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. 2014 Jan;42(1):370-9.
doi: 10.1093/nar/gkt881. Epub 2013 Oct 1.

Malaria parasites utilize both homologous recombination and alternative end joining pathways to maintain genome integrity

Laura A Kirkman  1 , Elizabeth A Lawrence, Kirk W Deitsch
Affiliations

Malaria parasites utilize both homologous recombination and alternative end joining pathways to maintain genome integrity

Laura A Kirkman et al. Nucleic Acids Res. 2014 Jan.

Abstract

Malaria parasites replicate asexually within their mammalian hosts as haploid cells and are subject to DNA damage from the immune response and chemotherapeutic agents that can significantly disrupt genomic integrity. Examination of the annotated genome of the parasite Plasmodium falciparum identified genes encoding core proteins required for the homologous recombination (HR) pathway for repairing DNA double-strand breaks (DSBs), but surprisingly none of the components of the canonical non-homologous end joining (C-NHEJ) pathway were identified. To better understand how malaria parasites repair DSBs and maintain genome integrity, we modified the yeast I-SceI endonuclease system to generate inducible, site-specific DSBs within the parasite's genome. Analysis of repaired genomic DNA showed that parasites possess both a typical HR pathway resulting in gene conversion events as well as an end joining (EJ) pathway for repair of DSBs when no homologous sequence is available. The products of EJ were limited in number and identical products were observed in multiple independent experiments. The repair junctions frequently contained short insertions also found in the surrounding sequences, suggesting the possibility of a templated repair process. We propose that an alternative end-joining pathway rather than C-NHEJ, serves as a primary method for repairing DSBs in malaria parasites.

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Figures

Figure 1.
Figure 1.
A system for inducing targeted, DSBs in P. falciparum. (a) On the left is shown the endogenous enoyl-acyl carrier reductase gene (ENR) on chromosome 6 (top) as well as the duplicated sequence containing the I-SceI site and inserted into the att site on chromosome 7 (bottom). Note that digestion with Not I/EcoR V results in a 5-kb fragment containing the GFP tag. On the right is shown the same sequences after induction of the I-SceI endonuclease. Cleavage and repair leads to gene conversion and loss of the Not I site, resulting in an 8-kb fragment containing the GFP tag after digestion with Not I/EcoR V. (b) The I-SceI recognition and cleavage site is shown. Note that cleavage results in a 4 bp 3′-overhang (blue or gray text). (c) Western blot showing stabilization of the I-SceI endonuclease upon treatment with Shield1. Extracts from parasites grown in the presence or absence of Shield1 were separated by SDS–PAGE, blotted onto nylon membrane and probed with antibodies specific for the destabilization domain that is fused to the endonuclease (top panel). To control for loading, the blot was stripped and reprobed with antibodies to the protein Pf39 (bottom panel). (d) Southern blot hybridized with a probe to GFP. Lanes 1, 2 and 3 show genomic DNA extracted from parasites after 0, 10 and 20 days of growth in the presence of Shield1, respectively.
Figure 2.
Figure 2.
HR-mediated repair leading to gene conversion events. A fragment from either the enoyl-acyl carrier reductase gene (PF3D7_0615100, ENR) on chromosome 6 (a) or from a var gene (PF3D7_0223500) within the subtelomeric region of chromosome 2 (c) was inserted at the att site on chromosome 7. Both duplicated fragments contained an I-SceI site inserted near the middle of the sequence. Break and repair of the duplicated fragments elicits repair by gene conversion resulting in removal of the I-SceI sites. (b) The sequence of the duplicated PF3D7_0615100 sequence after cleavage and repair. The arrowhead shows the location of the 18-bp I-SceI sequence prior to the gene conversion event. Nucleotides in bold and underlined indicate SNPs between the endogenous gene and the fragment inserted at the att site. (d) The sequence of the duplicated PF3D7_0223500 sequence after cleavage and repair. The arrowhead shows the location of the 18-bp I-SceI sequence prior to the gene conversion event.
Figure 3.
Figure 3.
EJ-mediated repair of a DSB. (a) A fragment from either the Gaussia luciferase gene or the enoyl-acyl carrier reductase (ENR) gene containing either 2% or 20% sequence divergence from the endogenous copy was inserted into the att site on chromosome 7. All of the inserted fragments contained the I-SceI recognition site near the middle of the insertion. Induction of the I-SceI endonuclease with Shield1 led to a DSB and repair, resulting in disruption of the recognition site. Note that repair by EJ results in the Not I site being maintained. (b) The sequences of the repair products for Guassia luciferase are shown. Both strands of each DNA molecule are represented. The original I-SceI site is displayed on top, with the break shown as a gap in the sequence and the single-stranded overhangs shown in blue/gray type. Two products of repair are shown. Note that the single stranded stretches have been deleted in the repair products and an AA doublet inserted (highlighted). Also note that a single C was deleted in product 2. (c) The sequences of the repair products for ENR fragments are shown. The original I-SceI site is displayed on top, with both strands of the DNA molecule shown and the single-strand overhangs shown in blue/gray. Two products of repair are shown, one observed when the ENR sequence diverged from the endogenous copy by 2% and the second when the sequence diverged by 20%. Base pairs that have been synthesized as part of the repair process are highlighted. Note that the TTAT on the top strand was likely newly synthesized during the repair process since it resides downstream of the inserted AACC stretch. Potential template sequences for repair are underlined with light blue/gray and the corresponding duplicated sequence in dark blue/gray.
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