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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.


Related in: MedlinePlus

PTR1 gene amplification of L. infantum methotrexate (MTX) resistant cells.L. infantum cells were selected for MTX resistance, and their chromosomes were separated by pulsed-field gel electrophoresis using a separation range between 150 kb and 1500 kb, transferred on membranes then hybridized with a PTR1 probe. MTX-resistant clones resistant to 1600 nM MTX derived from the WT (A), the HYG/NEO MRE11−/− cells (B), the HYG/PUR-MRE11WT cells (C) and the HYG/PUR-MRE11H210Y cells (D). Lanes 0 are parasites without drug selection.
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pgen-1004805-g006: PTR1 gene amplification of L. infantum methotrexate (MTX) resistant cells.L. infantum cells were selected for MTX resistance, and their chromosomes were separated by pulsed-field gel electrophoresis using a separation range between 150 kb and 1500 kb, transferred on membranes then hybridized with a PTR1 probe. MTX-resistant clones resistant to 1600 nM MTX derived from the WT (A), the HYG/NEO MRE11−/− cells (B), the HYG/PUR-MRE11WT cells (C) and the HYG/PUR-MRE11H210Y cells (D). Lanes 0 are parasites without drug selection.

Mentions: We compared the ability of the MRE11 mutants and WT cells to generate extrachromosomal linear amplicons. We selected clones of wild-type cells and of HYG/NEO MRE11−/− for MTX resistance in a stepwise manner (up to 1600 nM, a 16-fold increase in resistance compared to starting parent cells), a drug known to select for PTR1 linear DNA amplifications [13], [18], [28]. Leishmania chromosomes extracted from ten MTX resistant clones derived from either WT or HYG/NEO MRE11−/− parasites were separated by pulse field gel electrophoresis (PFGE) and hybridized with a PTR1 probe. Ethidium bromide stained gels already indicated that some linear amplicons smaller than the smallest chromosome were present in some resistant clones derived from WT but not in the MTX resistant MRE11−/− mutants (Figure S4). Hybridization data revealed that all ten MTX resistant clones derived from WT cells displayedPTR1 linear amplicons of varying size of 125 kb, 250 kb, 450 kb and 565 kb (Figure 6A). Clones 6 and 7 also gave rise to PTR1 circular amplicons, as suggested from the hybridizing smears (Figure 6A). The situation was drastically different in the HYG/NEO MRE11−/− parasites selected for MTX resistance. We observed only one resistant clone from the MRE11 mutant with a faint hybridization signal corresponding to a PTR1 linear amplification (Figure 6B, clone 1), while a PTR1 circular amplification was present in clones 4 and 5 derived from the MRE11−/− mutant (Figure 6B). Clone 3 displayed a hybridization signal at around 1150 kb (Figure 6B) which could correspond to either a very large linear amplicon or to a chromosomal translocation. The difference in formation of linear amplicons between WT and MRE11−/− mutant was found to be significant (p<0,01). We also selected the add back strains HYG/PUR-MRE11WT and HYG/PUR-MRE11H210Y for MTX resistance for testing for the specificity of the phenotype and for assessing the role of the MRE11-exonuclease activity in the generation of linear amplicons. While the MRE11−/− mutants had a decreased capacity to generate linear amplicons after MTX selection (Figure 6B), nine out of ten MTX resistant clones derived from the HYG/PUR-MRE11WT add back strain had PTR1 linear amplicons (Figure 6C, clones 1, 2, 4–10). Similar to the mutants derived from the wild-type cells (Figure 6A), four different PTR1 linear amplicons of 125, 250, 450 and 565 kb (Figure 6C) were present and four clones derived from HYG/PUR-MRE11WT had additional PTR1 circular amplicons (Figure 6C, clones 1, 2, 6 and 7). This phenotype reversion was not observed when the MRE11−/− cells were complemented with MRE11WT as part of an episomal construct. Clones derived from the latter transfectants and selected for MTX resistance were similar to the MRE11−/− mutants with no PTR1 linear amplicons (Figure S5). The results were even more surprising with the MTX resistant clones derived from HYG/PUR-MRE11H210Y. Strikingly all mutants had circular amplifications and the PTR1 hybridization intensity was in general much higher suggesting a higher copy number of the circles. Four clones derived from this add-back revertant also had a PTR1 linear amplicon (Figure 6D, clones 2, 7, 9, 10).


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)

PTR1 gene amplification of L. infantum methotrexate (MTX) resistant cells.L. infantum cells were selected for MTX resistance, and their chromosomes were separated by pulsed-field gel electrophoresis using a separation range between 150 kb and 1500 kb, transferred on membranes then hybridized with a PTR1 probe. MTX-resistant clones resistant to 1600 nM MTX derived from the WT (A), the HYG/NEO MRE11−/− cells (B), the HYG/PUR-MRE11WT cells (C) and the HYG/PUR-MRE11H210Y cells (D). Lanes 0 are parasites without drug selection.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004805-g006: PTR1 gene amplification of L. infantum methotrexate (MTX) resistant cells.L. infantum cells were selected for MTX resistance, and their chromosomes were separated by pulsed-field gel electrophoresis using a separation range between 150 kb and 1500 kb, transferred on membranes then hybridized with a PTR1 probe. MTX-resistant clones resistant to 1600 nM MTX derived from the WT (A), the HYG/NEO MRE11−/− cells (B), the HYG/PUR-MRE11WT cells (C) and the HYG/PUR-MRE11H210Y cells (D). Lanes 0 are parasites without drug selection.
Mentions: We compared the ability of the MRE11 mutants and WT cells to generate extrachromosomal linear amplicons. We selected clones of wild-type cells and of HYG/NEO MRE11−/− for MTX resistance in a stepwise manner (up to 1600 nM, a 16-fold increase in resistance compared to starting parent cells), a drug known to select for PTR1 linear DNA amplifications [13], [18], [28]. Leishmania chromosomes extracted from ten MTX resistant clones derived from either WT or HYG/NEO MRE11−/− parasites were separated by pulse field gel electrophoresis (PFGE) and hybridized with a PTR1 probe. Ethidium bromide stained gels already indicated that some linear amplicons smaller than the smallest chromosome were present in some resistant clones derived from WT but not in the MTX resistant MRE11−/− mutants (Figure S4). Hybridization data revealed that all ten MTX resistant clones derived from WT cells displayedPTR1 linear amplicons of varying size of 125 kb, 250 kb, 450 kb and 565 kb (Figure 6A). Clones 6 and 7 also gave rise to PTR1 circular amplicons, as suggested from the hybridizing smears (Figure 6A). The situation was drastically different in the HYG/NEO MRE11−/− parasites selected for MTX resistance. We observed only one resistant clone from the MRE11 mutant with a faint hybridization signal corresponding to a PTR1 linear amplification (Figure 6B, clone 1), while a PTR1 circular amplification was present in clones 4 and 5 derived from the MRE11−/− mutant (Figure 6B). Clone 3 displayed a hybridization signal at around 1150 kb (Figure 6B) which could correspond to either a very large linear amplicon or to a chromosomal translocation. The difference in formation of linear amplicons between WT and MRE11−/− mutant was found to be significant (p<0,01). We also selected the add back strains HYG/PUR-MRE11WT and HYG/PUR-MRE11H210Y for MTX resistance for testing for the specificity of the phenotype and for assessing the role of the MRE11-exonuclease activity in the generation of linear amplicons. While the MRE11−/− mutants had a decreased capacity to generate linear amplicons after MTX selection (Figure 6B), nine out of ten MTX resistant clones derived from the HYG/PUR-MRE11WT add back strain had PTR1 linear amplicons (Figure 6C, clones 1, 2, 4–10). Similar to the mutants derived from the wild-type cells (Figure 6A), four different PTR1 linear amplicons of 125, 250, 450 and 565 kb (Figure 6C) were present and four clones derived from HYG/PUR-MRE11WT had additional PTR1 circular amplicons (Figure 6C, clones 1, 2, 6 and 7). This phenotype reversion was not observed when the MRE11−/− cells were complemented with MRE11WT as part of an episomal construct. Clones derived from the latter transfectants and selected for MTX resistance were similar to the MRE11−/− mutants with no PTR1 linear amplicons (Figure S5). The results were even more surprising with the MTX resistant clones derived from HYG/PUR-MRE11H210Y. Strikingly all mutants had circular amplifications and the PTR1 hybridization intensity was in general much higher suggesting a higher copy number of the circles. Four clones derived from this add-back revertant also had a PTR1 linear amplicon (Figure 6D, clones 2, 7, 9, 10).

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.


Related in: MedlinePlus