Interactions between BRCA2 and RAD51 for promoting homologous recombination in Leishmania infantum.

In most organisms, the primary function of homologous recombination (HR) is to allow genome protection by the faithful repair of DNA double-strand breaks. The vital step of HR is the search for sequence homology, mediated by the RAD51 recombinase, which is stimulated further by proteins mediators such as the tumor suppressor BRCA2. The biochemical interplay between RAD51 and BRCA2 is unknown in Leishmania or Trypanosoma. Here we show that the Leishmania infantum BRCA2 protein possesses several critical features important for the regulation of DNA recombination at the genetic and biochemical level. A BRCA2 null mutant, generated by gene disruption, displayed genomic instability and gene-targeting defects. Furthermore, cytological studies show that LiRAD51 can no longer localize to the nucleus in this mutant. The Leishmania RAD51 and BRCA2 interact together and the purified proteins bind single-strand DNA. Remarkably, LiBRCA2 is a recombination mediator that stimulates the invasion of a resected DNA double-strand break in an undamaged template by LiRAD51 to form a D-loop structure. Collectively, our data show that LiBRCA2 and LiRAD51 promote HR at the genetic and biochemical level in L. infantum, the causative agent of visceral leishmaniasis

Rôles de la recombinaison homologue dans la résistance aux drogues chez le parasite "Leishmania"

Tableau d’honneur de la Faculté des études supérieures et postdoctorales, 2015-2016Bien que l’on attribue généralement un effet néfaste à l’instabilité génomique, pour le parasite Leishmania, elle s’avère pourtant bénéfique. De façon étonnante, ce protozoaire possède la capacité de remanier le nombre de copies de ses gènes pour s’adapter à son environnement et résister aux drogues utilisées contre lui. La présence de séquences répétées identiques près des gènes essentiels à sa survie permet une recombinaison homologue (RH) efficace et entraîne subséquemment la formation d’une copie extrachromosomique supplémentaire du gène ciblé. Ce phénomène de réarrangement génique induit la formation d’amplicons circulaires ou linéaires et est, entre autres, responsable de la résistance aux agents thérapeutiques. À l’heure actuelle, les enzymes impliquées dans ce processus sont peu connues et l’émergence d’un nombre croissant de cas d’infections réfractaires aux traitements favorise l’étude des protéines médiant la RH dans des conditions de résistance. Il a été récemment montré que la formation d’amplicons circulaires est compromise en l’absence du gène LiRad51, mais n’est toutefois pas abolie. Cette observation suggère l’implication d’autres acteurs dans ce processus, alors que le mécanisme par lequel les amplicons linéaires sont formés demeure inconnu. Cette thèse présente la caractérisation biochimique et cellulaire des protéines clés de la RH impliquées dans l’amplification génique chez le parasite Leishmania infantum. Plus précisément, le rôle de LiBrca2, en tant que médiateur de LiRad51, est tout d'abord démontré en raison de son implication essentielle dans la localisation nucléaire de LiRad51 et dans la stimulation d’invasion de brins effectuée par la recombinase. De façon similaire, les paralogues de LiRad51 peuvent, possiblement grâce à leur capacité de lier et d’apparier de l’ADN, eux aussi, stimuler LiRad51 pour l’invasion d’un ADN simple-brin dans un duplex homologue. L’inactivation génique pour l’un d’entre eux (LiRad51-4) a montré un rôle considérable sur l'amplification circulaire, malgré le fait qu’aucune activité d’invasion n’a pu être observée en l’absence de LiRad51. Enfin, en accord avec son rôle bien établi chez l’humain, cette étude confirme que la protéine LiMre11 possède une activité exonucléase et qu’elle serait impliquée dans la production d’amplicons linéaires. Ces résultats mettent en évidence le potentiel thérapeutique des protéines de la réparation de l’ADN, domaine de recherche prometteur pour mieux lutter contre la leishmaniose.Genomic instability is usually known as a harmful hallmark, although in the parasite Leishmania this represents a beneficial feature. Surprisingly, this protozoan takes advantage of its ability to reorganize its genome for adapting itself to different environments as well as for drug resistance. In fact, the presence of identical repeats near essential genes allows homologous recombination (HR) between them, and subsequently causes the formation of an additional extrachromosomally copy of the targeted gene. This phenomenon of gene rearrangement induces circular or linear amplicons which leads to drug resistance. However, little is known about the enzymes involved in this process. In addition, the increasing number of infection cases refractory to treatment promotes the study of proteins mediating HR in these circumstances. It has been shown recently by gene inactivation that LiRad51 recombinase plays an important role in circular amplicon formation but was not essential. This observation suggested the presence of other players involved in this process while the mechanism by which linear amplicons are formed is still unknown. This thesis presents biochemical and cellular characterization of key HR proteins involved in extrachromosomal DNA amplification. We first determine the role of LiBrca2 as a mediator of LiRad51. In particular, LiBrca2 is involved in LiRad51 nuclear localisation and stimulates the homology search performed by the recombinase. Secondly, we show evidence that the LiRad51 paralogs help LiRad51 to promote HR through their capacity to bind and anneal DNA. Similary to LiBrca2, they can stimulate LiRad51 to invade a resected DNA double-strand break in an undamaged template to form a D-loop structure. However, they cannot perform this key step on their own. We succeed to inactivate one paralog (LiRad51-4) and provide insights on circular gene amplification. Finally, we show that LiMre11 harbors both DNA binding and exonuclease activities while inactivation of this gene led to a reduction of linear amplicon formation after drug selection. These results highlight a novel LiMre11-dependent pathway used by Leishmania to amplify stochastically portions of its genome. The relevance of DNA repair for drug resistance and its potential as a drug target represent a promising area of research for future treatments of leishmaniasis

GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. Protein purification protocols should combine efficiency, simplicity and cost effectiveness(1). Here, we describe the GST-His method as a new small-scale affinity purification system for recombinant proteins, based on a N-terminal Glutathione Sepharose Tag (GST)(2,3) and a C-terminal 10xHis tag(4), which are both fused to the protein of interest. The latter construct is used to generate baculoviruses, for infection of Sf9 infected cells for protein expression(5). GST is a rather long tag (29 kDa) which serves to ensure purification efficiency. However, it might influence physiological properties of the protein. Hence, it is subsequently cleaved off the protein using the PreScission enzyme(6). In order to ensure maximum purity and to remove the cleaved GST, we added a second affinity purification step based on the comparatively small His-Tag. Importantly, our technique is based on two different tags flanking the two ends of the protein, which is an efficient tool to remove degraded proteins and, therefore, enriches full-length proteins. The method presented here does not require an expensive instrumental setup, such as FPLC. Additionally, we incorporated MgCl(2) and ATP washes to remove heat shock protein impurities and nuclease treatment to abolish contaminating nucleic acids. In summary, the combination of two different tags flanking the N- and the C-terminal and the capability to cleave off one of the tags, guaranties the recovery of a highly purified and full-length protein of interest

GST-His purification: a two-step affinity purification protocol yielding full-length purified proteins.

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. Protein purification protocols should combine efficiency, simplicity and cost effectiveness. Here, we describe the GST-His method as a new small-scale affinity purification system for recombinant proteins, based on a N-terminal Glutathione Sepharose Tag (GST) and a C-terminal 10xHis tag, which are both fused to the protein of interest. The latter construct is used to generate baculoviruses, for infection of Sf9 infected cells for protein expression. GST is a rather long tag (29 kDa) which serves to ensure purification efficiency. However, it might influence physiological properties of the protein. Hence, it is subsequently cleaved off the protein using the PreScission enzyme. In order to ensure maximum purity and to remove the cleaved GST, we added a second affinity purification step based on the comparatively small His-Tag. Importantly, our technique is based on two different tags flanking the two ends of the protein, which is an efficient tool to remove degraded proteins and, therefore, enriches full-length proteins. The method presented here does not require an expensive instrumental setup, such as FPLC. Additionally, we incorporated MgCl2 and ATP washes to remove heat shock protein impurities and nuclease treatment to abolish contaminating nucleic acids. In summary, the combination of two different tags flanking the N- and the C-terminal and the capability to cleave off one of the tags, guaranties the recovery of a highly purified and full-length protein of interest

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

<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

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