DNA repair mechanisms and their biological roles in the malaria parasite Plasmodium falciparum

Abstract

Research into the complex genetic underpinnings of the malaria parasite Plasmodium falciparum is entering a new era with the arrival of site-specific genome engineering. Previously restricted only to model systems but now expanded to most laboratory organisms, and even to humans for experimental gene therapy studies, this technology allows researchers to rapidly generate previously unattainable genetic modifications. This technological advance is dependent on DNA double-strand break repair (DSBR), specifically homologous recombination in the case of Plasmodium. Our understanding of DSBR in malaria parasites, however, is based largely on assumptions and knowledge taken from other model systems, which do not always hold true in Plasmodium. Here we describe the causes of double-strand breaks, the mechanisms of DSBR, and the differences between model systems and P. falciparum. These mechanisms drive basic parasite functions, such as meiosis, antigen diversification, and copy number variation, and allow the parasite to continually evolve in the contexts of host immune pressure and drug selection. Finally, we discuss the new technologies that leverage DSBR mechanisms to accelerate genetic investigations into this global infectious pathogen.

FIG 1

The Plasmodium life cycle. A malaria infection begins with the transmission of a Plasmodium parasite via a female Anopheles mosquito host (left) to a human host (right). After the initial liver stage, the parasite begins its asexual intraerythrocytic cycle. Sexual forms, which develop from the intraerythrocytic parasites, can be transmitted to another mosquito. In the mosquito, parasites undergo meiotic and mitotic replication to form sporozoites, which can infect another human host. RBCs, red blood cells.

FIG 2

The Plasmodium asexual intraerythrocytic cycle. A haploid (1n) merozoite invades a red blood cell (RBC) and develops as the “ring” form from 0 h to about 24 h postinvasion, corresponding to the G₁ phase of the cell cycle. As the parasite transitions from rings to trophozoites, its metabolic activity increases in preparation for DNA replication. Prior to S phase and DNA replication, the parasite is still haploid, allowing for possible alternative EJ pathways. DNA replication produces multiple copies of the genome in an intact nucleus that does not undergo membrane degradation, providing homologous templates for HR. Plasmodium DNA replication is asynchronous and can produce a range of sister chromatids, up to about 24n. Nearing the end of the 48-h cycle, each genome is packaged into separate daughter merozoites, which then egress and invade another RBC.

FIG 3

Canonical models of homologous recombination, synthesis-dependent strand annealing, and break-induced replication. A double-strand break (DSB) in a chromosome (A) is first sensed and tethered together by the MRX-Sae2 complex (B). (C) Resection by Exo1 (gray “Pac-man”) or the STR-Dna2 complex (yellow and red triangles, respectively) exposes 3′ ssDNA tails, which are then bound by RPA (green dots). RPA is replaced by Rad51 (gray helices) (D), which forms a nucleoprotein filament that invades homologous templates (E). (F) Synthesis-dependent strand annealing (SDSA) uses the invading 3′ tail as a primer for DNA synthesis. Once sufficient homology is synthesized, the invading strand can bridge the DSB to restore chromosome integrity. (L) If the distal chromosome arm is lost, then break-induced replication can occur, with DNA synthesis continuing conservatively until it reaches the end of the chromosome arm. If both broken DNA ends are captured by a homologous template (G), then a double Holliday junction (dHJ) can be formed (H). This structure can be resolved in a number of ways. (I) Merging of both Holliday junctions by the STR complex (yellow triangles) dissolves the dHJ in a hemicatenane structure, which is unlinked by the Top3 topoisomerase. Cleavage of both Holliday junctions by structure-specific resolvases (purple triangles) can unlink the dHJ to produce either noncrossover (J) or crossover (K) products.

FIG 4

EJ pathways. (A) Nonhomologous end joining (NHEJ) involves the joining of broken DNA ends with little to no homology. A simplified model of NHEJ depicts the restoration of the original DNA sequence and introduction of small insertions (blue strands) or small deletions. (B) Microhomology-mediated end joining (MMEJ) relies on minimal resection by proteins shared with HR to expose short homologies of up to 25 bp, which can anneal, deleting any intervening sequence. (C) Single-strand annealing (SSA) involves longer stretches of resection. Rad52 (not shown) in yeast displaces RPA and anneals longer homologies, generating larger deletions.

FIG 5

Mechanisms of copy number variation (CNV). (A) Nonallelic homologous recombination (NAHR) can generate genome amplifications or deletions. In NAHR, a DSB is repaired using an incorrect template. Resolution of the dHJ intermediate forms a crossover, which leads to a duplication of a segment (amplicon) on one sister and a deletion on the other. (B) Microhomology-mediated break-induced replication (MMBIR) can generate amplifications or deletions. DNA replication fork progression through a nick will generate a single-ended DSB. Microhomology can then anneal to microhomology on a replicating sister chromatid and undergo BIR to recapitulate the remaining chromosome arm.

FIG 6

Established technologies for genome editing. (A) Single-site crossover (SSC) was developed to utilize a region homologous to a genomic locus carried on a transfected plasmid with a positive marker (green strands). SSC depends on a stochastic DSB in the genome to provide recombinogenic DNA ends that will use the plasmid region of homology as a template for repair. Formation of a dHJ and its resolution to yield a crossover product will lead to plasmid integration into the genome. (B) An apparent double crossover requires the parasite to incorporate the positive marker placed between two regions homologous to the gene of interest. Recombination (e.g., by SDSA) can copy the positive marker and introduce this into the genome while avoiding the negative marker (dark gray strands). An alternative scenario (not shown) involves two independent crossover events. In this case, the entire plasmid integrates into the genome by SSC and is followed by recombination between the homologous sequences flanking the negative marker, thereby looping it out. Selection for both positive and negative markers will select only for parasites that have incorporated the positive marker.