Resistance levels to 5-FU and MTX in <i>L. infantum</i> 263 WT and 5-FU resistant mutants.

*<p>Rev = revertant strain cultured without drug pressure over 30 passages;</p>**<p>(p<0,005).</p

Role of thymidine kinase (<i>LinJ21.1450</i>) in methotrexate susceptibility.

<p>Growth curves in the presence of methotrexate were determined for <i>L. infantum</i> wild-type cells (lines with circles) and the Lin5FU500.3 mutant (lines with squares), each transfected either with an empty vector (pSP72<i>αHYGα</i>) (black circles and black squares respectively) or with a thymidine kinase expression construct (pSP72<i>αHYGα</i>-<i>LinJ.21.1450</i>) (white circles and white squares respectively). Average of three independent biological replicates. Transfection of <i>TK</i> in the mutant led to MTX susceptibility that was statistically different than the mock control (p<0.05).</p

<i>DHFR-TS</i> amplification and resistance to 5-fluorouracil.

<p>(<b>A</b>). Comparative genomic hybridization (CGH) experiments between wild-type and Lin5FU500.2 cells. Grey, equal amount of DNA between the two strains; black, increased copy number of DNA in the mutant Lin5FU500.2. (<b>B</b>). Southern blots of total digested DNAs isolated from WT and each mutant strains at 0 and 30 passages without drug were hybridized to a specific <i>DHFR-TS</i> probe. The DNA was digested to discriminate the chromosomal copy (C*) from the amplified copy (A*). (<b>C</b>). Role of <i>DHFR-TS</i> in 5-FU resistance. Growth curves of wild-type <i>L. infantum</i> parasites transfected with the expression construct pSP72<i>αHYGα- DHFR-TS</i> (black squares) or with the empty vector pSP72<i>αHYGα</i> (white squares) or the Lin5FU500.2 revertant transfected with pSP72<i>αHYGα- DHFR-TS</i> (black circles) or pSP72<i>αHYGα</i> (white circles). Average of at least three independent experiments. Transfection of <i>DHFR-TS</i> led to 5FU resistance that was statistically significant compared to mock transfectants (p<0.05).</p

Transport activities of uracil and 5-fluorouracil in <i>L. infantum</i> wild-type cells and 5-FU resistant mutants.

<p>(<b>A</b>). Transport of 5-fluorouracil (black squares) and uracil (black triangles) in <i>L. infantum</i> WT cells and in Lin5FU500.4 mutant cells (5-fluorouracil (white squares) and uracil (white triangles)) after 1, 2, 5 and 10 minutes (<b>B</b>). Transport of [³H]-uracil (white bars) competed with 200× (grey bars) or 2000× ratio (black bars) of cold uracil (left panel) or 5-FU (right panel) after a 10 minutes incubation period. (<b>C</b>). Accumulation of [³H]-5-fluorouracil in <i>L. infantum</i> WT 263 strain and in the 5-FU mutants after 10 minutes. Average of three independent biological replicates. * (p<0.05).</p

Resistance to 5-fluorouracil in 5-FU resistant mutants genetically complemented with WT alleles.

<p>Resistance to 5-fluorouracil in 5-FU resistant mutants genetically complemented with WT alleles.</p

Pyrimidine metabolism and linkages with folate metabolism in <i>Leishmania</i>.

<p>Uracil, after its importation by a non-identified transporter, is metabolized into uridine monophosphate (UMP) by the action of the uracil phosphoribosyl transferase (UPRT, <i>LinJ.34.1110</i>) or into deoxy-uridine (dUrd) by the uridine phosphorylase (UP, <i>LinJ.10.1090</i>). The thymidine kinase (TK, <i>LinJ.21.1450</i>) is involved in synthesis of deoxy-uridine monophosphate (dUMP) but also deoxy-thymidine monophosphate (dTMP). The dUMP produced will lead to dTMP through the action of the bifunctional enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS). The drug 5-FU is also transported by the uracil transporter and can be metabolized by UPRT, UP and TK. Abbreviations: F, folate; DHF, dihydrofolate; THF, tetrahydrofolate; SHMTc, cytosolic serine hydroxymethyltransferase; U, uracil; 5FU, 5-fluorouracil; 5-FdUrd, 5-fluoro-deoxy-uridine; 5-FUMP, 5-fluoro-uridine monophosphate; 5-FdUMP, 5-fluoro-deoxy-uridine monophosphate.</p

Chromosomal Translocations in the Parasite <i>Leishmania</i> by a MRE11/RAD50-Independent Microhomology-Mediated End Joining Mechanism

<div><p>The parasite <i>Leishmania</i> often relies on gene rearrangements to survive stressful environments. However, safeguarding a minimum level of genome integrity is important for cell survival. We hypothesized that maintenance of genomic integrity in <i>Leishmania</i> would imply a leading role of the MRE11 and RAD50 proteins considering their role in DNA repair, chromosomal organization and protection of chromosomes ends in other organisms. Attempts to generate <i>RAD50</i> null mutants in a wild-type background failed and we provide evidence that this gene is essential. Remarkably, inactivation of <i>RAD50</i> was possible in a <i>MRE11</i> null mutant that we had previously generated, providing good evidence that <i>RAD50</i> may be dispensable in the absence of <i>MRE11</i>. Inactivation of the <i>MRE11</i> and <i>RAD50</i> genes led to a decreased frequency of homologous recombination and analysis of the null mutants by whole genome sequencing revealed several chromosomal translocations. Sequencing of the junction between translocated chromosomes highlighted microhomology sequences at the level of breakpoint regions. Sequencing data also showed a decreased coverage at subtelomeric locations in many chromosomes in the <i>MRE11</i>^<i>-/-</i><i>RAD50</i>^<i>-/-</i> parasites. This study demonstrates an MRE11-independent microhomology-mediated end-joining mechanism and a prominent role for MRE11 and RAD50 in the maintenance of genomic integrity. Moreover, we suggest the possible involvement of RAD50 in subtelomeric regions stability.</p></div

Translocation breakpoints mapping and genomic features at the breakpoints.

<p>Translocation breakpoints mapping and genomic features at the breakpoints.</p

Gene amplification and rearrangement in <i>L</i>.<i>infantum MRE11</i>^<i>-/-</i><i>RAD50</i>^<i>-/-</i> cells selected for methotrexate (MTX) resistant cells.

<p><b>(A, D)</b><i>L</i>. <i>infantum</i> WT clone (lane +), <b>(B, E)</b> <i>L</i>. <i>infantum MRE11</i>^<i>-/-</i> clone (lane +) and <b>(C, F)</b> <i>L</i>. <i>infantum MRE11</i>^<i>-/-</i><i>RAD50</i>^<i>-/-</i> clones (lanes 1–10) were selected for MTX resistance up to 1600nM MTX, their chromosomes were separated by pulsed-field gel electrophoresis using a separation range between 150kb and 1500kb, transferred on membranes then hybridized with <i>PTR1</i> <b>(A, B, C)</b> and <i>DHFR-TS</i> <b>(D, E, F)</b> probes. Lanes 0 and lanes—are parasites without drug selection.</p

Translocation between chromosome 08 and 17 in <i>L</i>. <i>infantum MRE11</i>^<i>-/-</i><i>RAD50</i>^<i>-/-</i> cells.

<p><b>(A)</b> Schematic representation of the translocation T 08–17 between chromosomes 08 and 17. <b>(B)</b> <i>L</i>. <i>infantum</i> chromosomes were separated by pulsed-field gel electrophoresis, transferred on membranes then hybridized with probes from LinJ.08.0280 (□), LinJ.08.0290 (■), LinJ.17.1130 (○) and LinJ.17.1140 (●). Lanes: 1, <i>L</i>. <i>infantum</i> WT; 2, <i>MRE11</i>^<i>-/-</i> and 3, <i>MRE11</i>^<i>-/-</i><i>RAD50</i>^<i>-/-</i>. <b>(C, D)</b> Log₂-transformed normalized read counts for non-overlapping 5 kb genomic windows on chromosomes 08 and 17. The Y-axis indicates log₂ fold change from an initial diploid state for chromosomes 08 and 17. Arrows indicate direction and breakpoints of the translocations. Blue, <i>L</i>. <i>infantum</i> 263 WT; orange, <i>LiMRE11</i>^<i>-/-</i> and green, <i>LiMRE11</i>^<i>-/-</i><i>RAD50</i>^<i>-/-</i>.</p