Plasmodium falciparum DNA repair dynamics reveal unique roles for TLS polymerases and PfRad51 in genome diversification.

The human malaria parasite, Plasmodium falciparum, faces unique DNA repair challenges; it is haploid, undergoes asynchronous mitosis termed schizogony, and lacks canonical non-homologous end joining (C-NHEJ). Yet, it has adapted DNA repair pathways that enable survival in distinct environments, including human erythrocytes and hepatocytes, as well as the mosquito vector. Plasmodium falciparum chromosomes are partitioned into a conserved core genome and highly diverse subtelomeric regions containing hypervariable, multicopy gene families, including var, which encodes a critical parasite virulence factor. The molecular mechanisms maintaining this chromosomal structure remain unclear. Here, we describe specific DNA repair pathways that distinguish hypervariable subtelomeric regions from the conserved core genome. By disrupting the DNA repair enzyme PfRad51 and TLS polymerases PfPolζ and PfRev1, we identified differential irradiation hypersensitivity across the cell cycle for TLSΔ parasites and uniform hypersensitivity for PfRad51Δ parasites, highlighting variable roles for these repair pathways. Repair of targeted double-strand breaks demonstrated that PfRad51 is essential for HR-mediated repair in the core genome, whereas a Rad51-independent, homology-directed repair pathway was observed in subtelomeric regions. This previously unidentified alternative repair pathway was independent of TLS polymerases. We propose that these differential DNA repair responses maintain the unique structure that defines P. falciparum chromosomes.