Super-resolution imaging of RAD51 and DMC1 in DNA repair foci reveals dynamic distribution patterns in meiotic prophase

English version

Autoři: Johan A. Slotman ^aff001; Maarten W. Paul ^aff001; Fabrizia Carofiglio ^aff003; H. Martijn de Gruiter ^aff001; Tessa Vergroesen ^aff003; Lieke Koornneef ^aff003; Wiggert A. van Cappellen ^aff001; Adriaan B. Houtsmuller ^aff001; Willy M. Baarends ^aff003
Působiště autorů: Erasmus Optical Imaging Centre, Department of Pathology, Erasmus MC—University Medical Center, Rotterdam, The Netherlands ^aff001; Department of Pathology, Erasmus MC—University Medical Center, Rotterdam, The Netherlands ^aff002; Department of Developmental Biology, Erasmus MC—University Medical Center, Rotterdam, The Netherlands ^aff003
Vyšlo v časopise: Super-resolution imaging of RAD51 and DMC1 in DNA repair foci reveals dynamic distribution patterns in meiotic prophase. PLoS Genet 16(6): e32767. doi:10.1371/journal.pgen.1008595
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pgen.1008595

Souhrn

The recombinase RAD51, and its meiosis-specific paralog DMC1 localize at DNA double-strand break (DSB) sites in meiotic prophase. While both proteins are required during meiotic prophase, their spatial organization during meiotic DSB repair is not fully understood. Using super-resolution microscopy on mouse spermatocyte nuclei, we aimed to define their relative position at DSB foci, and how these vary in time. We show that a large fraction of meiotic DSB repair foci (38%) consisted of a single RAD51 nanofocus and a single DMC1 nanofocus (D1R1 configuration) that were partially overlapping with each other (average center-center distance around 70 nm). The vast majority of the rest of the foci had a similar large RAD51 and DMC1 nanofocus, but in combination with additional smaller nanofoci (D2R1, D1R2, D2R2, or DxRy configuration) at an average distance of around 250 nm. As prophase progressed, less D1R1 and more D2R1 foci were observed, where the large RAD51 nanofocus in the D2R1 foci elongated and gradually oriented towards the distant small DMC1 nanofocus. D1R2 foci frequency was relatively constant, and the single DMC1 nanofocus did not elongate, but was frequently observed between the two RAD51 nanofoci in early stages. D2R2 foci were rare (<10%) and nearest neighbour analyses also did not reveal cofoci formation between D1R1 foci. However, overall, foci localized nonrandomly along the SC, and the frequency of the distance distributions peaked at 800 nm, indicating interference and/or a preferred distance between two ends of a DSB. DMC1 nanofoci where somewhat further away from the axial or lateral elements of the synaptonemal complex (SC, connecting the chromosomal axes of homologs) compared to RAD51 nanofoci. In the absence of the transverse filament of the SC, early configurations were more prominent, and RAD51 nanofocus elongation occurred only transiently. This in-depth analysis of single cell landscapes of RAD51 and DMC1 accumulation patterns at DSB repair sites at super-resolution revealed the variability of foci composition, and defined functional consensus configurations that change over time.

Klíčová slova:

Built structures – DNA repair – Homologous chromosomes – Meiotic prophase – Recombinant proteins – Simulation and modeling – Spermatocytes – Prophase

Zdroje

1. Baudat F, Manova K, Yuen JP, Jasin M, Keeney S. Chromosome synapsis defects and sexually dimorphic meiotic progression in mice lacking spo11. Molecular cell. 2000;6(5):989–98. doi: 10.1016/s1097-2765(00)00098-8 11106739

2. Romanienko PJ, Camerini-Otero RD. The mouse spo11 gene is required for meiotic chromosome synapsis. Molecular cell. 2000;6(5):975–87. doi: 10.1016/s1097-2765(00)00097-6 11106738

3. Robert T, Nore A, Brun C, Maffre C, Crimi B, Bourbon HM, et al. The TopoVIB-Like protein family is required for meiotic DNA double-strand break formation. Science. 2016;351(6276):943–9. doi: 10.1126/science.aad5309 26917764

4. Hunter N. Meiotic Recombination: The Essence of Heredity. Cold Spring Harbor perspectives in biology. 2015;7(12).

5. Wright WD, Shah SS, Heyer WD. Homologous recombination and the repair of DNA double-strand breaks. The Journal of biological chemistry. 2018;293(27):10524–35. doi: 10.1074/jbc.TM118.000372 29599286

6. Inagaki A, Schoenmakers S, Baarends WM. DNA double strand break repair, chromosome synapsis and transcriptional silencing in meiosis. Epigenetics. 2010;5(4):255–66. doi: 10.4161/epi.5.4.11518 20364103

7. Ribeiro J, Abby E, Livera G, Martini E. RPA homologs and ssDNA processing during meiotic recombination. Chromosoma. 2016;125(2):265–76. doi: 10.1007/s00412-015-0552-7 26520106

8. Moens PB, Kolas NK, Tarsounas M, Marcon E, Cohen PE, Spyropoulos B. The time course and chromosomal localization of recombination-related proteins at meiosis in the mouse are compatible with models that can resolve the early DNA-DNA interactions without reciprocal recombination. Journal of cell science. 2002;115(Pt 8):1611–22. 11950880

9. Habu T, Taki T, West A, Nishimune Y, Morita T. The mouse and human homologs of DMC1, the yeast meiosis-specific homologous recombination gene, have a common unique form of exon -⁠ skipped transcript in meiosis. Nucleic acids research. 1996;24(3):470–7. doi: 10.1093/nar/24.3.470 8602360

10. Moens PB, Chen DJ, Shen Z, Kolas N, Tarsounas M, Heng HHQ. Rad51 immunocytology in rat and mouse spermatocytes and oocytes. Chromosoma. 1997;106 : 207–15. doi: 10.1007/s004120050241 9254722

11. Tarsounas M, Morita T, Pearlman RE, Moens PB. RAD51 and DMC1 form mixed complexes associated with mouse meiotic chromosome cores and synaptonemal complexes. The Journal of cell biology. 1999;147(2):207–20. doi: 10.1083/jcb.147.2.207 10525529

12. Kurzbauer MT, Uanschou C, Chen D, Schlogelhofer P. The recombinases DMC1 and RAD51 are functionally and spatially separated during meiosis in Arabidopsis. Plant Cell. 2012;24(5):2058–70. doi: 10.1105/tpc.112.098459 22589466

13. Brown MS, Grubb J, Zhang A, Rust MJ, Bishop DK. Small Rad51 and Dmc1 Complexes Often Co-occupy Both Ends of a Meiotic DNA Double Strand Break. PLoS Genet. 2015;11(12):e1005653. doi: 10.1371/journal.pgen.1005653 26719980

14. Fraune J, Schramm S, Alsheimer M, Benavente R. The mammalian synaptonemal complex: protein components, assembly and role in meiotic recombination. Experimental cell research. 2012;318(12):1340–6. doi: 10.1016/j.yexcr.2012.02.018 22394509

15. de Vries FA, de Boer E, van den Bosch M, Baarends WM, Ooms M, Yuan L, et al. Mouse Sycp1 functions in synaptonemal complex assembly, meiotic recombination, and XY body formation. Genes & development. 2005;19(11):1376–89.

16. Billings T, Sargent EE, Szatkiewicz JP, Leahy N, Kwak IY, Bektassova N, et al. Patterns of recombination activity on mouse chromosome 11 revealed by high resolution mapping. PloS one. 2010;5(12):e15340. doi: 10.1371/journal.pone.0015340 21170346

17. de Boer E, Dietrich AJ, Hoog C, Stam P, Heyting C. Meiotic interference among MLH1 foci requires neither an intact axial element structure nor full synapsis. Journal of cell science. 2007;120(Pt 5):731–6. doi: 10.1242/jcs.003186 17298983

18. de Boer E, Stam P, Dietrich AJ, Pastink A, Heyting C. Two levels of interference in mouse meiotic recombination. Proceedings of the National Academy of Sciences of the United States of America. 2006;103(25):9607–12. doi: 10.1073/pnas.0600418103 16766662

19. Cole F, Kauppi L, Lange J, Roig I, Wang R, Keeney S, et al. Homeostatic control of recombination is implemented progressively in mouse meiosis. Nature cell biology. 2012;14(4):424–30. doi: 10.1038/ncb2451 22388890

20. Carofiglio F, Inagaki A, de Vries S, Wassenaar E, Schoenmakers S, Vermeulen C, et al. SPO11-Independent DNA Repair Foci and Their Role in Meiotic Silencing. PLoS Genet. 2013;9(6):e1003538. doi: 10.1371/journal.pgen.1003538 23754961

21. Paul MW, de Gruiter HM, Lin Z, Baarends WM, van Cappellen WA, Houtsmuller AB, et al. SMoLR: visualization and analysis of single-molecule localization microscopy data in R. BMC Bioinformatics. 2019;20(1):30. doi: 10.1186/s12859-018-2578-3 30646838

22. Boateng KA, Bellani MA, Gregoretti IV, Pratto F, Camerini-Otero RD. Homologous pairing preceding SPO11-mediated double-strand breaks in mice. Developmental cell. 2013;24(2):196–205. doi: 10.1016/j.devcel.2012.12.002 23318132

23. Hamer G, Gell K, Kouznetsova A, Novak I, Benavente R, Hoog C. Characterization of a novel meiosis-specific protein within the central element of the synaptonemal complex. Journal of cell science. 2006;119(Pt 19):4025–32. doi: 10.1242/jcs.03182 16968740

24. Hamer G, Wang H, Bolcun-Filas E, Cooke HJ, Benavente R, Hoog C. Progression of meiotic recombination requires structural maturation of the central element of the synaptonemal complex. Journal of cell science. 2008;121(Pt 15):2445–51. doi: 10.1242/jcs.033233 18611960

25. Schramm S, Fraune J, Naumann R, Hernandez-Hernandez A, Hoog C, Cooke HJ, et al. A novel mouse synaptonemal complex protein is essential for loading of central element proteins, recombination, and fertility. PLoS Genet. 2011;7(5):e1002088. doi: 10.1371/journal.pgen.1002088 21637789

26. Sanchez H, Paul MW, Grosbart M, van Rossum-Fikkert SE, Lebbink JH, Kanaar R, et al. Architectural plasticity of human BRCA2-RAD51 complexes in DNA break repair. Nucleic acids research. 2017;45(8):4507–18. doi: 10.1093/nar/gkx084 28168276

27. Haas KT, Lee M, Esposito A, Venkitaraman AR. Single-molecule localization microscopy reveals molecular transactions during RAD51 filament assembly at cellular DNA damage sites. Nucleic acids research. 2018;46(5):2398–416. doi: 10.1093/nar/gkx1303 29309696

28. Mikhaylova M, Cloin BM, Finan K, van den Berg R, Teeuw J, Kijanka MM, et al. Resolving bundled microtubules using anti-tubulin nanobodies. Nature communications. 2015;6 : 7933. doi: 10.1038/ncomms8933 26260773

29. Pleiner T, Bates M, Gorlich D. A toolbox of anti-mouse and anti-rabbit IgG secondary nanobodies. The Journal of cell biology. 2018;217(3):1143–54. doi: 10.1083/jcb.201709115 29263082

30. Ristic D, Modesti M, van der Heijden T, van Noort J, Dekker C, Kanaar R, et al. Human Rad51 filaments on double -⁠ and single-stranded DNA: correlating regular and irregular forms with recombination function. Nucleic Acids Res. 2005;33(10):3292–302. doi: 10.1093/nar/gki640 15944450

31. Lange J, Yamada S, Tischfield SE, Pan J, Kim S, Zhu X, et al. The Landscape of Mouse Meiotic Double-Strand Break Formation, Processing, and Repair. Cell. 2016;167(3):695–708 e16. doi: 10.1016/j.cell.2016.09.035 27745971

32. Hinch AG, Zhang G, Becker PW, Moralli D, Hinch R, Davies B, et al. Factors influencing meiotic recombination revealed by whole-genome sequencing of single sperm. Science. 2019;363(6433).

33. Woglar A, Villeneuve AM. Dynamic Architecture of DNA Repair Complexes and the Synaptonemal Complex at Sites of Meiotic Recombination. Cell. 2018;173(7):1678–91 e16. doi: 10.1016/j.cell.2018.03.066 29754818

34. Howard-Till RA, Lukaszewicz A, Loidl J. The recombinases Rad51 and Dmc1 play distinct roles in DNA break repair and recombination partner choice in the meiosis of Tetrahymena. PLoS Genet. 2011;7(3):e1001359. doi: 10.1371/journal.pgen.1001359 21483758

35. Brown MS, Bishop DK. DNA strand exchange and RecA homologs in meiosis. Cold Spring Harbor perspectives in biology. 2014;7(1):a016659. doi: 10.1101/cshperspect.a016659 25475089

36. Faieta M, Di Cecca S, de Rooij DG, Luchetti A, Murdocca M, Di Giacomo M, et al. A surge of late-occurring meiotic double-strand breaks rescues synapsis abnormalities in spermatocytes of mice with hypomorphic expression of SPO11. Chromosoma. 2015.

37. Gray S, Allison RM, Garcia V, Goldman AS, Neale MJ. Positive regulation of meiotic DNA double-strand break formation by activation of the DNA damage checkpoint kinase Mec1(ATR). Open biology. 2013;3(7):130019. doi: 10.1098/rsob.130019 23902647

38. Kauppi L, Barchi M, Lange J, Baudat F, Jasin M, Keeney S. Numerical constraints and feedback control of double-strand breaks in mouse meiosis. Genes & development. 2013;27(8):873–86.

39. Peters AH, Plug AW, van Vugt MJ, de Boer P. A drying-down technique for the spreading of mammalian meiocytes from the male and female germline. Chromosome research: an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology. 1997;5(1):66–8.

40. Essers J, Hendriks RW, Wesoly J, Beerens CE, Smit B, Hoeijmakers JH, et al. Analysis of mouse Rad54 expression and its implications for homologous recombination. DNA repair. 2002;1(10):779–93. doi: 10.1016/s1568-7864(02)00110-6 12531026

41. van de Linde S, Loschberger A, Klein T, Heidbreder M, Wolter S, Heilemann M, et al. Direct stochastic optical reconstruction microscopy with standard fluorescent probes. Nat Protoc. 2011;6(7):991–1009. doi: 10.1038/nprot.2011.336 21720313

42. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nature methods. 2012;9(7):676–82. doi: 10.1038/nmeth.2019 22743772

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