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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

Fluorescence recovery after photobleaching analysis after DNA damage induction in UV-irradiated cells.LiMRE11-GFP is recruited to DNA damage sites in human MRE11-deficient cells (ATLD), as the human MRE11-GFP.
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pgen-1004805-g004: Fluorescence recovery after photobleaching analysis after DNA damage induction in UV-irradiated cells.LiMRE11-GFP is recruited to DNA damage sites in human MRE11-deficient cells (ATLD), as the human MRE11-GFP.

Mentions: We generated a LiMRE11-GFP fusion construct that was transfected in L. infantum cells. However, we could never achieve a high copy number of the plasmid (overexpression of MRE11 can be toxic to the cell, see below) and fluorescence levels were too low for analysis. We then turned to a heterologous system to study LiMRE11 in vivo. DNA constructs encoding the fusion protein LiMRE11WT-GFP and the human counterpart hMRE11WT-GFP were transfected in human ATLD cells, which are deficient for hMRE11 [43]. After laser-induced DNA damage in these cells, we detected a localized fluorescent foci representative of the recruitment of LiMRE11WT-GFP in micro-irradiated nuclear regions (Figure 4, upper panels), similar to what was observed for the human MRE11WT-GFP fusion protein (Figure 4, bottom panels). Among 24 ATLD cells micro-irradiated, we observed a recruitment of LiMRE11 to DNA damages sites in 75% of the cases, while the human homolog was recruited in 100% of the cells. No recruitment was observed for the control GFP alone. These observations confirmed the ability of the Leishmania MRE11 protein to be recruited at DNA damage sites, in a heterologous cellular model. Altogether, these results show that LiMRE11 display similar localization properties as the human enzyme.


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)

Fluorescence recovery after photobleaching analysis after DNA damage induction in UV-irradiated cells.LiMRE11-GFP is recruited to DNA damage sites in human MRE11-deficient cells (ATLD), as the human MRE11-GFP.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004805-g004: Fluorescence recovery after photobleaching analysis after DNA damage induction in UV-irradiated cells.LiMRE11-GFP is recruited to DNA damage sites in human MRE11-deficient cells (ATLD), as the human MRE11-GFP.
Mentions: We generated a LiMRE11-GFP fusion construct that was transfected in L. infantum cells. However, we could never achieve a high copy number of the plasmid (overexpression of MRE11 can be toxic to the cell, see below) and fluorescence levels were too low for analysis. We then turned to a heterologous system to study LiMRE11 in vivo. DNA constructs encoding the fusion protein LiMRE11WT-GFP and the human counterpart hMRE11WT-GFP were transfected in human ATLD cells, which are deficient for hMRE11 [43]. After laser-induced DNA damage in these cells, we detected a localized fluorescent foci representative of the recruitment of LiMRE11WT-GFP in micro-irradiated nuclear regions (Figure 4, upper panels), similar to what was observed for the human MRE11WT-GFP fusion protein (Figure 4, bottom panels). Among 24 ATLD cells micro-irradiated, we observed a recruitment of LiMRE11 to DNA damages sites in 75% of the cases, while the human homolog was recruited in 100% of the cells. No recruitment was observed for the control GFP alone. These observations confirmed the ability of the Leishmania MRE11 protein to be recruited at DNA damage sites, in a heterologous cellular model. Altogether, these results show that LiMRE11 display similar localization properties as the human enzyme.

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