation of a PfRad51-BRCA2 interaction
    • No demonstration that a Plasmodium BRC repeat binds PfRad51
  3. 2013 High

    The catalytic activity of PfRad51 and its regulation were undefined; in vitro reconstitution established ATPase and strand-exchange activity and identified stimulatory (PfRad54, PfRPA1L) and inhibitory (PfRPA1S) partners.

    Evidence In vitro ATPase and strand-exchange assays with recombinant proteins, comet assay, and expression analysis in P. falciparum

    PMID:23631919

    Open questions at the time
    • Partner interactions shown biochemically but not validated in vivo
    • Stoichiometry and filament architecture not resolved
  4. 2014 High

    Whether PfRad51 catalytic function is required for parasite DSB repair and survival was answered by an active-site dominant-negative mutant that abolished repair and reduced parasite burden, establishing HR as the dominant DSB repair route.

    Evidence K143R dominant-negative mutagenesis in transgenic P. berghei, mouse infection model, yeast surrogate genetics, and in vitro disruption assay

    PMID:25145341

    Open questions at the time
    • Dominant-negative rather than clean loss-of-function in P. falciparum
    • Contribution of specific partners in vivo not dissected
  5. 2016 Medium

    How subtelomeric DSBs are repaired was probed by sequencing repair products, showing HR competes with de novo telomere addition and that pathway choice is dictated by local sequence.

    Evidence SMRT sequencing of subtelomeric repair products from spontaneous and induced DSBs in P. falciparum

    PMID:28790200

    Open questions at the time
    • HR involvement inferred from product sequences without direct PfRad51 disruption
    • Exact PfRad51 dependence at subtelomeres not tested here
  6. 2019 High

    Whether PfRad51 is druggable was tested with B02, which blocked its enzymatic activities and foci formation, killed parasites, and synergized with front-line antimalarials, nominating PfRad51 as a chemotherapeutic target.

    Evidence In silico docking, in vitro ATPase and strand-exchange assays, immunofluorescence of PfRad51 foci, growth inhibition and drug synergy assays in P. falciparum 3D7 and Dd2

    PMID:30936202

    Open questions at the time
    • Filament-disruption mechanism proposed but not structurally confirmed
    • B02 selectivity for PfRad51 versus host RAD51 not established
  7. 2025 High

    Whether PfRad51 is genuinely essential for HR and whether all DSBs depend on it was resolved by knockout, showing it is required for core-genome HR and uniform irradiation resistance but dispensable for a distinct subtelomeric homology-directed pathway.

    Evidence PfRad51 genetic knockout, irradiation hypersensitivity across the erythrocytic cycle, and targeted DSB repair-product sequencing in P. falciparum (peer-reviewed; also as bioRxiv preprint)

    PMID:40667286 PMID:41277688

    Open questions at the time
    • Molecular identity of the Rad51-independent subtelomeric pathway not defined
    • Mechanistic basis of pathway partitioning by genomic region unresolved
  8. 2025 Low

    Whether PfRad51 acts beyond nuclear HR was raised by a physical interaction with a mitochondrial exonuclease, hinting at a mitochondrial DSB repair role.

    Evidence Pull-down assay with recombinant PfExomit2 and immunofluorescence localization in P. falciparum

    PMID:41243319

    Open questions at the time
    • Single pull-down without reciprocal or functional validation
    • No evidence PfRad51 localizes to or acts within mitochondria

Open questions

Synthesis pass · forward-looking unresolved questions
  • The molecular identity of the PfRad51-independent subtelomeric homology-directed repair machinery, and whether PfRad51 has a bona fide functional role in mitochondrial DSB repair, remain open.
  • Effectors of Rad51-independent subtelomeric repair unidentified
  • Functional consequence of PfExomit2 interaction untested
  • In vivo relevance of BRCA2/BRC-repeat prediction unconfirmed

Mechanism profile

Synthesis pass · controlled-vocabulary classification
Partners
PFEXOMIT2 (MITOCHONDRIAL EXONUCLEASE; PHYSICAL PULL-DOWN INTERACTION, UNVALIDATED) [#6]PFRAD54 (ACCELERATES PFRAD51-MEDIATED HOMOLOGOUS PAIRING) [#1]PFRPA1L (STIMULATES PFRAD51 STRAND EXCHANGE) [#1]PFRPA1S (NEGATIVELY REGULATES PFRPA1L) [#1]

Evidence

Reading pass · 9 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2003 PfRad51 (PF3D7_1107400) encodes a 351 amino acid polypeptide sharing 66–75% sequence identity within the catalytic region with Rad51 from human, yeast, and other protozoan parasites. Its transcript and protein are up-regulated in intra-erythrocytic P. falciparum cultures in response to the DNA damaging agent methyl methanesulfonate (MMS), implicating it in recombinational DNA repair. Gene identification, sequence analysis, and expression analysis (RT-PCR, Western blot) of MMS-treated P. falciparum cultures International journal for parasitology Medium 14527521
2013 PfRad51 (PF3D7_1107400) exhibits ATPase activity and promotes DNA strand exchange in vitro. PfRad54 accelerates PfRad51-mediated pairing between ssDNA and homologous linear dsDNA in the presence of CaCl2. PfRPA1L (a replication protein A homologue) stimulates PfRad51-mediated homologous pairing and strand exchange, while PfRPA1S negatively regulates PfRPA1L activity in a dose-dependent manner. In vivo, PfRad51 transcript and protein are up-regulated upon MMS-induced DNA damage. In vitro ATPase assay, in vitro strand exchange assay with recombinant proteins, comet assay for in vivo DNA damage, RT-PCR and Western blot for expression mBio High 23631919
2014 A dominant negative mutant of PfRad51 (PF3D7_1107400), PfRad51(K143R), when expressed in Plasmodium berghei, abolishes DSB repair as shown by hypersensitivity to DNA-damaging agents. Mice infected with mutant parasites lived significantly longer with markedly reduced parasite burden. In a yeast surrogate model, PfRad51(K143R) completely inhibited DNA repair, gene conversion, and gene targeting. Biochemically, very low levels of mutant protein were sufficient to disrupt wild-type PfRad51 activity, consistent with a dominant negative mechanism. Homologous recombination (HR) was established as the most important DSB repair pathway in Plasmodium. Dominant negative mutagenesis (K143R), transgenic P. berghei line, DNA damage hypersensitivity assay, mouse infection model, yeast surrogate genetic assay, in vitro biochemical disruption assay Molecular microbiology High 25145341
2003 Based on the crystal structure of the human BRC4-RAD51 complex, sequence fingerprint analysis predicts that the Plasmodium RAD51 orthologue (PF3D7_1107400) retains the interaction interface required for binding BRC repeat motifs of BRCA2, whereas yeast and archaeal/bacterial RAD51 orthologues and human RAD51 paralogues (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3, DMC1) do not fulfil the criteria. A BRCA2 homologue containing BRC repeats was identified in two Plasmodium species. Crystal structure of human BRC4-RAD51 complex used to define interaction fingerprints; comparative sequence analysis applied to Plasmodium RAD51 DNA repair Low 12967658
2019 The small molecule B02 docks with high affinity to the predicted tertiary structure of PfRad51 (PF3D7_1107400), blocks its ATPase and strand-exchange activities in vitro, abrogates formation of PfRad51 foci on damaged DNA, and inhibits P. falciparum growth (IC50 8 µM in 3D7, 3 µM in Dd2). B02 synergizes with artemisinin and chloroquine (reducing their IC50 values by 15- and 8-fold, respectively) and causes accumulation of unrepaired parasitic DNA. Disruption of homomeric PfRad51 interactions was proposed as the mechanism, consistent with B02 blocking filament formation. In silico docking, in vitro ATPase assay, in vitro strand exchange assay, immunofluorescence microscopy of PfRad51 foci, parasite growth inhibition assay, drug synergy assay, DNA damage accumulation assay The Journal of biological chemistry High 30936202
2016 In P. falciparum, homologous recombination (HR) and de novo telomere addition compete to repair DSBs in subtelomeric regions, with the pathway used determined by the DNA sequences immediately surrounding the break. Both pathways were observed in response to spontaneous and experimentally induced DSBs, demonstrating that HR (in which PfRad51 participates) is active in repairing DSBs within subtelomeric regions. Single-molecule real-time (SMRT) sequencing of subtelomeric repair products from spontaneous and induced DSBs in P. falciparum culture mBio Medium 28790200
2025 PfRad51 (PF3D7_1107400) interacts with PfExomit2, a mitochondria-localised exonuclease, in pull-down assays, suggesting that PfRad51 is a component of the mitochondrial double-strand break repair (DSBR) pathway in P. falciparum. Pull-down assay with recombinant proteins; immunofluorescence localization of interacting partners The FEBS journal Low 41243319
2025 Knockout of PfRad51 (PF3D7_1107400) in P. falciparum causes uniform hypersensitivity to irradiation across the erythrocytic cell cycle. PfRad51 is essential for HR-mediated repair of targeted DSBs in the conserved core genome. In contrast, when DSBs are induced in subtelomeric regions, a Rad51-independent, homology-directed repair pathway operates, indicating that PfRad51 is dispensable for subtelomeric DSB repair. Genetic knockout (PfRad51Δ), irradiation hypersensitivity assay across the cell cycle, targeted DSB induction with sequencing of repair products Nucleic acids research High 41277688
2025 Knockout of PfRad51 (PF3D7_1107400) in P. falciparum causes uniform irradiation hypersensitivity across the erythrocytic cycle and is essential for HR-mediated repair in the core genome, while a Rad51-independent homology-directed repair pathway operates in subtelomeric regions (preprint version of the peer-reviewed paper). Genetic knockout, irradiation sensitivity assay, targeted DSB repair sequencing bioRxivpreprint Medium 40667286

Source papers

Stage 0 corpus · 23 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2000 The bacterial replicative helicase DnaB evolved from a RecA duplication. Genome research 162 10645945
2003 Sequence fingerprints in BRCA2 and RAD51: implications for DNA repair and cancer. DNA repair 100 12967658
1986 Rapid repeated DNA replication during microgametogenesis and DNA synthesis in young zygotes of Plasmodium berghei. Transactions of the Royal Society of Tropical Medicine and Hygiene 61 3088783
1987 Plasmodium species: flow cytometry and microfluorometry assessments of DNA content and synthesis. Experimental parasitology 57 2440713
2016 DNA damage regulation and its role in drug-related phenotypes in the malaria parasites. Scientific reports 56 27033103
1968 DNA, RNA , and protein synthesis in erythrocytic forms of Plasmodium knowlesi. The American journal of tropical medicine and hygiene 56 4971014
1979 Plasmodium-infected blood cells analyzed and sorted by flow fluorimetry with the deoxyribonucleic acid binding dye 33258 Hoechst. The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 52 87413
1975 Circular mitochondrial DNA from the avian malarial parasite Plasmodium lophurae. Biochimica et biophysica acta 52 1125317
1994 Maternal inheritance of extrachromosomal DNA in malaria parasites. Molecular and biochemical parasitology 48 7935632
1985 Nuclear and mitochondrial DNA of the primate malarial parasite Plasmodium knowlesi. Molecular and biochemical parasitology 48 2985981
1988 DNA synthesis in gametocytes of Plasmodium falciparum. Parasitology 47 3283664
2005 DNA-[adenine] methylation in lower eukaryotes. Biochemistry. Biokhimiia 42 15948708
2003 Identification and molecular characterisation of DNA damaging agent induced expression of Plasmodium falciparum recombination protein PfRad51. International journal for parasitology 31 14527521
1987 From Laveran's discovery to DNA probes: new trends in diagnosis of malaria. Lancet (London, England) 31 2892060
2004 Molecular players of homologous recombination in protozoan parasites: implications for generating antigenic variation. Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases 30 15157626
2017 Chromosome End Repair and Genome Stability in Plasmodium falciparum. mBio 29 28790200
2019 A small-molecule inhibitor of the DNA recombinase Rad51 from Plasmodium falciparum synergizes with the antimalarial drugs artemisinin and chloroquine. The Journal of biological chemistry 27 30936202
2013 Opposing roles for two molecular forms of replication protein A in Rad51-Rad54-mediated DNA recombination in Plasmodium falciparum. mBio 27 23631919
2017 Homologous Recombination in Protozoan Parasites and Recombinase Inhibitors. Frontiers in microbiology 22 28936205
2014 Dominant negative mutant of Plasmodium Rad51 causes reduced parasite burden in host by abrogating DNA double-strand break repair. Molecular microbiology 19 25145341
2025 Plasmodium mitochondrial exodeoxyribonucleases, Exomit1 and Exomit2, are conserved within alveolates with Exomit1 essential for the establishment of blood-stage infection. The FEBS journal 1 41243319
2025 Plasmodium falciparum DNA repair dynamics reveal unique roles for TLS polymerases and PfRad51 in genome diversification. bioRxiv : the preprint server for biology 0 40667286
2025 Plasmodium falciparum DNA repair dynamics reveal unique roles for TLS polymerases and PfRad51 in genome diversification. Nucleic acids research 0 41277688