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Formation of linear amplicons with inverted duplications in Leishmania requires the MRE11 nuclease.

Laffitte MC, Genois MM, Mukherjee A, Légaré D, Masson JY, Ouellette M - PLoS Genet. (2014)

Bottom Line: Inactivation of the LiMRE11 gene led to parasites with enhanced sensitivity to DNA damaging agents.The MRE11(-/-) parasites had a reduced capacity to form linear amplicons after drug selection, and the reintroduction of an MRE11 allele led to parasites regaining their capacity to generate linear amplicons, but only when MRE11 had an active nuclease activity.These results highlight a novel MRE11-dependent pathway used by Leishmania to amplify portions of its genome to respond to a changing environment.

View Article: PubMed Central - PubMed

Affiliation: Centre de Recherche en Infectiologie du CHU de Québec, Quebec City, Québec, Canada.

ABSTRACT
Extrachromosomal DNA amplification is frequent in the protozoan parasite Leishmania selected for drug resistance. The extrachromosomal amplified DNA is either circular or linear, and is formed at the level of direct or inverted homologous repeated sequences that abound in the Leishmania genome. The RAD51 recombinase plays an important role in circular amplicons formation, but the mechanism by which linear amplicons are formed is unknown. We hypothesized that the Leishmania infantum DNA repair protein MRE11 is required for linear amplicons following rearrangements at the level of inverted repeats. The purified LiMRE11 protein showed both DNA binding and exonuclease activities. Inactivation of the LiMRE11 gene led to parasites with enhanced sensitivity to DNA damaging agents. The MRE11(-/-) parasites had a reduced capacity to form linear amplicons after drug selection, and the reintroduction of an MRE11 allele led to parasites regaining their capacity to generate linear amplicons, but only when MRE11 had an active nuclease activity. These results highlight a novel MRE11-dependent pathway used by Leishmania to amplify portions of its genome to respond to a changing environment.

No MeSH data available.


Detection of gene rearrangements leading to PTR1 containing amplicons using PCR assays.(A) Schematic representation of inverted repeated sequences (arrows depicted as A, A′, B, B′, C, C′, D, D′, E, E′) and direct repeats (F, F′) at the PTR1 chromosomal locus on chromosome 23. Arrowheads (depicted as a, a′, b, b′, c, c′, d, d′, e, e′, f and f′) indicate position and orientation of PCR primers that were used to detect amplicon junctions. (B–E) PCR amplification of newly formed amplicon junctions in ten MTX-resistant clones derived either from WT (B), HYG/NEO MRE11−/− (C), HYG/PUR-MRE11WT (D) and HYG/PUR-MRE11H210Y (E).
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pgen-1004805-g007: Detection of gene rearrangements leading to PTR1 containing amplicons using PCR assays.(A) Schematic representation of inverted repeated sequences (arrows depicted as A, A′, B, B′, C, C′, D, D′, E, E′) and direct repeats (F, F′) at the PTR1 chromosomal locus on chromosome 23. Arrowheads (depicted as a, a′, b, b′, c, c′, d, d′, e, e′, f and f′) indicate position and orientation of PCR primers that were used to detect amplicon junctions. (B–E) PCR amplification of newly formed amplicon junctions in ten MTX-resistant clones derived either from WT (B), HYG/NEO MRE11−/− (C), HYG/PUR-MRE11WT (D) and HYG/PUR-MRE11H210Y (E).

Mentions: Previous data has indicated that linear amplicons are constituted of inverted duplications rearranged at the level of IRs with the formation of a new junction that can be amplified by PCR (see Figure 1). The diversity in size of linear amplicons observed in Figure 6A and 6C would suggest that different IRs were used and we tested for the presence of IRs in the chromosome 23 that could lead to PTR1 amplicons with size (125, 250, 450 and 565 kb) consistent with what observed in the blots. We detected a potential of 5 such IRs with size ranging from 440 to 790 bp and with a minimum of 85% identity (Figure 7A), a finding consistent with our demonstration that low copy repeated sequences are widespread throughout the genomes [19]. We performed PCR assays using five different pairs of primers recognizing the five different pairs of IRs under the principle shown in Figure 1. Amplification of the GAPDH gene was also done as a control. In the ten MTX resistant clones derived from WT cells and the HYG/PUR-MRE11WT add back strain, we detected several junctions by PCR (Figure 7B and D). The junction formed following a rearrangement at the level of IRs AA′ was the most frequently observed rearrangement, but junctions BB′, EE′ were also detected frequently while the junctions DD′ and CC′ were detected once in clones derived respectively from the WT or add back strains (Figures 7B and 7D). In clones in which we detected circles by Southern blots (Figure 6), we also obtained a positive signal for junction FF′ where the repeats are in direct orientation (Figure 7). There was a general good agreement between the number of amplicons detected by southern blots and PCR although PCR was more sensitive. For example, we could not detect a linear amplicon in clone 2 in MRE11−/− (Figure 6B) but it had a positive PCR reaction for the junction AA′ (Figure 7C).


Formation of linear amplicons with inverted duplications in Leishmania requires the MRE11 nuclease.

Laffitte MC, Genois MM, Mukherjee A, Légaré D, Masson JY, Ouellette M - PLoS Genet. (2014)

Detection of gene rearrangements leading to PTR1 containing amplicons using PCR assays.(A) Schematic representation of inverted repeated sequences (arrows depicted as A, A′, B, B′, C, C′, D, D′, E, E′) and direct repeats (F, F′) at the PTR1 chromosomal locus on chromosome 23. Arrowheads (depicted as a, a′, b, b′, c, c′, d, d′, e, e′, f and f′) indicate position and orientation of PCR primers that were used to detect amplicon junctions. (B–E) PCR amplification of newly formed amplicon junctions in ten MTX-resistant clones derived either from WT (B), HYG/NEO MRE11−/− (C), HYG/PUR-MRE11WT (D) and HYG/PUR-MRE11H210Y (E).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4256157&req=5

pgen-1004805-g007: Detection of gene rearrangements leading to PTR1 containing amplicons using PCR assays.(A) Schematic representation of inverted repeated sequences (arrows depicted as A, A′, B, B′, C, C′, D, D′, E, E′) and direct repeats (F, F′) at the PTR1 chromosomal locus on chromosome 23. Arrowheads (depicted as a, a′, b, b′, c, c′, d, d′, e, e′, f and f′) indicate position and orientation of PCR primers that were used to detect amplicon junctions. (B–E) PCR amplification of newly formed amplicon junctions in ten MTX-resistant clones derived either from WT (B), HYG/NEO MRE11−/− (C), HYG/PUR-MRE11WT (D) and HYG/PUR-MRE11H210Y (E).
Mentions: Previous data has indicated that linear amplicons are constituted of inverted duplications rearranged at the level of IRs with the formation of a new junction that can be amplified by PCR (see Figure 1). The diversity in size of linear amplicons observed in Figure 6A and 6C would suggest that different IRs were used and we tested for the presence of IRs in the chromosome 23 that could lead to PTR1 amplicons with size (125, 250, 450 and 565 kb) consistent with what observed in the blots. We detected a potential of 5 such IRs with size ranging from 440 to 790 bp and with a minimum of 85% identity (Figure 7A), a finding consistent with our demonstration that low copy repeated sequences are widespread throughout the genomes [19]. We performed PCR assays using five different pairs of primers recognizing the five different pairs of IRs under the principle shown in Figure 1. Amplification of the GAPDH gene was also done as a control. In the ten MTX resistant clones derived from WT cells and the HYG/PUR-MRE11WT add back strain, we detected several junctions by PCR (Figure 7B and D). The junction formed following a rearrangement at the level of IRs AA′ was the most frequently observed rearrangement, but junctions BB′, EE′ were also detected frequently while the junctions DD′ and CC′ were detected once in clones derived respectively from the WT or add back strains (Figures 7B and 7D). In clones in which we detected circles by Southern blots (Figure 6), we also obtained a positive signal for junction FF′ where the repeats are in direct orientation (Figure 7). There was a general good agreement between the number of amplicons detected by southern blots and PCR although PCR was more sensitive. For example, we could not detect a linear amplicon in clone 2 in MRE11−/− (Figure 6B) but it had a positive PCR reaction for the junction AA′ (Figure 7C).

Bottom Line: Inactivation of the LiMRE11 gene led to parasites with enhanced sensitivity to DNA damaging agents.The MRE11(-/-) parasites had a reduced capacity to form linear amplicons after drug selection, and the reintroduction of an MRE11 allele led to parasites regaining their capacity to generate linear amplicons, but only when MRE11 had an active nuclease activity.These results highlight a novel MRE11-dependent pathway used by Leishmania to amplify portions of its genome to respond to a changing environment.

View Article: PubMed Central - PubMed

Affiliation: Centre de Recherche en Infectiologie du CHU de Québec, Quebec City, Québec, Canada.

ABSTRACT
Extrachromosomal DNA amplification is frequent in the protozoan parasite Leishmania selected for drug resistance. The extrachromosomal amplified DNA is either circular or linear, and is formed at the level of direct or inverted homologous repeated sequences that abound in the Leishmania genome. The RAD51 recombinase plays an important role in circular amplicons formation, but the mechanism by which linear amplicons are formed is unknown. We hypothesized that the Leishmania infantum DNA repair protein MRE11 is required for linear amplicons following rearrangements at the level of inverted repeats. The purified LiMRE11 protein showed both DNA binding and exonuclease activities. Inactivation of the LiMRE11 gene led to parasites with enhanced sensitivity to DNA damaging agents. The MRE11(-/-) parasites had a reduced capacity to form linear amplicons after drug selection, and the reintroduction of an MRE11 allele led to parasites regaining their capacity to generate linear amplicons, but only when MRE11 had an active nuclease activity. These results highlight a novel MRE11-dependent pathway used by Leishmania to amplify portions of its genome to respond to a changing environment.

No MeSH data available.