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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

Purification and DNA binding of the L. infantum MRE11 protein.(A) Alignment of L. infantum and human MRE11 proteins showing the conserved catalytic residue (H) that has been mutated in LiMRE11 (H210Y) to generate the LiMRE11H210Y mutated version and purification of LiMRE11WT and LiMRE11H210Y followed by SDS–PAGE separation. Purified proteins (150 ng) were loaded on an 8% SDS-PAGE, run then stained with Coomassie blue (GE Healthcare). Lane 1: molecular weight markers (Bio-Rad Laboratories); lane 2: purified LiMRE11WT; lane 3: purified LiMRE11H210Y. (B) LiMRE11WT and mutant H210Y can bind various DNA structures. Competition electrophoretic mobility shift assays were performed with LiMRE11WT (lanes 2–4) and LiMRE11H210Y (lanes 5–7) and 25 nM of ssDNA (SS), dsDNA (DS) and splayed arm (SA) substrates with increasing concentration of the proteins (0, 5, 10, 15 nM). (C) Quantification of the DNA binding signals of panel B.
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pgen-1004805-g002: Purification and DNA binding of the L. infantum MRE11 protein.(A) Alignment of L. infantum and human MRE11 proteins showing the conserved catalytic residue (H) that has been mutated in LiMRE11 (H210Y) to generate the LiMRE11H210Y mutated version and purification of LiMRE11WT and LiMRE11H210Y followed by SDS–PAGE separation. Purified proteins (150 ng) were loaded on an 8% SDS-PAGE, run then stained with Coomassie blue (GE Healthcare). Lane 1: molecular weight markers (Bio-Rad Laboratories); lane 2: purified LiMRE11WT; lane 3: purified LiMRE11H210Y. (B) LiMRE11WT and mutant H210Y can bind various DNA structures. Competition electrophoretic mobility shift assays were performed with LiMRE11WT (lanes 2–4) and LiMRE11H210Y (lanes 5–7) and 25 nM of ssDNA (SS), dsDNA (DS) and splayed arm (SA) substrates with increasing concentration of the proteins (0, 5, 10, 15 nM). (C) Quantification of the DNA binding signals of panel B.

Mentions: Since the critical catalytic residues of MRE11 are conserved in Leishmania[41] and the replacement of the histidine (H) at position 217 by a tyrosine (Y) is known to abolish the nuclease activity of the human MRE11 but not its nucleic acid binding property [42], we scrutinized the amino acid alignment between the human and Leishmania sequences and found the equivalent of human H217 at position 210 of LiMRE11 (Figure 2A, upper panel and Figure S1). We therefore produced a LiMRE11 mutated at the corresponding amino acid (LiMRE11H210Y) and used a two-step affinity purification procedure to purify LiMRE11WT and LiMRE11H210Y as described in Material and Methods (Figure 2A, lower panel).


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)

Purification and DNA binding of the L. infantum MRE11 protein.(A) Alignment of L. infantum and human MRE11 proteins showing the conserved catalytic residue (H) that has been mutated in LiMRE11 (H210Y) to generate the LiMRE11H210Y mutated version and purification of LiMRE11WT and LiMRE11H210Y followed by SDS–PAGE separation. Purified proteins (150 ng) were loaded on an 8% SDS-PAGE, run then stained with Coomassie blue (GE Healthcare). Lane 1: molecular weight markers (Bio-Rad Laboratories); lane 2: purified LiMRE11WT; lane 3: purified LiMRE11H210Y. (B) LiMRE11WT and mutant H210Y can bind various DNA structures. Competition electrophoretic mobility shift assays were performed with LiMRE11WT (lanes 2–4) and LiMRE11H210Y (lanes 5–7) and 25 nM of ssDNA (SS), dsDNA (DS) and splayed arm (SA) substrates with increasing concentration of the proteins (0, 5, 10, 15 nM). (C) Quantification of the DNA binding signals of panel B.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004805-g002: Purification and DNA binding of the L. infantum MRE11 protein.(A) Alignment of L. infantum and human MRE11 proteins showing the conserved catalytic residue (H) that has been mutated in LiMRE11 (H210Y) to generate the LiMRE11H210Y mutated version and purification of LiMRE11WT and LiMRE11H210Y followed by SDS–PAGE separation. Purified proteins (150 ng) were loaded on an 8% SDS-PAGE, run then stained with Coomassie blue (GE Healthcare). Lane 1: molecular weight markers (Bio-Rad Laboratories); lane 2: purified LiMRE11WT; lane 3: purified LiMRE11H210Y. (B) LiMRE11WT and mutant H210Y can bind various DNA structures. Competition electrophoretic mobility shift assays were performed with LiMRE11WT (lanes 2–4) and LiMRE11H210Y (lanes 5–7) and 25 nM of ssDNA (SS), dsDNA (DS) and splayed arm (SA) substrates with increasing concentration of the proteins (0, 5, 10, 15 nM). (C) Quantification of the DNA binding signals of panel B.
Mentions: Since the critical catalytic residues of MRE11 are conserved in Leishmania[41] and the replacement of the histidine (H) at position 217 by a tyrosine (Y) is known to abolish the nuclease activity of the human MRE11 but not its nucleic acid binding property [42], we scrutinized the amino acid alignment between the human and Leishmania sequences and found the equivalent of human H217 at position 210 of LiMRE11 (Figure 2A, upper panel and Figure S1). We therefore produced a LiMRE11 mutated at the corresponding amino acid (LiMRE11H210Y) and used a two-step affinity purification procedure to purify LiMRE11WT and LiMRE11H210Y as described in Material and Methods (Figure 2A, lower panel).

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