DS0405 - Génétique et génomique: relation génotype-phénotype, interactions génome-environnement, épigénétique

Resolution of meiotic recombination intermediates from yeast to mouse – Resolve

Resolution of meiotic recombination intermediates

Meiotic crossovers, necessary for gamete viability, are generated by the cleavage of a precursor recombination intermediate: the double Holliday junction (dHJ), by an uncharacterized meiotic “resolvase”.

General objective

A key question is to identify all actors directly involved in dHJ resolution, a reaction that requires two cleavage reactions each of two DNA strands, and to understand why the cleavage is made only asymmetrically, such that only crossovers are produced. This differs from the mechanism proposed for the canonical resolvases operating to repair recombination intermediates in somatic cells, where the resolution produces both crossover and non-crossover products. A wealth of converging genetic evidence points to the conserved MutL? complex, Mlh1-Mlh3, for being a key actor in the resolution of meiotic dHJ, but this complex shows no resolution activity in vitro, suggesting the need for additional partners. MutL? complex being a mismatch repair factor, another key issue is to determine how it is recruited to dHJ.<br />Our project is to explore the mechanism of crossover resolution and to identify the meiotic resolvase complex. For this, we will use a highly multidisciplinary approach combining yeast genetics, structural biochemistry, biochemistry and mouse genetics.

We will study in deep the localization of the MutL? complex in yeast meiotic cells, providing for the first time a high resolution map of meiotic crossover sites. Next, we will combine efforts to decipher the mechanism of dHJ resolution by MutL?. First, we will determine in yeast meiotic cells the protein partners associated with MutL?. Next, we will determine the crystal structure of the whole MutL? together with a dHJ DNA substrate, and use biochemical and biophysical assays with various types of DNA substrates and study the dHJ cleavage reaction in vitro by MutL?. Finally, we will use mouse genetics and genome engineering to test the functional conservation of our findings regarding the role of MutL? partners by building separation of function mutants in mice.

Through the characterization of the meiotic partners of Mlh1, we uncovered a new function of Mlh1, within the MutLB complex, in regulating the extent of meiotic recombination intermediates.

Together, this ambitious and comprehensive study of the MutL? complex in meiosis should uncover how the Mutl? complex promotes double Holliday junction resolution in meiosis, which guarantees gamete viability, fertility and diversity of the progeny. A major outcome of this project will be to rationalize meiosis dysfunctions observed in humans and associated with sterility or chromosomal abnormalities such as Trisomy 21.

Publication:
Duroc, Y., Kumar, R., Ranjha, L., Adam, C., Guérois, R., Md Muntaz, K., Marsolier-Kergoat, M.-C., Dingli, F., Laureau, R., Loew, D., Llorente, B., Charbonnier, J.-B., Cejka, P. and Borde, V. (2017) Concerted action of the MutLß heterodimer and Mer3 helicase regulates the global extent of meiotic gene conversion. eLife 6, e21900.

Meiotic crossovers, necessary for gamete viability, are generated by the cleavage of a precursor recombination intermediate: the double Holliday junction (dHJ), by an uncharacterized meiotic “resolvase”. A key question is to identify all actors directly involved in dHJ resolution, a reaction that requires two cleavage reactions each of two DNA strands, and to understand why the cleavage is made only asymmetrically, such that only crossovers are produced. This differs from the mechanism proposed for the canonical resolvases operating to repair recombination intermediates in somatic cells, where the resolution produces both crossover and non-crossover products. A wealth of converging genetic evidence points to the conserved MutLg complex, Mlh1-Mlh3, for being a key actor in the resolution of meiotic dHJ, but this complex shows no resolution activity in vitro, suggesting the need for additional partners. MutLg complex being a mismatch repair factor, another key issue is to determine how it is recruited to dHJ.
Our project is to explore the mechanism of crossover resolution and to identify the meiotic resolvase complex. For this, we will use a highly multidisciplinary approach combining yeast genetics, structural biochemistry, biochemistry and mouse genetics. We will study in deep the localization of the MutL? complex in yeast meiotic cells, providing for the first time a high resolution map of meiotic crossover sites. Next, we will combine efforts to decipher the mechanism of dHJ resolution by MutLg. First, we will determine in yeast meiotic cells the protein partners associated with MutL?. Next, we will determine the crystal structure of the whole MutLg together with a dHJ DNA substrate, and use biochemical and biophysical assays with various types of DNA substrates and study the dHJ cleavage reaction in vitro by MutLg. Finally, we will use mouse genetics and genome engineering to test the functional conservation of our findings regarding the role of MutLg partners by building separation of function mutants in mice.
Together, this ambitious and comprehensive study of the MutLg complex in meiosis should uncover how the Mutlg complex promotes double Holliday junction resolution in meiosis, which guarantees gamete viability, fertility and diversity of the progeny. A major outcome of this project will be to rationalize meiosis dysfunctions observed in humans and associated with sterility or chromosomal abnormalities such as Trisomy 21.






Project coordination

Valerie BORDE (INSTITUT CURIE - SECT DE RECHERCHE)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

CNRS Institut de Génétique Humaine CNRS UPR1142
University of Zurich Institute of Molecular Cancer Research
CEA/Saclay Institute of Biology and Technologies of Saclay SB2SM
IC INSTITUT CURIE - SECT DE RECHERCHE

Help of the ANR 517,297 euros
Beginning and duration of the scientific project: September 2015 - 36 Months

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