RPA1

PF3D7_0409600 is the large subunit of P. falciparum replication protein A and constitutes the major single-stranded DNA binding activity in the parasite, an activity expressed in a cell cycle-dependent manner that peaks in late trophozoites and schizonts, coinciding with the onset of chromosomal DNA replication (PMID:11880371). It is a short-type RPA1, lacking the N-terminal protein interaction domain found in higher-eukaryote RPA1 orthologues (PMID:11880371). Functionally, recombinant PF3D7_0409600 substitutes for bacterial SSB to initiate homologous pairing and stimulate PfRad51-PfRad54-mediated strand exchange in vitro, a stimulatory function that is negatively regulated in a dose-dependent manner by the short-type RPA1 paralogue PfRPA1S; upon methyl methanesulfonate-induced DNA damage in live parasites, PF3D7_0409600 transcript and protein are upregulated alongside PfRad51, PfRad54, and PfRPA1S (PMID:23631919). Beyond its ssDNA-binding and strand-exchange stimulatory roles, no further mechanistic detail for PF3D7_0409600 has been characterized in the available corpus.

1. 2002 Medium

   Whether P. falciparum possesses a dedicated single-stranded DNA binding protein and how its activity is timed relative to replication was unknown; this work identified PF3D7_0409600 as the parasite's principal ssDNA-binding factor and linked its activity to the replicative cell cycle.

   Evidence Affinity purification and mass spectrometry identifying a ~55 kDa factor, EMSA for ssDNA binding, and Northern/RT-PCR for cell-cycle transcript timing in P. falciparum extracts

   PMID:11880371

   Open questions at the time

   • Direct role in replication fork progression not tested in vivo
   • Absence of the N-terminal interaction domain leaves partner recruitment mechanism unresolved
   • No structural characterization of the ssDNA-binding mode
2. 2013 Medium

   Whether PF3D7_0409600 participates in homologous recombination and how its activity is controlled was unknown; reconstitution showed it stimulates Rad51-Rad54 strand exchange and is antagonized by its short-type paralogue, placing it in the parasite's DNA damage response.

   Evidence In vitro strand exchange assay with recombinant proteins, dose-response inhibition by PfRPA1S, comet assay for MMS-induced damage, and RT-PCR/western blot for damage-induced upregulation in live P. falciparum

   PMID:23631919

   Open questions at the time

   • In vivo requirement for HR (e.g. genetic knockout/knockdown) not established
   • Molecular basis of PfRPA1S dose-dependent inhibition not defined
   • Physical interaction between PF3D7_0409600 and Rad51/Rad54 not directly demonstrated

• How PF3D7_0409600 is recruited to sites of replication and repair in the absence of the canonical N-terminal interaction domain, and its genetic essentiality in the parasite, remain unresolved.
• No genetic loss-of-function data for PF3D7_0409600
• No defined protein interactome or complex architecture
• No structural model of the ssDNA-bound or Rad51-stimulating state