{"gene":"PF3D7_0419300","run_date":"2026-09-07T10:55:51","timeline":{"discoveries":[{"year":2003,"finding":"PfRad51 (PF3D7_0419300) transcript and protein expression are up-regulated in intra-erythrocytic P. falciparum in response to the DNA-damaging agent methyl methanesulfonate (MMS), indicating functional involvement in recombinational repair.","method":"RT-PCR and Western blot of in vitro P. falciparum cultures treated with MMS","journal":"International journal for parasitology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — expression up-regulation demonstrated by two complementary methods (transcript and protein), single lab; functional inference from response to DNA damage","pmids":["14527521"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2005,"finding":"Recombinant PfRad51 (PF3D7_0419300) catalyzes DNA strand-exchange reaction in vitro without requiring ATP hydrolysis, and also catalyzes ssDNA-dependent ATP hydrolysis with kcat values similar to human Rad51.","method":"In vitro strand-exchange assay and ATPase assay using purified recombinant PfRad51 expressed in E. coli","journal":"Molecular and biochemical parasitology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct in vitro enzymatic reconstitution of both strand-exchange and ATPase activities with purified protein; two orthogonal biochemical assays in a single study","pmids":["15610817"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2013,"finding":"PfRad54 accelerates PfRad51 (PF3D7_0419300)-mediated pairing between ssDNA and homologous linear dsDNA in vitro in the presence of CaCl2, establishing PfRad54 as a positive cofactor for PfRad51 strand-exchange activity.","method":"In vitro DNA pairing/strand-exchange assay with recombinant PfRad51 and PfRad54","journal":"mBio","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct in vitro biochemical reconstitution with purified proteins showing functional interaction; complemented by in vivo comet assay evidence","pmids":["23631919"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2013,"finding":"PfRPA1L stimulates PfRad51 (PF3D7_0419300)-mediated homologous pairing and strand-exchange activity (functioning like bacterial SSB), while PfRPA1S negatively regulates PfRPA1L function in a dose-dependent manner, thus indirectly suppressing PfRad51 activity.","method":"In vitro strand-exchange assay with recombinant PfRad51, PfRPA1L, and PfRPA1S; in vivo comet assay for DNA damage","journal":"mBio","confidence":"High","confidence_rationale":"Tier 1 / Moderate — biochemical reconstitution with purified proteins demonstrating opposing regulatory roles; dose-response relationship established; two orthogonal methods (in vitro assay + in vivo comet assay)","pmids":["23631919"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2014,"finding":"A dominant-negative ATPase-dead mutant PfRad51(K143R) introduced into Plasmodium berghei disrupts HR-mediated DSB repair, rendering parasites hypersensitive to DNA-damaging agents and reducing parasite burden in mice; in yeast, the same mutant completely inhibits DNA repair, gene conversion, and gene targeting even at very low expression levels.","method":"Transgenic P. berghei expressing PfRad51(K143R); mouse infection model; yeast complementation/gene targeting assay; DNA damage sensitivity assay","journal":"Molecular microbiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — dominant-negative mutagenesis with multiple phenotypic readouts (hypersensitivity, parasite burden, yeast HR assays); mechanistic dissection via ATPase-dead mutant validated in two model systems","pmids":["25145341"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""},{"species":"Plasmodium berghei","strain":""}]},{"year":2019,"finding":"The small molecule B02 inhibits the ATPase and strand-exchange activities of PfRad51 (PF3D7_0419300) in vitro, abrogates formation of PfRad51 foci on damaged chromosomal DNA, and likely blocks homomeric interactions of PfRad51, leading to accumulation of unrepaired DNA and synergistic antimalarial activity with artemisinin and chloroquine.","method":"In vitro ATPase and strand-exchange assays; immunofluorescence foci assay; in silico docking; parasite growth inhibition assay (IC50); drug synergy analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — direct enzymatic inhibition assays combined with cellular foci assay and drug synergy experiments; multiple orthogonal methods in one study","pmids":["30936202"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":"3D7"},{"species":"Plasmodium falciparum","strain":"Dd2"}]},{"year":2021,"finding":"PfHsp90 physically interacts with PfRad51 (PF3D7_0419300) and promotes its UV irradiation-induced DNA repair activity by controlling PfRad51 protein stability; inhibition of PfHsp90 with 17-AAG accelerates proteasomal degradation of PfRad51 and sensitizes parasites to DNA damage.","method":"Co-immunoprecipitation (physical interaction); proteasome inhibitor experiment; parasite DNA damage sensitivity assay; drug synergy analysis with B02","journal":"Antimicrobial agents and chemotherapy","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP interaction demonstrated; proteasomal degradation shown pharmacologically; functional consequence (DNA repair activity and drug sensitivity) established; single lab","pmids":["34097485"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":"3D7"},{"species":"Plasmodium falciparum","strain":"Dd2"}]},{"year":2022,"finding":"P. berghei BRCA2 BRC repeats interact with PbRad51 (ortholog of PF3D7_0419300) as demonstrated by mammalian two-hybrid assay, establishing that the BRCA2–Rad51 interaction is conserved in Plasmodium.","method":"Mammalian two-hybrid assay; BRCA2 knockout parasite phenotyping in mice and mosquitoes","journal":"Parasites & vectors","confidence":"Medium","confidence_rationale":"Tier 3 / Weak — protein–protein interaction demonstrated by two-hybrid assay only; single study, single method for the interaction itself","pmids":["35804459"],"is_preprint":false,"attribution":[{"species":"Plasmodium berghei","strain":""}]},{"year":2020,"finding":"PfBlm (RecQ helicase) interacts with PfRad51 (PF3D7_0419300) as a component of the DSB repair pathway in P. falciparum.","method":"Co-immunoprecipitation/pulldown demonstrating physical interaction between PfBlm and PfRad51","journal":"mSphere","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single pulldown interaction reported, limited mechanistic detail about PfRad51's specific role in the interaction from the abstract","pmids":["33239368"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2023,"finding":"Loss of PfRad51 (PF3D7_0419300) recombinase function reduces recombination events among var gene sequences over 100 generations, demonstrating that PfRad51-mediated HR drives antigenic diversification at var loci.","method":"Whole-genome sequencing of PfRad51-proficient vs. PfRad51-deficient parasite lines over ~100 generations; comparison of var sequence rearrangement rates","journal":"Frontiers in molecular biosciences","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — loss-of-function with defined molecular phenotype (reduced var recombination) measured by WGS; single lab, single study","pmids":["37593128"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2025,"finding":"PfRad51 (PF3D7_0419300) is essential for HR-mediated repair of DSBs in the conserved core genome of P. falciparum; PfRad51 knockout parasites show uniform hypersensitivity to irradiation across the erythrocytic cycle, whereas a Rad51-independent homology-directed repair pathway operates specifically in subtelomeric regions.","method":"PfRad51 knockout parasite lines; targeted DSB assay; irradiation hypersensitivity assay across the cell cycle; comparison with TLS polymerase knockouts","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 2 / Moderate — clean knockout with defined cellular phenotype; targeted DSB repair assay distinguishes chromosomal domains; multiple orthogonal readouts in a single peer-reviewed study","pmids":["41277688"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2025,"finding":"PfRad51 (PF3D7_0419300) interacts with the mitochondria-targeted exonuclease PfExomit2, suggesting PfRad51 is a component of the mitochondrial DSB repair pathway.","method":"Pull-down assay demonstrating interaction between PfExomit2 and PfRad51","journal":"The FEBS journal","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single pulldown interaction; mitochondrial localization of PfRad51 itself not directly confirmed in the abstract","pmids":["41243319"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"current_model":"PF3D7_0419300 (PfRad51) is the central recombinase of P. falciparum that catalyzes ATP-independent DNA strand exchange and ssDNA-dependent ATP hydrolysis; it is stimulated by PfRad54 and PfRPA1L, negatively regulated by PfRPA1S, stabilized by PfHsp90 (which prevents its proteasomal degradation), and is essential for HR-mediated DSB repair in the conserved core genome—including repair of damage caused by artemisinin—while a Rad51-independent homology-directed repair pathway handles DSBs in subtelomeric regions; PfRad51-driven HR also mediates antigenic diversification at var loci, and its physical interactions with BRCA2 BRC repeats, PfBlm helicase, and PfExomit2 exonuclease further integrate it into multiple DNA repair contexts."},"narrative":{"mechanistic_narrative":"PF3D7_0419300 (PfRad51) is the central recombinase driving homologous-recombination (HR) repair of DNA double-strand breaks in Plasmodium falciparum, with its expression induced by DNA-damaging insult [PMID:14527521] and its activity essential for repair of breaks in the conserved core genome [PMID:41277688]. As purified recombinant protein, PfRad51 catalyzes DNA strand exchange without requiring ATP hydrolysis and performs ssDNA-dependent ATP hydrolysis at rates comparable to human Rad51 [PMID:15610817]. Its recombinase activity is tuned by accessory factors: PfRad54 accelerates strand exchange as a positive cofactor [PMID:23631919], PfRPA1L stimulates homologous pairing while PfRPA1S antagonizes this stimulation to suppress PfRad51 activity [PMID:23631919], and PfHsp90 physically binds PfRad51 to maintain its stability and prevent proteasomal degradation [PMID:34097485]. The ATPase function is mechanistically required in vivo: a dominant-negative ATPase-dead mutant abolishes HR-mediated repair and sensitizes parasites to DNA damage [PMID:25145341], and the inhibitor B02 blocks both enzymatic activities and PfRad51 focus formation, synergizing with antimalarials [PMID:30936202]. Beyond genome maintenance, PfRad51-mediated HR generates recombination among var genes, driving antigenic diversification [PMID:37593128], while a Rad51-independent homology-directed pathway handles subtelomeric breaks [PMID:41277688]. Physical partnerships with BRCA2 BRC repeats [PMID:35804459], PfBlm helicase [PMID:33239368], and the mitochondrial exonuclease PfExomit2 [PMID:41243319] place PfRad51 within multiple repair contexts.","teleology":[{"year":2003,"claim":"Establishing whether PfRad51 participates in DNA repair at all, its induction by a DNA-damaging agent provided the first functional link to recombinational repair in the parasite.","evidence":"RT-PCR and Western blot of MMS-treated intra-erythrocytic P. falciparum cultures","pmids":["14527521"],"confidence":"Medium","gaps":["Expression up-regulation is correlative, not a direct demonstration of repair activity","Does not establish substrate or mechanism"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2005,"claim":"To determine whether PfRad51 is a bona fide recombinase, purified protein was tested biochemically, confirming ATP-independent strand exchange and ssDNA-dependent ATPase activity comparable to human Rad51.","evidence":"In vitro strand-exchange and ATPase assays with recombinant PfRad51 from E. coli","pmids":["15610817"],"confidence":"High","gaps":["In vitro reconstitution does not establish in vivo requirement","Role of ATPase activity in the cell not defined"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2013,"claim":"To identify regulators of PfRad51's recombinase activity, reconstitution with candidate cofactors showed PfRad54 accelerates strand exchange and defined a PfRPA1L/PfRPA1S positive/negative regulatory axis.","evidence":"In vitro DNA pairing/strand-exchange assays with recombinant PfRad51, PfRad54, PfRPA1L, PfRPA1S, plus in vivo comet assay","pmids":["23631919"],"confidence":"High","gaps":["Structural basis of cofactor interactions unresolved","In vivo stoichiometry and timing of regulation unknown"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2014,"claim":"To test whether ATPase activity is mechanistically required in vivo, a dominant-negative ATPase-dead mutant was shown to disrupt HR-mediated repair and reduce parasite burden, linking enzymatic function to parasite fitness.","evidence":"Transgenic P. berghei expressing PfRad51(K143R), mouse infection model, and yeast HR/gene-targeting assays","pmids":["25145341"],"confidence":"High","gaps":["Dominant-negative approach does not equate to clean loss of function","Mechanism of dominant interference not detailed"],"attribution":[{"species":"Plasmodium falciparum","strain":""},{"species":"Plasmodium berghei","strain":""}]},{"year":2019,"claim":"To validate PfRad51 as a druggable antimalarial target, the small molecule B02 was shown to inhibit its enzymatic activities and focus formation and to synergize with existing antimalarials.","evidence":"In vitro ATPase/strand-exchange assays, immunofluorescence foci assay, in silico docking, and drug-synergy growth assays in strains 3D7 and Dd2","pmids":["30936202"],"confidence":"High","gaps":["B02 specificity for PfRad51 over other targets not fully excluded","Proposed block of homomeric interactions not structurally demonstrated"],"attribution":[{"species":"Plasmodium falciparum","strain":"3D7"},{"species":"Plasmodium falciparum","strain":"Dd2"}]},{"year":2021,"claim":"To explain how PfRad51 protein levels are maintained during damage response, PfHsp90 was found to physically bind PfRad51 and prevent its proteasomal degradation, coupling chaperone function to repair capacity.","evidence":"Co-immunoprecipitation, proteasome-inhibitor experiments, DNA-damage sensitivity assays, and B02 synergy in strains 3D7 and Dd2","pmids":["34097485"],"confidence":"Medium","gaps":["Single-lab Co-IP without reciprocal structural mapping of the interaction","Whether Hsp90 also regulates PfRad51 activity beyond stability unclear"],"attribution":[{"species":"Plasmodium falciparum","strain":"3D7"},{"species":"Plasmodium falciparum","strain":"Dd2"}]},{"year":2020,"claim":"To broaden the PfRad51 interaction network in DSB repair, PfBlm helicase was shown to physically associate with PfRad51.","evidence":"Co-immunoprecipitation/pulldown between PfBlm and PfRad51","pmids":["33239368"],"confidence":"Low","gaps":["Single pulldown without reciprocal validation","Functional consequence of the PfBlm-PfRad51 interaction for repair not defined","Direct vs. indirect interaction not distinguished"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2022,"claim":"To test conservation of the BRCA2-Rad51 axis, P. berghei BRCA2 BRC repeats were shown to interact with the PbRad51 ortholog, indicating the loading regulator is conserved in Plasmodium.","evidence":"Mammalian two-hybrid assay with BRCA2 BRC repeats, plus BRCA2 knockout phenotyping in mice and mosquitoes","pmids":["35804459"],"confidence":"Medium","gaps":["Interaction shown by a single two-hybrid method in a heterologous system","Interaction demonstrated for the P. berghei ortholog, not directly for PF3D7_0419300","Functional effect on PfRad51 loading not measured"],"attribution":[{"species":"Plasmodium berghei","strain":""}]},{"year":2023,"claim":"To determine a physiological role of PfRad51-mediated HR beyond genome maintenance, loss of recombinase function was shown to reduce var gene recombination, implicating PfRad51 in antigenic diversification.","evidence":"Whole-genome sequencing of PfRad51-proficient vs. -deficient lines over ~100 generations comparing var rearrangement rates","pmids":["37593128"],"confidence":"Medium","gaps":["Single-lab, single-study observation","Mechanistic link between recombinase activity and var-specific rearrangement not detailed"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2025,"claim":"To define which genomic regions depend on PfRad51, knockout parasites revealed PfRad51 is essential for core-genome DSB repair while a Rad51-independent pathway handles subtelomeric breaks, partitioning repair by chromosomal domain.","evidence":"PfRad51 knockout lines, targeted DSB assay, cell-cycle-resolved irradiation hypersensitivity, and comparison with TLS polymerase knockouts","pmids":["41277688"],"confidence":"High","gaps":["Molecular identity of the Rad51-independent subtelomeric pathway not established","Factors directing domain-specific pathway choice unknown"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2025,"claim":"To extend PfRad51's repair contexts to the organelle, PfRad51 was found to interact with the mitochondria-targeted exonuclease PfExomit2, hinting at a role in mitochondrial DSB repair.","evidence":"Pull-down assay between PfExomit2 and PfRad51","pmids":["41243319"],"confidence":"Low","gaps":["Single pulldown without reciprocal validation","Mitochondrial localization of PfRad51 itself not directly confirmed","Functional role in mitochondrial repair not demonstrated"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":null,"claim":"The molecular identity of the Rad51-independent subtelomeric repair pathway and the structural basis by which cofactors (Rad54, RPA1L/S, BRCA2, Hsp90) coordinate PfRad51 filament assembly remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of PfRad51 filaments or cofactor complexes","Mechanism of chromosomal-domain-specific pathway choice unknown","Direct evidence for PfRad51 in mitochondrial repair lacking"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":[],"partners":["PFRAD54 (POSITIVE COFACTOR OF STRAND EXCHANGE) [#2]","PFRPA1L (STIMULATORY) [#3]","PFRPA1S (NEGATIVE REGULATOR VIA PFRPA1L) [#3]","PFHSP90 (STABILIZES AGAINST PROTEASOMAL DEGRADATION) [#6]","BRCA2 BRC REPEATS (CONSERVED INTERACTION, SHOWN FOR P. BERGHEI ORTHOLOG) [#7]","PFBLM RECQ HELICASE [#8]","PFEXOMIT2 MITOCHONDRIAL EXONUCLEASE [#11]"],"other_free_text":["Recombinase activity mechanistically requires ATPase function in vivo, demonstrated by the dominant-negative K143R ATPase-dead mutant [#4].","Pharmacologically inhibited by the small molecule B02, which blocks enzymatic activity and focus formation and synergizes with artemisinin and chloroquine [#5].","PfRad51-dependent core-genome DSB repair is distinct from a Rad51-independent homology-directed pathway operating in subtelomeric regions [#10]."]}},"prefetch_data":{},"corpus_meta":[{"pmid":"10645945","id":"PMC_10645945","title":"The bacterial replicative helicase DnaB evolved from a RecA duplication.","date":"2000","source":"Genome 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biology","url":"https://pubmed.ncbi.nlm.nih.gov/40667286","citation_count":0,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["RAD51","PfRAD51"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":12767,"output_tokens":3361,"usd":0.044358,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9182,"output_tokens":4611,"usd":0.080592,"stage2_stop_reason":"end_turn"},"total_usd":0.12495,"stage1_batch_id":"msgbatch_01DbiymDmubkPpYvdbAu5a7V","stage2_batch_id":"msgbatch_012TbrWvMRH129T7PQ4SmvrJ","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2003,\n      \"finding\": \"PfRad51 (PF3D7_0419300) transcript and protein expression are up-regulated in intra-erythrocytic P. falciparum in response to the DNA-damaging agent methyl methanesulfonate (MMS), indicating functional involvement in recombinational repair.\",\n      \"method\": \"RT-PCR and Western blot of in vitro P. falciparum cultures treated with MMS\",\n      \"journal\": \"International journal for parasitology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — expression up-regulation demonstrated by two complementary methods (transcript and protein), single lab; functional inference from response to DNA damage\",\n      \"pmids\": [\"14527521\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Recombinant PfRad51 (PF3D7_0419300) catalyzes DNA strand-exchange reaction in vitro without requiring ATP hydrolysis, and also catalyzes ssDNA-dependent ATP hydrolysis with kcat values similar to human Rad51.\",\n      \"method\": \"In vitro strand-exchange assay and ATPase assay using purified recombinant PfRad51 expressed in E. coli\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct in vitro enzymatic reconstitution of both strand-exchange and ATPase activities with purified protein; two orthogonal biochemical assays in a single study\",\n      \"pmids\": [\"15610817\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"PfRad54 accelerates PfRad51 (PF3D7_0419300)-mediated pairing between ssDNA and homologous linear dsDNA in vitro in the presence of CaCl2, establishing PfRad54 as a positive cofactor for PfRad51 strand-exchange activity.\",\n      \"method\": \"In vitro DNA pairing/strand-exchange assay with recombinant PfRad51 and PfRad54\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct in vitro biochemical reconstitution with purified proteins showing functional interaction; complemented by in vivo comet assay evidence\",\n      \"pmids\": [\"23631919\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"PfRPA1L stimulates PfRad51 (PF3D7_0419300)-mediated homologous pairing and strand-exchange activity (functioning like bacterial SSB), while PfRPA1S negatively regulates PfRPA1L function in a dose-dependent manner, thus indirectly suppressing PfRad51 activity.\",\n      \"method\": \"In vitro strand-exchange assay with recombinant PfRad51, PfRPA1L, and PfRPA1S; in vivo comet assay for DNA damage\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — biochemical reconstitution with purified proteins demonstrating opposing regulatory roles; dose-response relationship established; two orthogonal methods (in vitro assay + in vivo comet assay)\",\n      \"pmids\": [\"23631919\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"A dominant-negative ATPase-dead mutant PfRad51(K143R) introduced into Plasmodium berghei disrupts HR-mediated DSB repair, rendering parasites hypersensitive to DNA-damaging agents and reducing parasite burden in mice; in yeast, the same mutant completely inhibits DNA repair, gene conversion, and gene targeting even at very low expression levels.\",\n      \"method\": \"Transgenic P. berghei expressing PfRad51(K143R); mouse infection model; yeast complementation/gene targeting assay; DNA damage sensitivity assay\",\n      \"journal\": \"Molecular microbiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — dominant-negative mutagenesis with multiple phenotypic readouts (hypersensitivity, parasite burden, yeast HR assays); mechanistic dissection via ATPase-dead mutant validated in two model systems\",\n      \"pmids\": [\"25145341\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"},\n        {\"species\": \"Plasmodium berghei\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"The small molecule B02 inhibits the ATPase and strand-exchange activities of PfRad51 (PF3D7_0419300) in vitro, abrogates formation of PfRad51 foci on damaged chromosomal DNA, and likely blocks homomeric interactions of PfRad51, leading to accumulation of unrepaired DNA and synergistic antimalarial activity with artemisinin and chloroquine.\",\n      \"method\": \"In vitro ATPase and strand-exchange assays; immunofluorescence foci assay; in silico docking; parasite growth inhibition assay (IC50); drug synergy analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — direct enzymatic inhibition assays combined with cellular foci assay and drug synergy experiments; multiple orthogonal methods in one study\",\n      \"pmids\": [\"30936202\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Plasmodium falciparum\", \"strain\": \"3D7\"},\n        {\"species\": \"Plasmodium falciparum\", \"strain\": \"Dd2\"}\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"PfHsp90 physically interacts with PfRad51 (PF3D7_0419300) and promotes its UV irradiation-induced DNA repair activity by controlling PfRad51 protein stability; inhibition of PfHsp90 with 17-AAG accelerates proteasomal degradation of PfRad51 and sensitizes parasites to DNA damage.\",\n      \"method\": \"Co-immunoprecipitation (physical interaction); proteasome inhibitor experiment; parasite DNA damage sensitivity assay; drug synergy analysis with B02\",\n      \"journal\": \"Antimicrobial agents and chemotherapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP interaction demonstrated; proteasomal degradation shown pharmacologically; functional consequence (DNA repair activity and drug sensitivity) established; single lab\",\n      \"pmids\": [\"34097485\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Plasmodium falciparum\", \"strain\": \"3D7\"},\n        {\"species\": \"Plasmodium falciparum\", \"strain\": \"Dd2\"}\n      ]\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"P. berghei BRCA2 BRC repeats interact with PbRad51 (ortholog of PF3D7_0419300) as demonstrated by mammalian two-hybrid assay, establishing that the BRCA2–Rad51 interaction is conserved in Plasmodium.\",\n      \"method\": \"Mammalian two-hybrid assay; BRCA2 knockout parasite phenotyping in mice and mosquitoes\",\n      \"journal\": \"Parasites & vectors\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Weak — protein–protein interaction demonstrated by two-hybrid assay only; single study, single method for the interaction itself\",\n      \"pmids\": [\"35804459\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"PfBlm (RecQ helicase) interacts with PfRad51 (PF3D7_0419300) as a component of the DSB repair pathway in P. falciparum.\",\n      \"method\": \"Co-immunoprecipitation/pulldown demonstrating physical interaction between PfBlm and PfRad51\",\n      \"journal\": \"mSphere\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single pulldown interaction reported, limited mechanistic detail about PfRad51's specific role in the interaction from the abstract\",\n      \"pmids\": [\"33239368\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Loss of PfRad51 (PF3D7_0419300) recombinase function reduces recombination events among var gene sequences over 100 generations, demonstrating that PfRad51-mediated HR drives antigenic diversification at var loci.\",\n      \"method\": \"Whole-genome sequencing of PfRad51-proficient vs. PfRad51-deficient parasite lines over ~100 generations; comparison of var sequence rearrangement rates\",\n      \"journal\": \"Frontiers in molecular biosciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — loss-of-function with defined molecular phenotype (reduced var recombination) measured by WGS; single lab, single study\",\n      \"pmids\": [\"37593128\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PfRad51 (PF3D7_0419300) is essential for HR-mediated repair of DSBs in the conserved core genome of P. falciparum; PfRad51 knockout parasites show uniform hypersensitivity to irradiation across the erythrocytic cycle, whereas a Rad51-independent homology-directed repair pathway operates specifically in subtelomeric regions.\",\n      \"method\": \"PfRad51 knockout parasite lines; targeted DSB assay; irradiation hypersensitivity assay across the cell cycle; comparison with TLS polymerase knockouts\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean knockout with defined cellular phenotype; targeted DSB repair assay distinguishes chromosomal domains; multiple orthogonal readouts in a single peer-reviewed study\",\n      \"pmids\": [\"41277688\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PfRad51 (PF3D7_0419300) interacts with the mitochondria-targeted exonuclease PfExomit2, suggesting PfRad51 is a component of the mitochondrial DSB repair pathway.\",\n      \"method\": \"Pull-down assay demonstrating interaction between PfExomit2 and PfRad51\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single pulldown interaction; mitochondrial localization of PfRad51 itself not directly confirmed in the abstract\",\n      \"pmids\": [\"41243319\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"PF3D7_0419300 (PfRad51) is the central recombinase of P. falciparum that catalyzes ATP-independent DNA strand exchange and ssDNA-dependent ATP hydrolysis; it is stimulated by PfRad54 and PfRPA1L, negatively regulated by PfRPA1S, stabilized by PfHsp90 (which prevents its proteasomal degradation), and is essential for HR-mediated DSB repair in the conserved core genome—including repair of damage caused by artemisinin—while a Rad51-independent homology-directed repair pathway handles DSBs in subtelomeric regions; PfRad51-driven HR also mediates antigenic diversification at var loci, and its physical interactions with BRCA2 BRC repeats, PfBlm helicase, and PfExomit2 exonuclease further integrate it into multiple DNA repair contexts.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PF3D7_0419300 (PfRad51) is the central recombinase driving homologous-recombination (HR) repair of DNA double-strand breaks in Plasmodium falciparum, with its expression induced by DNA-damaging insult [#0] and its activity essential for repair of breaks in the conserved core genome [#10]. As purified recombinant protein, PfRad51 catalyzes DNA strand exchange without requiring ATP hydrolysis and performs ssDNA-dependent ATP hydrolysis at rates comparable to human Rad51 [#1]. Its recombinase activity is tuned by accessory factors: PfRad54 accelerates strand exchange as a positive cofactor [#2], PfRPA1L stimulates homologous pairing while PfRPA1S antagonizes this stimulation to suppress PfRad51 activity [#3], and PfHsp90 physically binds PfRad51 to maintain its stability and prevent proteasomal degradation [#6]. The ATPase function is mechanistically required in vivo: a dominant-negative ATPase-dead mutant abolishes HR-mediated repair and sensitizes parasites to DNA damage [#4], and the inhibitor B02 blocks both enzymatic activities and PfRad51 focus formation, synergizing with antimalarials [#5]. Beyond genome maintenance, PfRad51-mediated HR generates recombination among var genes, driving antigenic diversification [#9], while a Rad51-independent homology-directed pathway handles subtelomeric breaks [#10]. Physical partnerships with BRCA2 BRC repeats [#7], PfBlm helicase [#8], and the mitochondrial exonuclease PfExomit2 [#11] place PfRad51 within multiple repair contexts.\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"Establishing whether PfRad51 participates in DNA repair at all, its induction by a DNA-damaging agent provided the first functional link to recombinational repair in the parasite.\",\n      \"evidence\": \"RT-PCR and Western blot of MMS-treated intra-erythrocytic P. falciparum cultures\",\n      \"pmids\": [\"14527521\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Expression up-regulation is correlative, not a direct demonstration of repair activity\", \"Does not establish substrate or mechanism\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"To determine whether PfRad51 is a bona fide recombinase, purified protein was tested biochemically, confirming ATP-independent strand exchange and ssDNA-dependent ATPase activity comparable to human Rad51.\",\n      \"evidence\": \"In vitro strand-exchange and ATPase assays with recombinant PfRad51 from E. coli\",\n      \"pmids\": [\"15610817\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vitro reconstitution does not establish in vivo requirement\", \"Role of ATPase activity in the cell not defined\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"To identify regulators of PfRad51's recombinase activity, reconstitution with candidate cofactors showed PfRad54 accelerates strand exchange and defined a PfRPA1L/PfRPA1S positive/negative regulatory axis.\",\n      \"evidence\": \"In vitro DNA pairing/strand-exchange assays with recombinant PfRad51, PfRad54, PfRPA1L, PfRPA1S, plus in vivo comet assay\",\n      \"pmids\": [\"23631919\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of cofactor interactions unresolved\", \"In vivo stoichiometry and timing of regulation unknown\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"To test whether ATPase activity is mechanistically required in vivo, a dominant-negative ATPase-dead mutant was shown to disrupt HR-mediated repair and reduce parasite burden, linking enzymatic function to parasite fitness.\",\n      \"evidence\": \"Transgenic P. berghei expressing PfRad51(K143R), mouse infection model, and yeast HR/gene-targeting assays\",\n      \"pmids\": [\"25145341\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Dominant-negative approach does not equate to clean loss of function\", \"Mechanism of dominant interference not detailed\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}, {\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"To validate PfRad51 as a druggable antimalarial target, the small molecule B02 was shown to inhibit its enzymatic activities and focus formation and to synergize with existing antimalarials.\",\n      \"evidence\": \"In vitro ATPase/strand-exchange assays, immunofluorescence foci assay, in silico docking, and drug-synergy growth assays in strains 3D7 and Dd2\",\n      \"pmids\": [\"30936202\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"B02 specificity for PfRad51 over other targets not fully excluded\", \"Proposed block of homomeric interactions not structurally demonstrated\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"3D7\"}, {\"species\": \"Plasmodium falciparum\", \"strain\": \"Dd2\"}]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"To explain how PfRad51 protein levels are maintained during damage response, PfHsp90 was found to physically bind PfRad51 and prevent its proteasomal degradation, coupling chaperone function to repair capacity.\",\n      \"evidence\": \"Co-immunoprecipitation, proteasome-inhibitor experiments, DNA-damage sensitivity assays, and B02 synergy in strains 3D7 and Dd2\",\n      \"pmids\": [\"34097485\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab Co-IP without reciprocal structural mapping of the interaction\", \"Whether Hsp90 also regulates PfRad51 activity beyond stability unclear\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"3D7\"}, {\"species\": \"Plasmodium falciparum\", \"strain\": \"Dd2\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"To broaden the PfRad51 interaction network in DSB repair, PfBlm helicase was shown to physically associate with PfRad51.\",\n      \"evidence\": \"Co-immunoprecipitation/pulldown between PfBlm and PfRad51\",\n      \"pmids\": [\"33239368\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single pulldown without reciprocal validation\", \"Functional consequence of the PfBlm-PfRad51 interaction for repair not defined\", \"Direct vs. indirect interaction not distinguished\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"To test conservation of the BRCA2-Rad51 axis, P. berghei BRCA2 BRC repeats were shown to interact with the PbRad51 ortholog, indicating the loading regulator is conserved in Plasmodium.\",\n      \"evidence\": \"Mammalian two-hybrid assay with BRCA2 BRC repeats, plus BRCA2 knockout phenotyping in mice and mosquitoes\",\n      \"pmids\": [\"35804459\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Interaction shown by a single two-hybrid method in a heterologous system\", \"Interaction demonstrated for the P. berghei ortholog, not directly for PF3D7_0419300\", \"Functional effect on PfRad51 loading not measured\"],\n      \"attribution\": [{\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"To determine a physiological role of PfRad51-mediated HR beyond genome maintenance, loss of recombinase function was shown to reduce var gene recombination, implicating PfRad51 in antigenic diversification.\",\n      \"evidence\": \"Whole-genome sequencing of PfRad51-proficient vs. -deficient lines over ~100 generations comparing var rearrangement rates\",\n      \"pmids\": [\"37593128\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab, single-study observation\", \"Mechanistic link between recombinase activity and var-specific rearrangement not detailed\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"To define which genomic regions depend on PfRad51, knockout parasites revealed PfRad51 is essential for core-genome DSB repair while a Rad51-independent pathway handles subtelomeric breaks, partitioning repair by chromosomal domain.\",\n      \"evidence\": \"PfRad51 knockout lines, targeted DSB assay, cell-cycle-resolved irradiation hypersensitivity, and comparison with TLS polymerase knockouts\",\n      \"pmids\": [\"41277688\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular identity of the Rad51-independent subtelomeric pathway not established\", \"Factors directing domain-specific pathway choice unknown\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"To extend PfRad51's repair contexts to the organelle, PfRad51 was found to interact with the mitochondria-targeted exonuclease PfExomit2, hinting at a role in mitochondrial DSB repair.\",\n      \"evidence\": \"Pull-down assay between PfExomit2 and PfRad51\",\n      \"pmids\": [\"41243319\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single pulldown without reciprocal validation\", \"Mitochondrial localization of PfRad51 itself not directly confirmed\", \"Functional role in mitochondrial repair not demonstrated\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The molecular identity of the Rad51-independent subtelomeric repair pathway and the structural basis by which cofactors (Rad54, RPA1L/S, BRCA2, Hsp90) coordinate PfRad51 filament assembly remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of PfRad51 filaments or cofactor complexes\", \"Mechanism of chromosomal-domain-specific pathway choice unknown\", \"Direct evidence for PfRad51 in mitochondrial repair lacking\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0000150\", \"supporting_discovery_ids\": [1, 2, 3, 5]},\n      {\"term_id\": \"GO:0008094\", \"supporting_discovery_ids\": [1, 5]},\n      {\"term_id\": \"GO:0003684\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0000724\", \"supporting_discovery_ids\": [0, 4, 10]},\n      {\"term_id\": \"GO:0006310\", \"supporting_discovery_ids\": [9]},\n      {\"term_id\": \"GO:0006281\", \"supporting_discovery_ids\": [0, 6, 10]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"PfRad54 (positive cofactor of strand exchange) [#2]\",\n      \"PfRPA1L (stimulatory) [#3]\",\n      \"PfRPA1S (negative regulator via PfRPA1L) [#3]\",\n      \"PfHsp90 (stabilizes against proteasomal degradation) [#6]\",\n      \"BRCA2 BRC repeats (conserved interaction, shown for P. berghei ortholog) [#7]\",\n      \"PfBlm RecQ helicase [#8]\",\n      \"PfExomit2 mitochondrial exonuclease [#11]\"\n    ],\n    \"other_free_text\": [\n      \"Recombinase activity mechanistically requires ATPase function in vivo, demonstrated by the dominant-negative K143R ATPase-dead mutant [#4].\",\n      \"Pharmacologically inhibited by the small molecule B02, which blocks enzymatic activity and focus formation and synergizes with artemisinin and chloroquine [#5].\",\n      \"PfRad51-dependent core-genome DSB repair is distinct from a Rad51-independent homology-directed pathway operating in subtelomeric regions [#10].\"\n    ]\n  }\n}","audit_flag":{"gene":"PF3D7_0419300","tier":"IDENTITY_CHECK","verdict":"identity unverified","subtype":"names_other_cards","uniprot_band":"","rules_fired":"R12","issue":"No cited paper names this card; 11 of 11 cited papers name other catalogue genes (PF3D7_1107400, PF3D7_0409600, PF3D7_0508800). The summary may describe one of those genes."},"strains":[{"strain":"Pf3D7","gene_id":"PF3D7_0419300","name":null,"product":"RAD51 domain-containing protein","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PbANKA","gene_id":"PBANKA_0721400","name":null,"product":"conserved Plasmodium protein, unknown function","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PkH","gene_id":"PKNH_0512000","name":null,"product":"conserved Plasmodium protein, unknown function","uniprot_acc":null,"chromosome":null,"start_pos":null}],"gene_symbol":"","evaluation":null}