The role of bivalent ions in the regulation of D-loop extension mediated by DMC1 during meiotic recombination

Veronika Altmannova; Mario Spirek; Lucija Orlic; Atis Jēkabsons; Tereza Clarence; Adrian Henggeler; Jarmila Mlcouskova; Raphaël A.G. Chaleil; Joao Matos; Lumir Krejci

doi:10.1016/j.isci.2022.105439

The role of bivalent ions in the regulation of D-loop extension mediated by DMC1 during meiotic recombination

Veronika Altmannova (Corresponding author)
, Mario Spirek
, Lucija Orlic
, Atis Jēkabsons
, Tereza Clarence
, Adrian Henggeler
, Jarmila Mlcouskova
, Raphaël A.G. Chaleil
, Joao Matos
, Lumir Krejci (Corresponding author)

Publications: Contribution to journal › Article › Peer Reviewed

Abstract

During meiosis, programmed DNA double-strand breaks (DSBs) are repaired by homologous recombination. DMC1, a conserved recombinase, plays a central role in this process. DMC1 promotes DNA strand exchange between homologous chromosomes, thus creating the physical linkage between them. Its function is regulated not only by several accessory proteins but also by bivalent ions. Here, we show that whereas calcium ions in the presence of ATP cause a conformational change within DMC1, stimulating its DNA binding and D-loop formation, they inhibit the extension of the invading strand within the D-loop. Based on structural studies, we have generated mutants of two highly conserved amino acids – E162 and D317 – in human DMC1, which are deficient in calcium regulation. In vivo studies of their yeast homologues further showed that they exhibit severe defects in meiosis, thus emphasizing the importance of calcium ions in the regulation of DMC1 function and meiotic recombination.

Original language	English
Article number	105439
Journal	iScience
Volume	25
Issue number	11
DOIs	https://doi.org/10.1016/j.isci.2022.105439
Publication status	Published - 18 Nov 2022

Funding

We would like to thank Marek Sebesta and Martin Pacesa for purified proteins HOP2-MND1, RFC, polymerase δ, and PCNA, Jan Zalesak for initial cryo-EM study, and Alexandra Sisakova for technical support. We are also indebted to Nancy Hollingsworth for YEpFAT4-RAD51, YEpFAT4-RAD54-T132A, and pNH301 plasmids, and Neil Hunter for NHY1210 and NHY1215 strains. CIISB, Instruct-CZ Centre of Instruct-ERIC EU consortium, funded by MEYS CR infrastructure project LM2018127, is acknowledged for the financial support of the measurements at the CF Cryo-electron Microscopy and Tomography and Biophysical Techniques. Computational resources were supplied by the project “e-Infrastruktura CZ” (e-INFRA CZ LM2018140) supported by the Ministry of Education, Youth and Sports of the Czech Republic. We are also thankful to Biomolecular Interaction core facility of CEITEC and Loschmidt Laboratories for access to Prometheus NT.48 instrument and Chirascan CD spectrometer, respectively. We thank the BioOptics facility at MPL Vienna for their valuable support for the live-cell imaging experiment. We thank Michael Lichten for helpful comments on the manuscript. This work was supported by the Czech Science Foundation (GACR 21-22593X); the European Structural and Investment Funds, Operational Programme Research, Development and Education “Preclinical Progression of New Organic Compounds with Targeted Biological Activity” (Preclinprogress, CZ.02.1.01/0.0/0.0/16_025/0007381); Wellcome Trust Collaborative Grant (206292/E/17/Z); Masaryk University (MUNI/G/1594/2019); and the European Union's Horizon 2020 research and innovation programme under grant agreement No 812829. The Matos lab is supported by the Swiss National Science Foundation (155823 and 176108), European Research Council (101002629) and the University of Vienna. V.A. M.S. A.J. and T.C. performed the biochemical and biophysical experiments. J.M. and A.J. performed the EM analysis. V.A. L.K. and J.M. designed the yeast experiments. V.A. and M.S. performed the yeast genetics experiments. L.O. performed the physical analyses of recombination. L.O. and A.H performed and analyzed the live cell imaging experiments. A.H performed and analyzed the chromosome spreads. R.G. performed the molecular modelling. V.A. and L.K. wrote the paper with inputs and edits from all authors. The authors declare no competing interest. We would like to thank Marek Sebesta and Martin Pacesa for purified proteins HOP2-MND1, RFC, polymerase δ, and PCNA, Jan Zalesak for initial cryo-EM study, and Alexandra Sisakova for technical support. We are also indebted to Nancy Hollingsworth for YEpFAT4-RAD51, YEpFAT4-RAD54-T132A, and pNH301 plasmids, and Neil Hunter for NHY1210 and NHY1215 strains. CIISB, Instruct-CZ Centre of Instruct-ERIC EU consortium, funded by MEYS CR infrastructure project LM2018127 , is acknowledged for the financial support of the measurements at the CF Cryo-electron Microscopy and Tomography and Biophysical Techniques. Computational resources were supplied by the project “e-Infrastruktura CZ” (e-INFRA CZ LM2018140) supported by the Ministry of Education , Youth and Sports of the Czech Republic . We are also thankful to Biomolecular Interaction core facility of CEITEC and Loschmidt Laboratories for access to Prometheus NT.48 instrument and Chirascan CD spectrometer, respectively. We thank the BioOptics facility at MPL Vienna for their valuable support for the live-cell imaging experiment. We thank Michael Lichten for helpful comments on the manuscript. This work was supported by the Czech Science Foundation ( GACR 21-22593X ); the European Structural and Investment Funds, Operational Programme Research , Development and Education “ Preclinical Progression of New Organic Compounds with Targeted Biological Activity ” (Preclinprogress, CZ.02.1.01/0.0/0.0/16_025/0007381 ); Wellcome Trust Collaborative Grant ( 206292/E/17/Z ); Masaryk University ( MUNI/G/1594/2019 ); and the European Union's Horizon 2020 research and innovation programme under grant agreement No 812829 . The Matos lab is supported by the Swiss National Science Foundation ( 155823 and 176108 ), European Research Council ( 101002629 ) and the University of Vienna .

Austrian Fields of Science 2012

106023 Molecular biology

Keywords

Cell biology
Structural biology

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https://phaidra.univie.ac.at/o:1671465Licence: CC BY-NC-ND 4.0

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title = "The role of bivalent ions in the regulation of D-loop extension mediated by DMC1 during meiotic recombination",

abstract = "During meiosis, programmed DNA double-strand breaks (DSBs) are repaired by homologous recombination. DMC1, a conserved recombinase, plays a central role in this process. DMC1 promotes DNA strand exchange between homologous chromosomes, thus creating the physical linkage between them. Its function is regulated not only by several accessory proteins but also by bivalent ions. Here, we show that whereas calcium ions in the presence of ATP cause a conformational change within DMC1, stimulating its DNA binding and D-loop formation, they inhibit the extension of the invading strand within the D-loop. Based on structural studies, we have generated mutants of two highly conserved amino acids – E162 and D317 – in human DMC1, which are deficient in calcium regulation. In vivo studies of their yeast homologues further showed that they exhibit severe defects in meiosis, thus emphasizing the importance of calcium ions in the regulation of DMC1 function and meiotic recombination.",

keywords = "Cell biology, Structural biology",

author = "Veronika Altmannova and Mario Spirek and Lucija Orlic and Atis Jēkabsons and Tereza Clarence and Adrian Henggeler and Jarmila Mlcouskova and Chaleil, \{Rapha{\"e}l A.G.\} and Joao Matos and Lumir Krejci",

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AU - Altmannova, Veronika

AU - Spirek, Mario

AU - Orlic, Lucija

AU - Jēkabsons, Atis

AU - Clarence, Tereza

AU - Henggeler, Adrian

AU - Mlcouskova, Jarmila

AU - Chaleil, Raphaël A.G.

AU - Matos, Joao

AU - Krejci, Lumir

PY - 2022/11/18

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N2 - During meiosis, programmed DNA double-strand breaks (DSBs) are repaired by homologous recombination. DMC1, a conserved recombinase, plays a central role in this process. DMC1 promotes DNA strand exchange between homologous chromosomes, thus creating the physical linkage between them. Its function is regulated not only by several accessory proteins but also by bivalent ions. Here, we show that whereas calcium ions in the presence of ATP cause a conformational change within DMC1, stimulating its DNA binding and D-loop formation, they inhibit the extension of the invading strand within the D-loop. Based on structural studies, we have generated mutants of two highly conserved amino acids – E162 and D317 – in human DMC1, which are deficient in calcium regulation. In vivo studies of their yeast homologues further showed that they exhibit severe defects in meiosis, thus emphasizing the importance of calcium ions in the regulation of DMC1 function and meiotic recombination.

AB - During meiosis, programmed DNA double-strand breaks (DSBs) are repaired by homologous recombination. DMC1, a conserved recombinase, plays a central role in this process. DMC1 promotes DNA strand exchange between homologous chromosomes, thus creating the physical linkage between them. Its function is regulated not only by several accessory proteins but also by bivalent ions. Here, we show that whereas calcium ions in the presence of ATP cause a conformational change within DMC1, stimulating its DNA binding and D-loop formation, they inhibit the extension of the invading strand within the D-loop. Based on structural studies, we have generated mutants of two highly conserved amino acids – E162 and D317 – in human DMC1, which are deficient in calcium regulation. In vivo studies of their yeast homologues further showed that they exhibit severe defects in meiosis, thus emphasizing the importance of calcium ions in the regulation of DMC1 function and meiotic recombination.

KW - Cell biology

KW - Structural biology

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The role of bivalent ions in the regulation of D-loop extension mediated by DMC1 during meiotic recombination

Abstract

Funding

Austrian Fields of Science 2012

Keywords

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