Formation of Linear Amplicons with Inverted Duplications in <i>Leishmania</i> Requires the MRE11 Nuclease

<div><p>Extrachromosomal DNA amplification is frequent in the protozoan parasite <i>Leishmania</i> 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 <i>Leishmania</i> 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 <i>Leishmania infantum</i> 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 <i>LiMRE11</i> 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 <i>MRE11</i> 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 <i>Leishmania</i> to amplify portions of its genome to respond to a changing environment.</p></div

Detection of gene rearrangements leading to <i>PTR1</i> containing amplicons using PCR assays.

<p>(<b>A</b>) Schematic representation of inverted repeated sequences (arrows depicted as A, A′, B, B′, C, C′, D, D′, E, E′) and direct repeats (F, F′) at the <i>PTR1</i> chromosomal locus on chromosome 23. Arrowheads (depicted as a, a′, b, b′, c, c′, d, d′, e, e′, f and f′) indicate position and orientation of PCR primers that were used to detect amplicon junctions. (<b>B–E</b>) PCR amplification of newly formed amplicon junctions in ten MTX-resistant clones derived either from WT (<b>B</b>), <i>HYG/NEO MRE11^−/−</i> (<b>C</b>), <i>HYG/PUR-MRE11</i>^WT (<b>D</b>) and <i>HYG/PUR-MRE11</i>^H210Y (<b>E</b>).</p

<i>MRE11</i> gene inactivation in <i>L. infantum</i> and phenotypic analysis.

<p>(<b>A</b>) Schematic representation of the <i>MRE11</i> locus in <i>L. infantum</i> before and after integration of the inactivation cassettes neomycin phosphotransferase (5′-<i>NEO-3′</i>) and hygromycin phosphotransferase B (5′-<i>HYG-3′</i>) generating the double knockout strain <i>HYG/NEO MRE11^−/−</i>. A revertant was obtained by the integration of the re-expressing <i>MRE11</i>^WT or <i>MRE11</i>^H210Y puromycin cassettes (5′-<i>MRE11</i>^WT-α-<i>PUR-3′</i> and <i>5′-MRE11</i>^H210Y-<i>α-PUR-3′</i>) to replace the <i>NEO</i> allele, given respectively strains <i>HYG/PUR-MRE11</i>^WT and <i>HYG/PUR-MRE11</i>^H210Y. X, XhoI restriction sites. (<b>B</b>) Southern blot analysis with genomic DNAs digested with XhoI from the <i>L. infantum</i> WT strain (lanes 1 and 5) and recombinant clones of the double knockout <i>HYG/NEO MRE11^−/−</i> (lanes 2 and 6), <i>HYG/PUR-MRE11</i>^WT (lanes 3 and 7) and <i>HYG/PUR-MRE11</i>^H210Y parasites (lanes 4 and 8). Hybridizations with a probe covering either the 5′ or 3′ flanking region of <i>LiMRE11</i> are shown. (<b>C</b>) Growth retardation of promastigotes <i>MRE11</i> null mutants. <i>L. infantum</i> WT (white circles), <i>HYG/NEO MRE11^−/−</i> (black squares), <i>HYG/PUR-MRE11</i>^WT (black triangles), <i>HYG/PUR-MRE11</i>^H210Y (inverted white triangles). (<b>D</b>) Susceptibility to methylmethane sulfonate (MMS). <i>L. infantum</i> WT (white circles), <i>HYG/NEO MRE11^−/−</i> (black squares), <i>HYG/PUR-MRE11</i>^WT (black triangles), <i>HYG/PUR-MRE11</i>^H210Y (inverted white triangles).</p

Purification and DNA binding of the <i>L. infantum</i> MRE11 protein.

<p>(<b>A</b>) Alignment of <i>L. infantum</i> and human MRE11 proteins showing the conserved catalytic residue (H) that has been mutated in LiMRE11 (H210Y) to generate the LiMRE11^H210Y mutated version and purification of LiMRE11^WT and LiMRE11^H210Y 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 LiMRE11^WT; lane 3: purified LiMRE11^H210Y. (<b>B</b>) LiMRE11^WT and mutant H210Y can bind various DNA structures. Competition electrophoretic mobility shift assays were performed with LiMRE11^WT (lanes 2–4) and LiMRE11^H210Y (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). (<b>C</b>) Quantification of the DNA binding signals of panel <b>B</b>.</p

<i>PTR1</i> gene amplification of <i>L. infantum</i> methotrexate (MTX) resistant cells.

<p><i>L. infantum</i> cells were selected for MTX resistance, and their chromosomes were separated by pulsed-field gel electrophoresis using a separation range between 150 kb and 1500 kb, transferred on membranes then hybridized with a <i>PTR1</i> probe. MTX-resistant clones resistant to 1600 nM MTX derived from the WT (<b>A</b>), the <i>HYG/NEO MRE11^−/−</i> cells (<b>B</b>), the <i>HYG/PUR-MRE11</i>^WT cells (<b>C</b>) and the <i>HYG/PUR-MRE11</i>^H210Y cells (<b>D</b>). Lanes 0 are parasites without drug selection.</p

Fluorescence recovery after photobleaching analysis after DNA damage induction in UV-irradiated cells.

<p>LiMRE11-GFP is recruited to DNA damage sites in human MRE11-deficient cells (ATLD), as the human MRE11-GFP.</p

Potential mechanisms for the formation of extrachromosomal linear amplicons.

<p>(<b>1</b>) 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). (<b>2</b>) 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.</p