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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.


Exonuclease assays of MRE11 proteins.The Leishmania (A) and human (B) WT MRE11 proteins can perform DNA resection (lanes 1, 2, 3, 4) on dsDNA. Resection activity of the Leishmania MRE11H210Y (A) and human MRE11H217Y (B) (lanes 5, 6, 7). (C) Substrate specificity of LiMRE11WT for resection activity. Two hundred nM of blunt dsDNAs (lane 2), 3′ overhang (lane 3) and 5′overhang (lane 4) were incubated with 25 nM of purified LiMRE11WT protein and run on an 8% acrylamide/urea gel, followed by autoradiography.
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pgen-1004805-g003: Exonuclease assays of MRE11 proteins.The Leishmania (A) and human (B) WT MRE11 proteins can perform DNA resection (lanes 1, 2, 3, 4) on dsDNA. Resection activity of the Leishmania MRE11H210Y (A) and human MRE11H217Y (B) (lanes 5, 6, 7). (C) Substrate specificity of LiMRE11WT for resection activity. Two hundred nM of blunt dsDNAs (lane 2), 3′ overhang (lane 3) and 5′overhang (lane 4) were incubated with 25 nM of purified LiMRE11WT protein and run on an 8% acrylamide/urea gel, followed by autoradiography.

Mentions: We next tested whether purified LiMRE11WT and LiMRE11H210Y displayed exonuclease activity (Figure 3A), in comparison with human MRE11WT and hMRE11H217Y proteins (Figure 3B). Our findings suggest that LiMRE11WT is enzymatically active and can perform exonucleolytic degradation with a 3′ to 5′ polarity but it is less effective than the human MRE11WT protein in cleaving DNA into smaller fragments (Figure 3A–B, lanes 1–4). As expected, LiMRE11H210Y was unable to perform DNA resection (Figure 3A, lanes 5–7), similar to its human mutated counterpart (Figure 3B, lanes 5–7). The substrate specificity was also monitored by using 25 nM of LiMRE11WT protein with DS DNA, either blunt or with 3′ or 5′ overhang DNA structures (Figure 3C). The same extensive degradation was observed with DS DNA and 5′-overhang ends (Figure 3C, lane 2 and 4) while the protein was blocked by 3′-overhang extremities (Figure 3C, lane 3). LiMRE11WT also exhibits endonuclease activity, as shown by the 13 bp band found at the bottom of the gel.


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)

Exonuclease assays of MRE11 proteins.The Leishmania (A) and human (B) WT MRE11 proteins can perform DNA resection (lanes 1, 2, 3, 4) on dsDNA. Resection activity of the Leishmania MRE11H210Y (A) and human MRE11H217Y (B) (lanes 5, 6, 7). (C) Substrate specificity of LiMRE11WT for resection activity. Two hundred nM of blunt dsDNAs (lane 2), 3′ overhang (lane 3) and 5′overhang (lane 4) were incubated with 25 nM of purified LiMRE11WT protein and run on an 8% acrylamide/urea gel, followed by autoradiography.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004805-g003: Exonuclease assays of MRE11 proteins.The Leishmania (A) and human (B) WT MRE11 proteins can perform DNA resection (lanes 1, 2, 3, 4) on dsDNA. Resection activity of the Leishmania MRE11H210Y (A) and human MRE11H217Y (B) (lanes 5, 6, 7). (C) Substrate specificity of LiMRE11WT for resection activity. Two hundred nM of blunt dsDNAs (lane 2), 3′ overhang (lane 3) and 5′overhang (lane 4) were incubated with 25 nM of purified LiMRE11WT protein and run on an 8% acrylamide/urea gel, followed by autoradiography.
Mentions: We next tested whether purified LiMRE11WT and LiMRE11H210Y displayed exonuclease activity (Figure 3A), in comparison with human MRE11WT and hMRE11H217Y proteins (Figure 3B). Our findings suggest that LiMRE11WT is enzymatically active and can perform exonucleolytic degradation with a 3′ to 5′ polarity but it is less effective than the human MRE11WT protein in cleaving DNA into smaller fragments (Figure 3A–B, lanes 1–4). As expected, LiMRE11H210Y was unable to perform DNA resection (Figure 3A, lanes 5–7), similar to its human mutated counterpart (Figure 3B, lanes 5–7). The substrate specificity was also monitored by using 25 nM of LiMRE11WT protein with DS DNA, either blunt or with 3′ or 5′ overhang DNA structures (Figure 3C). The same extensive degradation was observed with DS DNA and 5′-overhang ends (Figure 3C, lane 2 and 4) while the protein was blocked by 3′-overhang extremities (Figure 3C, lane 3). LiMRE11WT also exhibits endonuclease activity, as shown by the 13 bp band found at the bottom of the gel.

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.