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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.


Potential mechanisms for the formation of extrachromosomal linear amplicons.(1) Single-strand hairpin formation (a) or single-strand break (SSB) (near the IRs) during replication followed by annealing of the IRs (b), 3′-5′ exonuclease digestion of the exposed end (c) and DNA synthesis of the upstream region (d) and of the second strand to form an inverted duplication (g). (2) Double-strand break (DSB) near the IRs (e) followed by 3′-5′ exonuclease digestion at the DNA break of one strand (f), annealing of the IRs to form an inverted duplication (d) and synthesis of the second strand to generate linear amplicon (g). The new junction formed during annealing of the IRs can be detected by PCR using specific primers. IRs, inverted repeated sequences; ss, single-strand; SSB, single-strand break; DSB, double-strand break; p1–p2, primer pair used to detect the new junction. White rectangle: telomeric sequences.
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pgen-1004805-g001: Potential mechanisms for the formation of extrachromosomal linear amplicons.(1) Single-strand hairpin formation (a) or single-strand break (SSB) (near the IRs) during replication followed by annealing of the IRs (b), 3′-5′ exonuclease digestion of the exposed end (c) and DNA synthesis of the upstream region (d) and of the second strand to form an inverted duplication (g). (2) Double-strand break (DSB) near the IRs (e) followed by 3′-5′ exonuclease digestion at the DNA break of one strand (f), annealing of the IRs to form an inverted duplication (d) and synthesis of the second strand to generate linear amplicon (g). The new junction formed during annealing of the IRs can be detected by PCR using specific primers. IRs, inverted repeated sequences; ss, single-strand; SSB, single-strand break; DSB, double-strand break; p1–p2, primer pair used to detect the new junction. White rectangle: telomeric sequences.

Mentions: The protozoan parasite Leishmania is responsible for a group of diseases named leishmaniasis, affecting approximately 12 million people worldwide. No vaccine is currently available against Leishmania and treatments mainly rely on chemotherapy [1], [2]. Pentavalent antimony is the main anti-leishmanial drug although treatment failure due to resistance has been reported in most endemic regions [3]–[7]. Locus amplification is a frequent resistance mechanism allowing the parasite to modulate gene copy number and increased gene expression. Indeed, the parasite Leishmania is an early diverging eukaryotic parasite with no control of gene expression at the level of transcription initiation [8]–[10] and amplification of DNA loci is one strategy to increase the expression of resistance genes. Resistance genes can be amplified as part of extrachromosomal circular DNAs (circular amplicons) or as inverted duplications (linear amplicons) under drug pressure [11]–[16]. Gene rearrangements leading to locus amplification always occur at the level of either homologous direct or inverted repeated (IRs) sequences leading respectively to circular or linear extrachromosomal amplification [14], [15], [17]–[19]. A model for the generation of linear amplicons is shown in Figure 1.


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)

Potential mechanisms for the formation of extrachromosomal linear amplicons.(1) Single-strand hairpin formation (a) or single-strand break (SSB) (near the IRs) during replication followed by annealing of the IRs (b), 3′-5′ exonuclease digestion of the exposed end (c) and DNA synthesis of the upstream region (d) and of the second strand to form an inverted duplication (g). (2) Double-strand break (DSB) near the IRs (e) followed by 3′-5′ exonuclease digestion at the DNA break of one strand (f), annealing of the IRs to form an inverted duplication (d) and synthesis of the second strand to generate linear amplicon (g). The new junction formed during annealing of the IRs can be detected by PCR using specific primers. IRs, inverted repeated sequences; ss, single-strand; SSB, single-strand break; DSB, double-strand break; p1–p2, primer pair used to detect the new junction. White rectangle: telomeric sequences.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004805-g001: Potential mechanisms for the formation of extrachromosomal linear amplicons.(1) Single-strand hairpin formation (a) or single-strand break (SSB) (near the IRs) during replication followed by annealing of the IRs (b), 3′-5′ exonuclease digestion of the exposed end (c) and DNA synthesis of the upstream region (d) and of the second strand to form an inverted duplication (g). (2) Double-strand break (DSB) near the IRs (e) followed by 3′-5′ exonuclease digestion at the DNA break of one strand (f), annealing of the IRs to form an inverted duplication (d) and synthesis of the second strand to generate linear amplicon (g). The new junction formed during annealing of the IRs can be detected by PCR using specific primers. IRs, inverted repeated sequences; ss, single-strand; SSB, single-strand break; DSB, double-strand break; p1–p2, primer pair used to detect the new junction. White rectangle: telomeric sequences.
Mentions: The protozoan parasite Leishmania is responsible for a group of diseases named leishmaniasis, affecting approximately 12 million people worldwide. No vaccine is currently available against Leishmania and treatments mainly rely on chemotherapy [1], [2]. Pentavalent antimony is the main anti-leishmanial drug although treatment failure due to resistance has been reported in most endemic regions [3]–[7]. Locus amplification is a frequent resistance mechanism allowing the parasite to modulate gene copy number and increased gene expression. Indeed, the parasite Leishmania is an early diverging eukaryotic parasite with no control of gene expression at the level of transcription initiation [8]–[10] and amplification of DNA loci is one strategy to increase the expression of resistance genes. Resistance genes can be amplified as part of extrachromosomal circular DNAs (circular amplicons) or as inverted duplications (linear amplicons) under drug pressure [11]–[16]. Gene rearrangements leading to locus amplification always occur at the level of either homologous direct or inverted repeated (IRs) sequences leading respectively to circular or linear extrachromosomal amplification [14], [15], [17]–[19]. A model for the generation of linear amplicons is shown in Figure 1.

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.