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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

MRE11 gene inactivation in L. infantum and phenotypic analysis.(A) Schematic representation of the MRE11 locus in L. infantum before and after integration of the inactivation cassettes neomycin phosphotransferase (5′-NEO-3′) and hygromycin phosphotransferase B (5′-HYG-3′) generating the double knockout strain HYG/NEO MRE11−/−. A revertant was obtained by the integration of the re-expressing MRE11WT or MRE11H210Y puromycin cassettes (5′-MRE11WT-α-PUR-3′ and 5′-MRE11H210Y-α-PUR-3′) to replace the NEO allele, given respectively strains HYG/PUR-MRE11WT and HYG/PUR-MRE11H210Y. X, XhoI restriction sites. (B) Southern blot analysis with genomic DNAs digested with XhoI from the L. infantum WT strain (lanes 1 and 5) and recombinant clones of the double knockout HYG/NEO MRE11−/− (lanes 2 and 6), HYG/PUR-MRE11WT (lanes 3 and 7) and HYG/PUR-MRE11H210Y parasites (lanes 4 and 8). Hybridizations with a probe covering either the 5′ or 3′ flanking region of LiMRE11 are shown. (C) Growth retardation of promastigotes MRE11  mutants. L. infantum WT (white circles), HYG/NEO MRE11−/− (black squares), HYG/PUR-MRE11WT (black triangles), HYG/PUR-MRE11H210Y (inverted white triangles). (D) Susceptibility to methylmethane sulfonate (MMS). L. infantum WT (white circles), HYG/NEO MRE11−/− (black squares), HYG/PUR-MRE11WT (black triangles), HYG/PUR-MRE11H210Y (inverted white triangles).
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pgen-1004805-g005: MRE11 gene inactivation in L. infantum and phenotypic analysis.(A) Schematic representation of the MRE11 locus in L. infantum before and after integration of the inactivation cassettes neomycin phosphotransferase (5′-NEO-3′) and hygromycin phosphotransferase B (5′-HYG-3′) generating the double knockout strain HYG/NEO MRE11−/−. A revertant was obtained by the integration of the re-expressing MRE11WT or MRE11H210Y puromycin cassettes (5′-MRE11WT-α-PUR-3′ and 5′-MRE11H210Y-α-PUR-3′) to replace the NEO allele, given respectively strains HYG/PUR-MRE11WT and HYG/PUR-MRE11H210Y. X, XhoI restriction sites. (B) Southern blot analysis with genomic DNAs digested with XhoI from the L. infantum WT strain (lanes 1 and 5) and recombinant clones of the double knockout HYG/NEO MRE11−/− (lanes 2 and 6), HYG/PUR-MRE11WT (lanes 3 and 7) and HYG/PUR-MRE11H210Y parasites (lanes 4 and 8). Hybridizations with a probe covering either the 5′ or 3′ flanking region of LiMRE11 are shown. (C) Growth retardation of promastigotes MRE11 mutants. L. infantum WT (white circles), HYG/NEO MRE11−/− (black squares), HYG/PUR-MRE11WT (black triangles), HYG/PUR-MRE11H210Y (inverted white triangles). (D) Susceptibility to methylmethane sulfonate (MMS). L. infantum WT (white circles), HYG/NEO MRE11−/− (black squares), HYG/PUR-MRE11WT (black triangles), HYG/PUR-MRE11H210Y (inverted white triangles).

Mentions: L. infantum MRE11 mutant parasites were generated by replacing the entire ORF (LinJ27.1790) with genes coding for the neomycin (NEO) and hygromycin (HYG) phosphotransferases. The two resistant markers were cloned between the 5′- and 3′-MRE11 flanking regions and targeting constructs were transfected independently in two rounds by electroporation. Southern blot analysis confirmed the homologous chromosomal integration of the two antibiotic markers in the MRE11 locus (Figures 5A and 5B). Genomic DNAs of the WT and the HYG/NEO MRE11−/− mutant were digested with XhoI, transferred onto membranes and hybridized. Hybridization with a probe recognizing the 5′UTR region of MRE11 yielded a 3 kb band in WT cells (Figure 5A and 5B-lane 1) while hybridization with a 3′UTR probe generated a 3,4 kb band as expected (Figure 5A and 5B, lane 5). In the HYG/NEO MRE11−/− strain, replacement of both MRE11 wild-type alleles by NEO and HYG led, as expected, to 4,7 kb and 4,9 kb bands respectively, with either UTR probes (Figure 5A and 5B, lanes 2 and 6).


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)

MRE11 gene inactivation in L. infantum and phenotypic analysis.(A) Schematic representation of the MRE11 locus in L. infantum before and after integration of the inactivation cassettes neomycin phosphotransferase (5′-NEO-3′) and hygromycin phosphotransferase B (5′-HYG-3′) generating the double knockout strain HYG/NEO MRE11−/−. A revertant was obtained by the integration of the re-expressing MRE11WT or MRE11H210Y puromycin cassettes (5′-MRE11WT-α-PUR-3′ and 5′-MRE11H210Y-α-PUR-3′) to replace the NEO allele, given respectively strains HYG/PUR-MRE11WT and HYG/PUR-MRE11H210Y. X, XhoI restriction sites. (B) Southern blot analysis with genomic DNAs digested with XhoI from the L. infantum WT strain (lanes 1 and 5) and recombinant clones of the double knockout HYG/NEO MRE11−/− (lanes 2 and 6), HYG/PUR-MRE11WT (lanes 3 and 7) and HYG/PUR-MRE11H210Y parasites (lanes 4 and 8). Hybridizations with a probe covering either the 5′ or 3′ flanking region of LiMRE11 are shown. (C) Growth retardation of promastigotes MRE11  mutants. L. infantum WT (white circles), HYG/NEO MRE11−/− (black squares), HYG/PUR-MRE11WT (black triangles), HYG/PUR-MRE11H210Y (inverted white triangles). (D) Susceptibility to methylmethane sulfonate (MMS). L. infantum WT (white circles), HYG/NEO MRE11−/− (black squares), HYG/PUR-MRE11WT (black triangles), HYG/PUR-MRE11H210Y (inverted white triangles).
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004805-g005: MRE11 gene inactivation in L. infantum and phenotypic analysis.(A) Schematic representation of the MRE11 locus in L. infantum before and after integration of the inactivation cassettes neomycin phosphotransferase (5′-NEO-3′) and hygromycin phosphotransferase B (5′-HYG-3′) generating the double knockout strain HYG/NEO MRE11−/−. A revertant was obtained by the integration of the re-expressing MRE11WT or MRE11H210Y puromycin cassettes (5′-MRE11WT-α-PUR-3′ and 5′-MRE11H210Y-α-PUR-3′) to replace the NEO allele, given respectively strains HYG/PUR-MRE11WT and HYG/PUR-MRE11H210Y. X, XhoI restriction sites. (B) Southern blot analysis with genomic DNAs digested with XhoI from the L. infantum WT strain (lanes 1 and 5) and recombinant clones of the double knockout HYG/NEO MRE11−/− (lanes 2 and 6), HYG/PUR-MRE11WT (lanes 3 and 7) and HYG/PUR-MRE11H210Y parasites (lanes 4 and 8). Hybridizations with a probe covering either the 5′ or 3′ flanking region of LiMRE11 are shown. (C) Growth retardation of promastigotes MRE11 mutants. L. infantum WT (white circles), HYG/NEO MRE11−/− (black squares), HYG/PUR-MRE11WT (black triangles), HYG/PUR-MRE11H210Y (inverted white triangles). (D) Susceptibility to methylmethane sulfonate (MMS). L. infantum WT (white circles), HYG/NEO MRE11−/− (black squares), HYG/PUR-MRE11WT (black triangles), HYG/PUR-MRE11H210Y (inverted white triangles).
Mentions: L. infantum MRE11 mutant parasites were generated by replacing the entire ORF (LinJ27.1790) with genes coding for the neomycin (NEO) and hygromycin (HYG) phosphotransferases. The two resistant markers were cloned between the 5′- and 3′-MRE11 flanking regions and targeting constructs were transfected independently in two rounds by electroporation. Southern blot analysis confirmed the homologous chromosomal integration of the two antibiotic markers in the MRE11 locus (Figures 5A and 5B). Genomic DNAs of the WT and the HYG/NEO MRE11−/− mutant were digested with XhoI, transferred onto membranes and hybridized. Hybridization with a probe recognizing the 5′UTR region of MRE11 yielded a 3 kb band in WT cells (Figure 5A and 5B-lane 1) while hybridization with a 3′UTR probe generated a 3,4 kb band as expected (Figure 5A and 5B, lane 5). In the HYG/NEO MRE11−/− strain, replacement of both MRE11 wild-type alleles by NEO and HYG led, as expected, to 4,7 kb and 4,9 kb bands respectively, with either UTR probes (Figure 5A and 5B, lanes 2 and 6).

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