Chromosome End Repair and Genome Stability in Plasmodium falciparum

Abstract

The human malaria parasite Plasmodium falciparum replicates within circulating red blood cells, where it is subjected to conditions that frequently cause DNA damage. The repair of DNA double-stranded breaks (DSBs) is thought to rely almost exclusively on homologous recombination (HR), due to a lack of efficient nonhomologous end joining. However, given that the parasite is haploid during this stage of its life cycle, the mechanisms involved in maintaining genome stability are poorly understood. Of particular interest are the subtelomeric regions of the chromosomes, which contain the majority of the multicopy variant antigen-encoding genes responsible for virulence and disease severity. Here, we show that parasites utilize a competitive balance between de novo telomere addition, also called "telomere healing," and HR to stabilize chromosome ends. Products of both repair pathways were observed in response to DSBs that occurred spontaneously during routine in vitro culture or resulted from experimentally induced DSBs, demonstrating that both pathways are active in repairing DSBs within subtelomeric regions and that the pathway utilized was determined by the DNA sequences immediately surrounding the break. In combination, these two repair pathways enable parasites to efficiently maintain chromosome stability while also contributing to the generation of genetic diversity.IMPORTANCE Malaria is a major global health threat, causing approximately 430,000 deaths annually. This mosquito-transmitted disease is caused by Plasmodium parasites, with infection with the species Plasmodium falciparum being the most lethal. Mechanisms underlying DNA repair and maintenance of genome integrity in P. falciparum are not well understood and represent a gap in our understanding of how parasites survive the hostile environment of their vertebrate and insect hosts. Our work examines DNA repair in real time by using single-molecule real-time (SMRT) sequencing focused on the subtelomeric regions of the genome that harbor the multicopy gene families important for virulence and the maintenance of infection. We show that parasites utilize two competing molecular mechanisms to repair double-strand breaks, homologous recombination and de novo telomere addition, with the pathway used being determined by the surrounding DNA sequence. In combination, these two pathways balance the need to maintain genome stability with the selective advantage of generating antigenic diversity.

Keywords: Plasmodium falciparum; chromosome stability; de novo telomere addition; gene conversion; homologous recombination; telomere healing.

FIG 1

Repair of double-strand breaks within subtelomeric chromosomal regions. (A) The typical structure of the subtelomeric regions of the chromosomes of P. falciparum. The core genome contains primarily single-copy housekeeping genes (gray), while the subtelomeric regions consists of large arrays of variant antigen-encoding genes of the var (blue), rif, stevor, and Pfmc-2TM (green) families. TAREs are positioned between the variant gene families and the telomere repeats. (B) The steps of de novo telomere addition, also called telomere healing. Step 1: typical chromosomes can be divided into a core genome containing housekeeping genes, telomere repeats at the extreme end of the chromosome, and intervening subtelomeric regions. A DSB within the subtelomeric region can be repaired by telomere healing. Step 2: the DSB is recognized by protein complexes that include exonuclease activity. In model organisms, both Exo1 and DNA2 have been implicated in the resection of DNA away from the telomere, revealing a single-stranded 3′ end. Step 3: when a single-strand sequence is revealed that can anneal to the template RNA of the telomerase complex, telomerase activity extends from the break, placing telomere repeats directly at this site. Step 4: repeated rounds of telomere addition result in a stable telomere and maintain genome integrity.

FIG 2

Telomere healing events identified in the genome of the parasite 3D7. (A) The five telomere healing events identified in the genome of the reference 3D7 sequence. The chromosome number is shown along with the gene structure of each subtelomeric region. For each gene, the gene family is indicated as well as the annotation number (provided by PlasmoDB.org). (B) Two additional telomere healing events identified in a clonal line of 3D7. The event that occurred on chromosome 2 (top) resulted in deletion of ~110 kb and led to truncation of the lsap2 gene (PF3D7_0202100), while the healing event on chromosome 3 resulted in loss of ~85 kb and added telomere repeats just downstream of a gene encoding a predicted alpha/beta-hydrolase (PF3D7_0301300).

FIG 3

Double-strand break repair within subtelomeric regions in response to X-ray irradiation. (A) Two products of telomere healing are shown. On chromosome 1 (top), telomere repeats were inserted into the coding region of the rif gene (Pf3D7_0101900) and on chromosome 2 (bottom), in which telomere repeats were inserted into the hypothetical gene Pf3D7_0221000. These events resulted in subtelomeric deletion of ~90 and ~100 kb, respectively. (B) Three products of repair by homologous recombination were also identified, two on chromosome 12 (top) and one on chromosome 13 (bottom). The two events on chromosome 12 resulted in the insertion of subtelomeric sequences from chromosomes 1 and 9; however, no coding regions were altered. The event on chromosome 13 resulted in the insertion of 25,493 bp of sequence from chromosome 9 and led to the creation of a new var gene that is a chimera of Pf3D7_1300100 (blue) and Pf3D7_0900100 (pink).

FIG 4

Sequence preference for telomere healing. (A) Model for telomere repeat additions at the site of a DSB. After exonuclease resection, a 3′ single-strand overhang is revealed. Single-stranded regions of sequence similarity can anneal to the template region of telomerase RNA (left). Telomerase activity can then extend by adding telomere repeat sequences directly to the chromosome end (right). Multiple rounds of repeat addition result in a lengthy telomere repeat region. (B) The nine examples of telomere healing identified in this study. Bold sequence portions indicate the original region of the chromosome, while italicized letters indicate the repeat sequences added by telomerase. The red sequence shows the template region of telomerase RNA and hypothetical annealing to the sequence where the healing event occurred. The blue G within the template indicates the base pair that is thought to specify either a C or T.

FIG 5

The sequences surrounding the break points of the three examples of DSB repair by homologous recombination identified in this study. (A and B) The recombination events that occurred within one of the subtelomeric regions of chromosome 12. The first (A) occurred within a region of 296 bp of sequence identity between chromosomes 9 and 1, while the second (B) occurred within a region of 127 bp of sequence identity between chromosomes 1 and 12. (C) A region of 27 bp of sequence identity spanning the break point of the recombination event found within a subtelomeric region of chromosome 13, leading to the creation of a chimeric var gene.

FIG 6

Model for the contribution of both telomere healing and homologous recombination in maintaining chromosome end stability in P. falciparum. (A) The occurrence of a DSB at a site of unique sequence within a subtelomeric region is stabilized by telomere healing. This results in a substantial deletion of the subtelomeric domain, including members of multicopy gene families and TAREs. (B) A subsequent DSB within a region that shares sequence identity with subtelomeric regions from other chromosomes can be repaired by HR, leading to reestablishment of the normal subtelomeric structure, including a full complement of multicopy genes and TAREs. Repair by HR can also result in chimeric genes, thereby contributing to the generation of diversity within the multicopy gene families.