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.

Biomolecular Modelling Laboratory The Francis Crick Institute London UK

Department of Biology Masaryk University Brno 62500 Czech Republic

International Clinical Research Center St Anne's University Hospital Brno 65691 Czech Republic

Max Perutz Labs University of Vienna Dr Bohr Gasse 9 1030 Vienna Austria

National Center for Biomolecular Research Masaryk University Brno 62500 Czech Republic

Zobrazit více v PubMed

Arter M., Hurtado-Nieves V., Oke A., Zhuge T., Wettstein R., Fung J.C., Blanco M.G., Matos J. Regulated crossing-over requires inactivation of yen1/GEN1 resolvase during meiotic prophase I. Dev. Cell. 2018;45:785–800. doi: 10.1016/j.devcel.2018.05.020. PubMed DOI PMC

Bishop D.K., Nikolski Y., Oshiro J., Chon J., Shinohara M., Chen X. High copy number suppression of the meiotic arrest caused by a dmc1 mutation: REC114 imposes an early recombination block and RAD54 promotes a DMC1-independent DSB repair pathway. Gene Cell. 1999;4:425–444. doi: 10.1046/j.1365-2443.1999.00273.x. PubMed DOI

Bishop D.K., Park D., Xu L., Kleckner N. DMC1: a meiosis-specific yeast homolog of E. coli recA required for recombination, synaptonemal complex formation, and cell cycle progression. Cell. 1992;69:439–456. doi: 10.1016/0092-8674(92)90446-j. PubMed DOI

Brown M.S., Bishop D.K. DNA strand exchange and RecA homologs in meiosis. Cold Spring Harb. Perspect. Biol. 2014;7:a016659. doi: 10.1101/cshperspect.a016659. PubMed DOI PMC

Bugreev D.V., Mazin A.V. Ca2+ activates human homologous recombination protein Rad51 by modulating its ATPase activity. Proc. Natl. Acad. Sci. USA. 2004;101:9988–9993. doi: 10.1073/pnas.0402105101. PubMed DOI PMC

Bugreev D.V., Golub E.I., Stasiak A.Z., Stasiak A., Mazin A.V. Activation of human meiosis-specific recombinase Dmc1 by Ca2+ J. Biol. Chem. 2005;280:26886–26895. doi: 10.1074/jbc.M502248200. PubMed DOI

Busygina V., Gaines W.A., Xu Y., Kwon Y., Williams G.J., Lin S.W., Chang H.Y., Chi P., Wang H.W., Sung P. Functional attributes of the Saccharomyces cerevisiae meiotic recombinase Dmc1. DNA Repair. 2013;12:707–712. doi: 10.1016/j.dnarep.2013.05.004. PubMed DOI PMC

Carroll J., Swann K., Whittingham D., Whitaker M. Spatiotemporal dynamics of intracellular [Ca2+]i oscillations during the growth and meiotic maturation of mouse oocytes. Development. 1994;120:3507–3517. PubMed

Chan Y.L., Brown M.S., Qin D., Handa N., Bishop D.K. The third exon of the budding yeast meiotic recombination gene HOP2 is required for calcium-dependent and recombinase Dmc1-specific stimulation of homologous strand assimilation. J. Biol. Chem. 2014;289:18076–18086. doi: 10.1074/jbc.M114.558601. PubMed DOI PMC

Chang H.Y., Liao C.Y., Su G.C., Lin S.W., Wang H.W., Chi P. Functional relationship of ATP hydrolysis, presynaptic filament stability, and homologous DNA pairing activity of the human meiotic recombinase DMC1. J. Biol. Chem. 2015;290:19863–19873. doi: 10.1074/jbc.M115.666289. PubMed DOI PMC

Cloud V., Chan Y.L., Grubb J., Budke B., Bishop D.K. Rad51 is an accessory factor for Dmc1-mediated joint molecule formation during meiosis. Science. 2012;337:1222–1225. doi: 10.1126/science.1219379. PubMed DOI PMC

Finkelstein J., Antony E., Hingorani M.M., O'Donnell M. Overproduction and analysis of eukaryotic multiprotein complexes in Escherichia coli using a dual-vector strategy. Anal. Biochem. 2003;319:78–87. doi: 10.1016/s0003-2697(03)00273-2. PubMed DOI

Goldstein A.L., McCusker J.H. Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae. Yeast. 1999;15:1541–1553. doi: 10.1002/(SICI)1097-0061. PubMed DOI

Grigaitis R., Ranjha L., Wild P., Kasaciunaite K., Ceppi I., Kissling V., Henggeler A., Susperregui A., Peter M., Seidel R., et al. Phosphorylation of the RecQ helicase Sgs1/BLM controls its DNA unwinding activity during meiosis and mitosis. Dev. Cell. 2020;53:706–723.e5. doi: 10.1016/j.devcel.2020.05.016. PubMed DOI

Grigaitis R., Susperregui A., Wild P., Matos J. Characterization of DNA helicases and nucleases from meiotic extracts of S. cerevisiae. Methods Cell Biol. 2018;144:371–388. doi: 10.1016/bs.mcb.2018.03.029. PubMed DOI

Grushcow J.M., Holzen T.M., Park K.J., Weinert T., Lichten M., Bishop D.K. Saccharomyces cerevisiae checkpoint genes MEC1, RAD17 and RAD24 are required for normal meiotic recombination partner choice. Genetics. 1999;153:607–620. PubMed PMC

Beigi Harchegani A., Irandoost A., Mirnamniha M., Rahmani H., Tahmasbpour E., Shahriary A. Possible mechanisms for the effects of calcium deficiency on male infertility. Int. J. Fertil. Steril. 2019;12:267–272. doi: 10.22074/ijfs.2019.5420. PubMed DOI PMC

Hunter N. Meiotic recombination: the essence of heredity. Cold Spring Harb. Perspect. Biol. 2015;7:a016618. doi: 10.1101/cshperspect.a016618. PubMed DOI PMC

Hunter N., Kleckner N. The single-end invasion: an asymmetric intermediate at the double-strand break to double-holliday junction transition of meiotic recombination. Cell. 2001;106:59–70. doi: 10.1016/s0092-8674(01)00430-5. PubMed DOI

Kagawa W., Kurumizaka H. From meiosis to postmeiotic events: uncovering the molecular roles of the meiosis-specific recombinase Dmc1. FEBS J. 2010;277:590–598. doi: 10.1111/j.1742-4658.2009.07503.x. PubMed DOI

Kaneuchi T., Sartain C.V., Takeo S., Horner V.L., Buehner N.A., Aigaki T., Wolfner M.F. Calcium waves occur as Drosophila oocytes activate. Proc. Natl. Acad. Sci. USA. 2015;112:791–796. doi: 10.1073/pnas.1420589112. PubMed DOI PMC

Kant C.R., Rao B.J., Sainis J.K. DNA binding and pairing activity of OsDmc1, a recombinase from rice. Plant Mol. Biol. 2005;57:1–11. doi: 10.1007/s11103-004-5828-x. PubMed DOI

Kelso A.A., Say A.F., Sharma D., Ledford L.L., Turchick A., Saski C.A., King A.V., Attaway C.C., Temesvari L.A., Sehorn M.G. Entamoeba histolytica Dmc1 catalyzes homologous DNA pairing and strand exchange that is stimulated by calcium and hop2-mnd1. PLoS One. 2015;10 doi: 10.1371/journal.pone.0139399. PubMed DOI PMC

Kim K.P., Weiner B.M., Zhang L., Jordan A., Dekker J., Kleckner N. Sister cohesion and structural axis components mediate homolog bias of meiotic recombination. Cell. 2010;143:924–937. doi: 10.1016/j.cell.2010.11.015. PubMed DOI PMC

Lao J.P., Cloud V., Huang C.C., Grubb J., Thacker D., Lee C.Y., Dresser M.E., Hunter N., Bishop D.K. Meiotic crossover control by concerted action of Rad51-Dmc1 in homolog template bias and robust homeostatic regulation. PLoS Genet. 2013;9 doi: 10.1371/journal.pgen.1003978. PubMed DOI PMC

Lee M.H., Chang Y.C., Hong E.L., Grubb J., Chang C.S., Bishop D.K., Wang T.F. Calcium ion promotes yeast Dmc1 activity via formation of long and fine helical filaments with single-stranded DNA. J. Biol. Chem. 2005;280:40980–40984. doi: 10.1074/jbc.M505896200. PubMed DOI

Lindgren A., Bungard D., Pierce M., Xie J., Vershon A., Winter E. The pachytene checkpoint in Saccharomyces cerevisiae requires the Sum1 transcriptional repressor. EMBO J. 2000;19:6489–6497. doi: 10.1093/emboj/19.23.6489. PubMed DOI PMC

Liu Y., Gaines W.A., Callender T., Busygina V., Oke A., Sung P., Fung J.C., Hollingsworth N.M. Down-regulation of Rad51 activity during meiosis in yeast prevents competition with Dmc1 for repair of double-strand breaks. PLoS Genet. 2014;10 doi: 10.1371/journal.pgen.1004005. PubMed DOI PMC

Loidl J., Klein F., Engebrecht J. Genetic and morphological approaches for the analysis of meiotic chromosomes in yeast. Methods Cell Biol. 1998;53:257–285. doi: 10.1016/s0091-679x(08)60882-1. PubMed DOI

Longtine M.S., McKenzie A., 3rd, Demarini D.J., Shah N.G., Wach A., Brachat A., Philippsen P., Pringle J.R. Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast. 1998;14:953–961. doi: 10.1002/(SICI)1097-0061. PubMed DOI

Luo S.C., Yeh H.Y., Lan W.H., Wu Y.M., Yang C.H., Chang H.Y., Su G.C., Lee C.Y., Wu W.J., Li H.W., et al. Identification of fidelity-governing factors in human recombinases DMC1 and RAD51 from cryo-EM structures. Nat. Commun. 2021;12:115. doi: 10.1038/s41467-020-20258-1. PubMed DOI PMC

Lydall D., Nikolsky Y., Bishop D.K., Weinert T. A meiotic recombination checkpoint controlled by mitotic checkpoint genes. Nature. 1996;383:840–843. doi: 10.1038/383840a0. PubMed DOI

Matos J., Blanco M.G., Maslen S., Skehel J.M., West S.C. Regulatory control of the resolution of DNA recombination intermediates during meiosis and mitosis. Cell. 2011;147:158–172. doi: 10.1016/j.cell.2011.08.032. PubMed DOI PMC

Matos J., Lipp J.J., Bogdanova A., Guillot S., Okaz E., Junqueira M., Shevchenko A., Zachariae W. Dbf4-dependent CDC7 kinase links DNA replication to the segregation of homologous chromosomes in meiosis I. Cell. 2008;135:662–678. doi: 10.1016/j.cell.2008.10.026. PubMed DOI

Matulova P., Marini V., Burgess R.C., Sisakova A., Kwon Y., Rothstein R., Sung P., Krejci L. Cooperativity of Mus81.Mms4 with Rad54 in the resolution of recombination and replication intermediates. J. Biol. Chem. 2009;284:7733–7745. doi: 10.1074/jbc.M806192200. PubMed DOI PMC

McDougall A., Sardet C. Function and characteristics of repetitive calcium waves associated with meiosis. Curr. Biol. 1995;5:318–328. doi: 10.1016/s0960-9822(95)00062-5. PubMed DOI

Nimonkar A.V., Dombrowski C.C., Siino J.S., Stasiak A.Z., Stasiak A., Kowalczykowski S.C. Saccharomyces cerevisiae Dmc1 and Rad51 proteins preferentially function with Tid1 and Rad54 proteins, respectively, to promote DNA strand invasion during genetic recombination. J. Biol. Chem. 2012;287:28727–28737. doi: 10.1074/jbc.M112.373290. PubMed DOI PMC

Niu H., Wan L., Busygina V., Kwon Y., Allen J.A., Li X., Kunz R.C., Kubota K., Wang B., Sung P., et al. Regulation of meiotic recombination via Mek1-mediated Rad54 phosphorylation. Mol. Cell. 2009;36:393–404. doi: 10.1016/j.molcel.2009.09.029. PubMed DOI PMC

Oh S.D., Jessop L., Lao J.P., Allers T., Lichten M., Hunter N. Stabilization and electrophoretic analysis of meiotic recombination intermediates in Saccharomyces cerevisiae. Methods Mol. Biol. 2009;557:209–234. doi: 10.1007/978-1-59745-527-5_14. PubMed DOI

Oh S.D., Lao J.P., Hwang P.Y.H., Taylor A.F., Smith G.R., Hunter N. BLM ortholog, Sgs1, prevents aberrant crossing-over by suppressing formation of multichromatid joint molecules. Cell. 2007;130:259–272. doi: 10.1016/j.cell.2007.05.035. PubMed DOI PMC

Pak J., Segall J. Role of Ndt80, Sum1, and Swe1 as targets of the meiotic recombination checkpoint that control exit from pachytene and spore formation in Saccharomyces cerevisiae. Mol. Cell Biol. 2002;22:6430–6440. doi: 10.1128/mcb.22.18.6430-6440.2002. PubMed DOI PMC

Passy S.I., Yu X., Li Z., Radding C.M., Masson J.Y., West S.C., Egelman E.H. Human Dmc1 protein binds DNA as an octameric ring. Proc. Natl. Acad. Sci. USA. 1999;96:10684–10688. doi: 10.1073/pnas.96.19.10684. PubMed DOI PMC

Petronczki M., Matos J., Mori S., Gregan J., Bogdanova A., Schwickart M., Mechtler K., Shirahige K., Zachariae W., Nasmyth K. Monopolar attachment of sister kinetochores at meiosis I requires casein kinase 1. Cell. 2006;126:1049–1064. doi: 10.1016/j.cell.2006.07.029. PubMed DOI

Petukhova G.V., Pezza R.J., Vanevski F., Ploquin M., Masson J.Y., Camerini-Otero R.D. The Hop2 and Mnd1 proteins act in concert with Rad51 and Dmc1 in meiotic recombination. Nat. Struct. Mol. Biol. 2005;12:449–453. doi: 10.1038/nsmb923. PubMed DOI

Pezza R.J., Petukhova G.V., Ghirlando R., Camerini-Otero R.D. Molecular activities of meiosis-specific proteins Hop2, Mnd1, and the Hop2-Mnd1 complex. J. Biol. Chem. 2006;281:18426–18434. doi: 10.1074/jbc.M601073200. PubMed DOI

Pezza R.J., Voloshin O.N., Vanevski F., Camerini-Otero R.D. Hop2/Mnd1 acts on two critical steps in Dmc1-promoted homologous pairing. Genes Dev. 2007;21:1758–1766. doi: 10.1101/gad.1562907. PubMed DOI PMC

Pittman D.L., Cobb J., Schimenti K.J., Wilson L.A., Cooper D.M., Brignull E., Handel M.A., Schimenti J.C. Meiotic prophase arrest with failure of chromosome synapsis in mice deficient for Dmc1, a germline-specific RecA homolog. Mol. Cell. 1998;1:697–705. doi: 10.1016/s1097-2765(00)80069-6. PubMed DOI

Qian X., He Y., Ma X., Fodje M.N., Grochulski P., Luo Y. Calcium stiffens archaeal Rad51 recombinase from Methanococcus voltae for homologous recombination. J. Biol. Chem. 2006;281:39380–39387. doi: 10.1074/jbc.M607785200. PubMed DOI

Qian X., He Y., Wu Y., Luo Y. Asp302 determines potassium dependence of a RadA recombinase from Methanococcus voltae. J. Mol. Biol. 2006;360:537–547. doi: 10.1016/j.jmb.2006.05.058. PubMed DOI

Rajanikant C., Kumbhakar M., Pal H., Rao B.J., Sainis J.K. DNA strand exchange activity of rice recombinase OsDmc1 monitored by fluorescence resonance energy transfer and the role of ATP hydrolysis. FEBS J. 2006;273:1497–1506. doi: 10.1111/j.1742-4658.2006.05170.x. PubMed DOI

Reitz D., Grubb J., Bishop D.K. A mutant form of Dmc1 that bypasses the requirement for accessory protein Mei5-Sae3 reveals independent activities of Mei5-Sae3 and Rad51 in Dmc1 filament stability. PLoS Genet. 2019;15 doi: 10.1371/journal.pgen.1008217. PubMed DOI PMC

Sakane I., Kamataki C., Takizawa Y., Nakashima M., Toki S., Ichikawa H., Ikawa S., Shibata T., Kurumizaka H. Filament formation and robust strand exchange activities of the rice DMC1A and DMC1B proteins. Nucleic Acids Res. 2008;36:4266–4276. doi: 10.1093/nar/gkn405. PubMed DOI PMC

Sasanuma H., Tawaramoto M.S., Lao J.P., Hosaka H., Sanda E., Suzuki M., Yamashita E., Hunter N., Shinohara M., Nakagawa A., Shinohara A. A new protein complex promoting the assembly of Rad51 filaments. Nat. Commun. 2013;4:1676. doi: 10.1038/ncomms2678. PubMed DOI PMC

Scherthan H., Wang H., Adelfalk C., White E.J., Cowan C., Cande W.Z., Kaback D.B. Chromosome mobility during meiotic prophase in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA. 2007;104:16934–16939. doi: 10.1073/pnas.0704860104. PubMed DOI PMC

Schwacha A., Kleckner N. Interhomolog bias during meiotic recombination: meiotic functions promote a highly differentiated interhomolog-only pathway. Cell. 1997;90:1123–1135. doi: 10.1016/s0092-8674(00)80378-5. PubMed DOI

Sebesta M., Burkovics P., Haracska L., Krejci L. Reconstitution of DNA repair synthesis in vitro and the role of polymerase and helicase activities. DNA Repair. 2011;10:567–576. doi: 10.1016/j.dnarep.2011.03.003. PubMed DOI PMC

Sehorn M.G., Sigurdsson S., Bussen W., Unger V.M., Sung P. Human meiotic recombinase Dmc1 promotes ATP-dependent homologous DNA strand exchange. Nature. 2004;429:433–437. doi: 10.1038/nature02563. PubMed DOI

Špírek M., Mlcoušková J., Belán O., Gyimesi M., Harami G.M., Molnár E., Novacek J., Kovács M., Krejci L. Human RAD51 rapidly forms intrinsically dynamic nucleoprotein filaments modulated by nucleotide binding state. Nucleic Acids Res. 2018;46:3967–3980. doi: 10.1093/nar/gky111. PubMed DOI PMC

Strynadka N.C., James M.N. Towards an understanding of the effects of calcium on protein structure and function. Curr. Opin. Struct. Biol. 1991;1:905–914. doi: 10.1016/0959-440x(91)90085-8. DOI

Suizu T., Tsutsumi H., Kawado A., Suginami K., Imayasu S., Murata K. Calcium ion influx during sporulation in the yeast Saccharomyces cerevisiae. Can. J. Microbiol. 1995;41:1035–1037. doi: 10.1139/m95-143. PubMed DOI

Tsubouchi H., Roeder G.S. The importance of genetic recombination for fidelity of chromosome pairing in meiosis. Dev. Cell. 2003;5:915–925. doi: 10.1016/s1534-5807(03)00357-5. PubMed DOI

Whitaker M., Patel R. Calcium and cell cycle control. Development. 1990;108:525–542. PubMed

Wu Y., Qian X., He Y., Moya I.A., Luo Y. Crystal structure of an ATPase-active form of Rad51 homolog from Methanococcus voltae. Insights into potassium dependence. J. Biol. Chem. 2005;280:722–728. doi: 10.1074/jbc.M411093200. PubMed DOI

Xu L., Weiner B.M., Kleckner N. Meiotic cells monitor the status of the interhomolog recombination complex. Genes Dev. 1997;11:106–118. doi: 10.1101/gad.11.1.106. PubMed DOI

Xue C., Molnarova L., Steinfeld J.B., Zhao W., Ma C., Spirek M., Kaniecki K., Kwon Y., Belan O., Krejci K., et al. Single-molecule visualization of human RECQ5 interactions with single-stranded DNA recombination intermediates. Nucleic Acids Research. 2021;49:285–305. doi: 10.1093/nar/gkaa1184. PubMed DOI PMC

Yoshida K., Kondoh G., Matsuda Y., Habu T., Nishimune Y., Morita T. The mouse RecA-like gene Dmc1 is required for homologous chromosome synapsis during meiosis. Mol. Cell. 1998;1:707–718. doi: 10.1016/s1097-2765(00)80070-2. PubMed DOI

Zhang K. Gctf: real-time CTF determination and correction. J. Struct. Biol. 2016;193:1–12. doi: 10.1016/j.jsb.2015.11.003. PubMed DOI PMC

Zhao W., Sung P. Significance of ligand interactions involving Hop2-Mnd1 and the RAD51 and DMC1 recombinases in homologous DNA repair and XX ovarian dysgenesis. Nucleic Acids Res. 2015;43:4055–4066. doi: 10.1093/nar/gkv259. PubMed DOI PMC

Zheng S.Q., Palovcak E., Armache J.P., Verba K.A., Cheng Y., Agard D.A. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods. 2017;14:331–332. doi: 10.1038/nmeth.4193. PubMed DOI PMC

Zivanov J., Nakane T., Forsberg B.O., Kimanius D., Hagen W.J., Lindahl E., Scheres S.H. New tools for automated high-resolution cryo-EM structure determination in RELION-3. Elife. 2018;7:e42166. doi: 10.7554/eLife.42166. PubMed DOI PMC