Translocation breakpoints mapping and genomic features at the breakpoints.

<p>Translocation breakpoints mapping and genomic features at the breakpoints.</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 between chromosome 12 and 06 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 12–06 between chromosomes 12 and 06. <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.12.0671 (■), LinJ.12.0690 (□), LinJ.06.0470 (○) and LinJ.06.0480 (●). 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> Reads were mapped to the <i>L</i>. <i>infantum</i> JPCM5 genome and log₂-transformed normalized read counts for non-overlapping 5 kb genomic windows are shown for chromosomes 12 and 06. Chromosome 12 is triploid in our <i>L</i>. <i>infantum</i> JPCM5 WT strain and a log₂ fold change of 0,5 would suggest that chromosome 12 is tetraploid in <i>L</i>. <i>infantum</i> 263 WT. 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

<i>RAD50</i> gene conditional inactivation in <i>L</i>. <i>infantum</i>.

<p><b>(A)</b> Schematic representation of the <i>RAD50</i> locus in <i>L</i>. <i>infantum</i> before and after integration of the inactivation cassettes blasticidin-S deaminase (5’-<i>BLAST-3’</i>), puromycin acetyltransferase (5’-<i>PURO-3’</i>) and transfection construct Psp-<i>NEO</i>-<i>RAD50</i>. S, SacI restriction sites. <b>(B, C)</b> Southern blot analysis with genomic DNAs digested with SacI were hybridized with probes covering either the 5’ flanking region of <i>RAD50</i> <b>(B)</b> or the <i>RAD50</i> ORF <b>(C). (D)</b> PCR analysis with primers set aa’ and bb’ the chromosomal copies of the <i>MRE11</i> and <i>RAD50</i> genes respectively. Lanes: 1, <i>L</i>.<i>infantum</i> WT; 2, WT Psp-<i>NEO-RAD50</i>; 3, WT Rev Psp-<i>NEO</i>-<i>RAD50</i>; 4, <i>RAD50</i>^<i>-/-</i> Psp-<i>NEO</i>-<i>RAD50</i>; 5, <i>RAD50</i>^<i>-/-</i> Rev Psp-<i>NEO</i>-<i>RAD50</i> grown for 55 passages in absence of G418; 6, <i>MRE11</i>^<i>-/-</i> and 7, <i>MRE11</i>^<i>-/-</i><i>RAD50</i>^<i>-/-</i>.</p

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

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

Translocation events in <i>L</i>. <i>infantum MRE11</i> and <i>RAD50</i> null mutants.

<p><b>(A, C, E)</b> Schematic representation of the translocations T 12–17 <b>(A)</b>, T 12–18 <b>(C)</b> and T 18–20 <b>(E)</b>. <b>(B, D, F)</b> <i>L</i>. <i>infantum</i> chromosomes were separated by pulsed-field gel electrophoresis, transferred on membranes then hybridized with probes from LinJ.17.1180 (■), LinJ.18.1520 (●), LinJ.20.1570 (▲). 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>.</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

<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

Sequence microhomology at translocation breakpoints.

<p>DNA sequences obtained from direct sequencing of the junctions T 12–06 <b>(A)</b>, T 12–18 <b>(B)</b>, T 18–20 <b>(C)</b> and T 08–17 <b>(D)</b>. Sequences of wild-type parasites found close to breakpoints were aligned to the respective chromosomes involved as well as the resulting hybrid chromosome. Microhomology sequences are highlighted in capital bold letters. Asterisk indicates that the sequence corresponds to the antisense strand.</p