Conditional knockout of RAD51-related genes in Leishmania major reveals a critical role for homologous recombination during genome replication

Autoři: Jeziel D. Damasceno aff001;  João Reis-Cunha aff002;  Kathryn Crouch aff001;  Dario Beraldi aff001;  Craig Lapsley aff001;  Luiz R. O. Tosi aff003;  Daniella Bartholomeu aff002;  Richard McCulloch aff001
Působiště autorů: The Wellcome Centre for Integrative Parasitology, University of Glasgow, Institute of Infection, Immunity and Inflammation, Sir Graeme Davies Building, 120 University Place, Glasgow, United Kingdom aff001;  Laboratório de Imunologia e Genômica de Parasitos, Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brasil aff002;  Department of Cell and Molecular Biology, Ribeirão Preto Medical School, University of São Paulo; Ribeirão Preto, SP, Brazil aff003
Vyšlo v časopise: Conditional knockout of RAD51-related genes in Leishmania major reveals a critical role for homologous recombination during genome replication. PLoS Genet 16(7): e32767. doi:10.1371/journal.pgen.1008828
Kategorie: Research Article
doi: 10.1371/journal.pgen.1008828


Homologous recombination (HR) has an intimate relationship with genome replication, both during repair of DNA lesions that might prevent DNA synthesis and in tackling stalls to the replication fork. Recent studies led us to ask if HR might have a more central role in replicating the genome of Leishmania, a eukaryotic parasite. Conflicting evidence has emerged regarding whether or not HR genes are essential, and genome-wide mapping has provided evidence for an unorthodox organisation of DNA replication initiation sites, termed origins. To answer this question, we have employed a combined CRISPR/Cas9 and DiCre approach to rapidly generate and assess the effect of conditional ablation of RAD51 and three RAD51-related proteins in Leishmania major. Using this approach, we demonstrate that loss of any of these HR factors is not immediately lethal but in each case growth slows with time and leads to DNA damage and accumulation of cells with aberrant DNA content. Despite these similarities, we show that only loss of RAD51 or RAD51-3 impairs DNA synthesis and causes elevated levels of genome-wide mutation. Furthermore, we show that these two HR factors act in distinct ways, since ablation of RAD51, but not RAD51-3, has a profound effect on DNA replication, causing loss of initiation at the major origins and increased DNA synthesis at subtelomeres. Our work clarifies questions regarding the importance of HR to survival of Leishmania and reveals an unanticipated, central role for RAD51 in the programme of genome replication in a microbial eukaryote.

Klíčová slova:

Cell cycle and cell division – DNA – DNA extraction – DNA replication – DNA synthesis – Homologous recombination – Leishmania – Polymerase chain reaction


1. Scully R, Panday A, Elango R, Willis NA. DNA double-strand break repair-pathway choice in somatic mammalian cells. Nature reviews Molecular cell biology. 2019. doi: 10.1038/s41580-019-0152-0 31263220.

2. Bell JC, Kowalczykowski SC. RecA: Regulation and Mechanism of a Molecular Search Engine. Trends in biochemical sciences. 2016;41(6):491–507. doi: 10.1016/j.tibs.2016.04.002 27156117; PubMed Central PMCID: PMC4892382.

3. Sullivan MR, Bernstein KA. RAD-ical New Insights into RAD51 Regulation. Genes (Basel). 2018;9(12). doi: 10.3390/genes9120629 30551670; PubMed Central PMCID: PMC6316741.

4. Zhang S, Wang L, Tao Y, Bai T, Lu R, Zhang T, et al. Structural basis for the functional role of the Shu complex in homologous recombination. Nucleic Acids Res. 2017;45(22):13068–79. doi: 10.1093/nar/gkx992 29069504; PubMed Central PMCID: PMC5727457.

5. Gaines WA, Godin SK, Kabbinavar FF, Rao T, VanDemark AP, Sung P, et al. Promotion of presynaptic filament assembly by the ensemble of S. cerevisiae Rad51 paralogues with Rad52. Nature communications. 2015;6:7834. doi: 10.1038/ncomms8834 26215801; PubMed Central PMCID: PMC4525180.

6. Taylor MRG, Spirek M, Chaurasiya KR, Ward JD, Carzaniga R, Yu X, et al. Rad51 Paralogs Remodel Pre-synaptic Rad51 Filaments to Stimulate Homologous Recombination. Cell. 2015;162(2):271–86. doi: 10.1016/j.cell.2015.06.015 26186187; PubMed Central PMCID: PMC4518479.

7. Rosenbaum JC, Bonilla B, Hengel SR, Mertz TM, Herken BW, Kazemier HG, et al. The Rad51 paralogs facilitate a novel DNA strand specific damage tolerance pathway. Nature communications. 2019;10(1):3515. doi: 10.1038/s41467-019-11374-8 31383866; PubMed Central PMCID: PMC6683157.

8. Saxena S, Somyajit K, Nagaraju G. XRCC2 Regulates Replication Fork Progression during dNTP Alterations. Cell reports. 2018;25(12):3273–82 e6. doi: 10.1016/j.celrep.2018.11.085 30566856.

9. Pond KW, de Renty C, Yagle MK, Ellis NA. Rescue of collapsed replication forks is dependent on NSMCE2 to prevent mitotic DNA damage. PLoS genetics. 2019;15(2):e1007942. doi: 10.1371/journal.pgen.1007942 30735491; PubMed Central PMCID: PMC6383951.

10. Malacaria E, Pugliese GM, Honda M, Marabitti V, Aiello FA, Spies M, et al. Rad52 prevents excessive replication fork reversal and protects from nascent strand degradation. Nature communications. 2019;10(1):1412. doi: 10.1038/s41467-019-09196-9 30926821; PubMed Central PMCID: PMC6441034.

11. Bhat KP, Krishnamoorthy A, Dungrawala H, Garcin EB, Modesti M, Cortez D. RADX Modulates RAD51 Activity to Control Replication Fork Protection. Cell reports. 2018;24(3):538–45. doi: 10.1016/j.celrep.2018.06.061 30021152; PubMed Central PMCID: PMC6086571.

12. Petermann E, Orta ML, Issaeva N, Schultz N, Helleday T. Hydroxyurea-Stalled Replication Forks Become Progressively Inactivated and Require Two Different RAD51-Mediated Pathways for Restart and Repair. MolCell. 2010;37(4):492–502.

13. Somyajit K, Saxena S, Babu S, Mishra A, Nagaraju G. Mammalian RAD51 paralogs protect nascent DNA at stalled forks and mediate replication restart. Nucleic Acids Res. 2015;43(20):9835–55. doi: 10.1093/nar/gkv880 26354865; PubMed Central PMCID: PMC4787763.

14. Kogoma T, von Meyenburg K. The origin of replication, oriC, and the dnaA protein are dispensable in stable DNA replication (sdrA) mutants of Escherichia coli K-12. The EMBO journal. 1983;2(3):463–8. 11894964; PubMed Central PMCID: PMC555155.

15. Asai T, Sommer S, Bailone A, Kogoma T. Homologous recombination-dependent initiation of DNA replication from DNA damage-inducible origins in Escherichia coli. The EMBO journal. 1993;12(8):3287–95. 8344265; PubMed Central PMCID: PMC413596.

16. Asai T, Kogoma T. D-loops and R-loops: alternative mechanisms for the initiation of chromosome replication in Escherichia coli. Journal of bacteriology. 1994;176(7):1807–12. doi: 10.1128/jb.176.7.1807-1812.1994 8144445; PubMed Central PMCID: PMC205281.

17. Kogoma T. Stable DNA replication: interplay between DNA replication, homologous recombination, and transcription. Microbiology and molecular biology reviews: MMBR. 1997;61(2):212–38. 9184011; PubMed Central PMCID: PMC232608.

18. Hawkins M, Malla S, Blythe MJ, Nieduszynski CA, Allers T. Accelerated growth in the absence of DNA replication origins. Nature. 2013;503(7477):544–7. doi: 10.1038/nature12650 24185008; PubMed Central PMCID: PMC3843117.

19. Piazza A, Heyer WD. Homologous Recombination and the Formation of Complex Genomic Rearrangements. Trends in cell biology. 2019;29(2):135–49. doi: 10.1016/j.tcb.2018.10.006 30497856; PubMed Central PMCID: PMC6402879.

20. Lee CS, Haber JE. Mating-type Gene Switching in Saccharomyces cerevisiae. Microbiology spectrum. 2015;3(2):MDNA3-0013-2014. doi: 10.1128/microbiolspec.MDNA3-0013-2014 26104712.

21. McCulloch R, Morrison LJ, Hall JP. DNA Recombination Strategies During Antigenic Variation in the African Trypanosome. Microbiology spectrum. 2015;3(2):MDNA3-0016–2014. doi: 10.1128/microbiolspec.MDNA3-0016-2014 26104717.

22. Trenaman A, Hartley C, Prorocic M, Passos-Silva DG, van den Hoek M, Nechyporuk-Zloy V, et al. Trypanosoma brucei BRCA2 acts in a life cycle-specific genome stability process and dictates BRC repeat number-dependent RAD51 subnuclear dynamics. Nucleic Acids Res. 2013;41(2):943–60. doi: 10.1093/nar/gks1192 23222131; PubMed Central PMCID: PMC3553974.

23. Hartley CL, McCulloch R. Trypanosoma brucei BRCA2 acts in antigenic variation and has undergone a recent expansion in BRC repeat number that is important during homologous recombination. MolMicrobiol. 2008;68(5):1237–51.

24. Moraes Barros RR, Marini MM, Antonio CR, Cortez DR, Miyake AM, Lima FM, et al. Anatomy and evolution of telomeric and subtelomeric regions in the human protozoan parasite Trypanosoma cruzi. BMC Genomics. 2012;13:229. doi: 10.1186/1471-2164-13-229 22681854; PubMed Central PMCID: PMC3418195.

25. Weatherly DB, Peng D, Tarleton RL. Recombination-driven generation of the largest pathogen repository of antigen variants in the protozoan Trypanosoma cruzi. BMC Genomics. 2016;17(1):729. doi: 10.1186/s12864-016-3037-z 27619017; PubMed Central PMCID: PMC5020489.

26. Alves CL, Repoles BM, da Silva MS, Mendes IC, Marin PA, Aguiar PHN, et al. The recombinase Rad51 plays a key role in events of genetic exchange in Trypanosoma cruzi. Scientific reports. 2018;8(1):13335. doi: 10.1038/s41598-018-31541-z 30190603; PubMed Central PMCID: PMC6127316.

27. Laffitte MC, Leprohon P, Hainse M, Legare D, Masson JY, Ouellette M. Chromosomal Translocations in the Parasite Leishmania by a MRE11/RAD50-Independent Microhomology-Mediated End Joining Mechanism. PLoS genetics. 2016;12(6):e1006117. doi: 10.1371/journal.pgen.1006117 27314941; PubMed Central PMCID: PMC4912120.

28. Laffitte MN, Leprohon P, Papadopoulou B, Ouellette M. Plasticity of the Leishmania genome leading to gene copy number variations and drug resistance. F1000Res. 2016;5:2350. doi: 10.12688/f1000research.9218.1 27703673; PubMed Central PMCID: PMC5031125.

29. Genois MM, Plourde M, Ethier C, Roy G, Poirier GG, Ouellette M, et al. Roles of Rad51 paralogs for promoting homologous recombination in Leishmania infantum. Nucleic Acids Res. 2015;43(5):2701–15. doi: 10.1093/nar/gkv118 25712090; PubMed Central PMCID: PMC4357719.

30. Ubeda JM, Raymond F, Mukherjee A, Plourde M, Gingras H, Roy G, et al. Genome-wide stochastic adaptive DNA amplification at direct and inverted DNA repeats in the parasite Leishmania. PLoS biology. 2014;12(5):e1001868. doi: 10.1371/journal.pbio.1001868 24844805; PubMed Central PMCID: PMC4028189.

31. Laffitte MC, Genois MM, Mukherjee A, Legare D, Masson JY, Ouellette M. Formation of linear amplicons with inverted duplications in Leishmania requires the MRE11 nuclease. PLoS genetics. 2014;10(12):e1004805. doi: 10.1371/journal.pgen.1004805 25474106; PubMed Central PMCID: PMC4256157.

32. Genois MM, Paquet ER, Laffitte MC, Maity R, Rodrigue A, Ouellette M, et al. DNA repair pathways in trypanosomatids: from DNA repair to drug resistance. Microbiology and molecular biology reviews: MMBR. 2014;78(1):40–73. doi: 10.1128/MMBR.00045-13 24600040; PubMed Central PMCID: PMC3957735.

33. Lachaud L, Bourgeois N, Kuk N, Morelle C, Crobu L, Merlin G, et al. Constitutive mosaic aneuploidy is a unique genetic feature widespread in the Leishmania genus. Microbes and infection / Institut Pasteur. 2014;16(1):61–6. doi: 10.1016/j.micinf.2013.09.005 24120456.

34. Bussotti G, Gouzelou E, Cortes Boite M, Kherachi I, Harrat Z, Eddaikra N, et al. Leishmania Genome Dynamics during Environmental Adaptation Reveal Strain-Specific Differences in Gene Copy Number Variation, Karyotype Instability, and Telomeric Amplification. mBio. 2018;9(6). doi: 10.1128/mBio.01399-18 30401775; PubMed Central PMCID: PMC6222132.

35. Dumetz F, Imamura H, Sanders M, Seblova V, Myskova J, Pescher P, et al. Modulation of Aneuploidy in Leishmania donovani during Adaptation to Different In Vitro and In Vivo Environments and Its Impact on Gene Expression. mBio. 2017;8(3). doi: 10.1128/mBio.00599-17 28536289; PubMed Central PMCID: PMC5442457.

36. Prieto Barja P, Pescher P, Bussotti G, Dumetz F, Imamura H, Kedra D, et al. Haplotype selection as an adaptive mechanism in the protozoan pathogen Leishmania donovani. Nat Ecol Evol. 2017;1(12):1961–9. doi: 10.1038/s41559-017-0361-x 29109466.

37. Zackay A, Cotton JA, Sanders M, Hailu A, Nasereddin A, Warburg A, et al. Genome wide comparison of Ethiopian Leishmania donovani strains reveals differences potentially related to parasite survival. PLoS genetics. 2018;14(1):e1007133. doi: 10.1371/journal.pgen.1007133 29315303; PubMed Central PMCID: PMC5777657.

38. Ubeda JM, Legare D, Raymond F, Ouameur AA, Boisvert S, Rigault P, et al. Modulation of gene expression in drug resistant Leishmania is associated with gene amplification, gene deletion and chromosome aneuploidy. Genome biology. 2008;9(7):R115. doi: 10.1186/gb-2008-9-7-r115 18638379; PubMed Central PMCID: PMC2530873.

39. Marques CA, Dickens NJ, Paape D, Campbell SJ, McCulloch R. Genome-wide mapping reveals single-origin chromosome replication in Leishmania, a eukaryotic microbe. Genome biology. 2015;16(1):230. doi: 10.1186/s13059-015-0788-9 26481451.

40. Lombrana R, Alvarez A, Fernandez-Justel JM, Almeida R, Poza-Carrion C, Gomes F, et al. Transcriptionally Driven DNA Replication Program of the Human Parasite Leishmania major. Cell reports. 2016;16(6):1774–86. doi: 10.1016/j.celrep.2016.07.007 27477279.

41. Marques CA, McCulloch R. Conservation and Variation in Strategies for DNA Replication of Kinetoplastid Nuclear Genomes. Current genomics. 2018;19(2):98–109. doi: 10.2174/1389202918666170815144627 29491738; PubMed Central PMCID: PMC5814967.

42. McKean PG, Keen JK, Smith DF, Benson FE. Identification and characterisation of a RAD51 gene from Leishmania major. MolBiochemParasitol. 2001;115(2):209–16.

43. Genois MM, Mukherjee A, Ubeda JM, Buisson R, Paquet E, Roy G, et al. Interactions between BRCA2 and RAD51 for promoting homologous recombination in Leishmania infantum. Nucleic Acids Res. 2012;40(14):6570–84. doi: 10.1093/nar/gks306 22505581; PubMed Central PMCID: PMC3413117.

44. Proudfoot C, McCulloch R. Distinct roles for two RAD51-related genes in Trypanosoma brucei antigenic variation. Nucleic Acids Res. 2005;33(21):6906–19. doi: 10.1093/nar/gki996 16326865

45. Proudfoot C, McCulloch R. Trypanosoma brucei DMC1 does not act in DNA recombination, repair or antigenic variation in bloodstream stage cells. MolBiochemParasitol. 2006;145(2):245–53.

46. Dobson R, Stockdale C, Lapsley C, Wilkes J, McCulloch R. Interactions among Trypanosoma brucei RAD51 paralogues in DNA repair and antigenic variation. MolMicrobiol. 2011;81(2):434–56.

47. Peacock L, Ferris V, Sharma R, Sunter J, Bailey M, Carrington M, et al. Identification of the meiotic life cycle stage of Trypanosoma brucei in the tsetse fly. Proceedings of the National Academy of Sciences of the United States of America. 2011;108(9):3671–6. doi: 10.1073/pnas.1019423108 21321215; PubMed Central PMCID: PMC3048101.

48. Zhang WW, Lypaczewski P, Matlashewski G. Optimized CRISPR-Cas9 Genome Editing for Leishmania and Its Use To Target a Multigene Family, Induce Chromosomal Translocation, and Study DNA Break Repair Mechanisms. mSphere. 2017;2(1). doi: 10.1128/mSphere.00340-16 28124028; PubMed Central PMCID: PMC5244264.

49. McCulloch R, Barry JD. A role for RAD51 and homologous recombination in Trypanosoma brucei antigenic variation. Genes & development. 1999;13(21):2875–88. doi: 10.1101/gad.13.21.2875 10557214; PubMed Central PMCID: PMC317127.

50. Liu B, Wang J, Yaffe N, Lindsay ME, Zhao Z, Zick A, et al. Trypanosomes have six mitochondrial DNA helicases with one controlling kinetoplast maxicircle replication. MolCell. 2009;35(4):490–501.

51. Byrd AK, Raney KD. Structure and function of Pif1 helicase. Biochemical Society transactions. 2017;45(5):1159–71. doi: 10.1042/BST20170096 28900015; PubMed Central PMCID: PMC5870758.

52. Deegan TD, Baxter J, Ortiz Bazan MA, Yeeles JTP, Labib KPM. Pif1-Family Helicases Support Fork Convergence during DNA Replication Termination in Eukaryotes. Molecular cell. 2019;74(2):231–44 e9. doi: 10.1016/j.molcel.2019.01.040 30850330; PubMed Central PMCID: PMC6477153.

53. Glover L, Horn D. Trypanosomal histone gammaH2A and the DNA damage response. MolBiochemParasitol. 2012;183(1):78–83.

54. Tiengwe C, Marcello L, Farr H, Dickens N, Kelly S, Swiderski M, et al. Genome-wide analysis reveals extensive functional interaction between DNA replication initiation and transcription in the genome of Trypanosoma brucei. Cell reports. 2012;2(1):185–97. doi: 10.1016/j.celrep.2012.06.007 22840408; PubMed Central PMCID: PMC3607257.

55. Muller CA, Nieduszynski CA. Conservation of replication timing reveals global and local regulation of replication origin activity. Genome research. 2012;22(10):1953–62. doi: 10.1101/gr.139477.112 22767388; PubMed Central PMCID: PMC3460190.

56. Conway C, Proudfoot C, Burton P, Barry JD, McCulloch R. Two pathways of homologous recombination in Trypanosoma brucei. Molecular microbiology. 2002;45(6):1687–700. doi: 10.1046/j.1365-2958.2002.03122.x 12354234.

57. Glover L, Jun J, Horn D. Microhomology-mediated deletion and gene conversion in African trypanosomes. Nucleic Acids Res. 2011;39(4):1372–80. doi: 10.1093/nar/gkq981 20965968

58. Glover L, McCulloch R, Horn D. Sequence homology and microhomology dominate chromosomal double-strand break repair in African trypanosomes. Nucleic Acids Res. 2008;36(8):2608–18. doi: 10.1093/nar/gkn104 18334531; PubMed Central PMCID: PMC2377438.

59. Zhang WW, Matlashewski G. CRISPR-Cas9-Mediated Genome Editing in Leishmania donovani. mBio. 2015;6(4):e00861. doi: 10.1128/mBio.00861-15 26199327; PubMed Central PMCID: PMC4513079.

60. Sollelis L, Ghorbal M, MacPherson CR, Martins RM, Kuk N, Crobu L, et al. First efficient CRISPR-Cas9-mediated genome editing in Leishmania parasites. Cellular microbiology. 2015;17(10):1405–12. doi: 10.1111/cmi.12456 25939677.

61. Lander N, Li ZH, Niyogi S, Docampo R. CRISPR/Cas9-Induced Disruption of Paraflagellar Rod Protein 1 and 2 Genes in Trypanosoma cruzi Reveals Their Role in Flagellar Attachment. mBio. 2015;6(4):e01012. doi: 10.1128/mBio.01012-15 26199333; PubMed Central PMCID: PMC4513075.

62. Peng D, Kurup SP, Yao PY, Minning TA, Tarleton RL. CRISPR-Cas9-mediated single-gene and gene family disruption in Trypanosoma cruzi. mBio. 2015;6(1):e02097–14. doi: 10.1128/mBio.02097-14 25550322; PubMed Central PMCID: PMC4281920.

63. Iantorno SA, Durrant C, Khan A, Sanders MJ, Beverley SM, Warren WC, et al. Gene Expression in Leishmania Is Regulated Predominantly by Gene Dosage. mBio. 2017;8(5). doi: 10.1128/mBio.01393-17 28900023; PubMed Central PMCID: PMC5596349.

64. Costantino L, Koshland D. Genome-wide Map of R-Loop-Induced Damage Reveals How a Subset of R-Loops Contributes to Genomic Instability. Molecular cell. 2018;71(4):487–97 e3. doi: 10.1016/j.molcel.2018.06.037 30078723; PubMed Central PMCID: PMC6264797.

65. Briggs E, Hamilton G, Crouch K, Lapsley C, McCulloch R. Genome-wide mapping reveals conserved and diverged R-loop activities in the unusual genetic landscape of the African trypanosome genome. Nucleic Acids Res. 2018;46(22):11789–805. doi: 10.1093/nar/gky928 30304482; PubMed Central PMCID: PMC6294496.

66. Kim HS. Genome-wide function of MCM-BP in Trypanosoma brucei DNA replication and transcription. Nucleic Acids Res. 2019;47(2):634–47. doi: 10.1093/nar/gky1088 30407533; PubMed Central PMCID: PMC6344857.

67. Santosa V, Martha S, Hirose N, Tanaka K. The fission yeast minichromosome maintenance (MCM)-binding protein (MCM-BP), Mcb1, regulates MCM function during prereplicative complex formation in DNA replication. The Journal of biological chemistry. 2013;288(10):6864–80. doi: 10.1074/jbc.M112.432393 23322785; PubMed Central PMCID: PMC3591596.

68. Nishiyama A, Frappier L, Mechali M. MCM-BP regulates unloading of the MCM2-7 helicase in late S phase. Genes & development. 2011;25(2):165–75. doi: 10.1101/gad.614411 21196493; PubMed Central PMCID: PMC3022262.

69. Marques CA, Tiengwe C, Lemgruber L, Damasceno JD, Scott A, Paape D, et al. Diverged composition and regulation of the Trypanosoma brucei origin recognition complex that mediates DNA replication initiation. Nucleic Acids Res. 2016;44(10):4763–84. doi: 10.1093/nar/gkw147 26951375; PubMed Central PMCID: PMC4889932.

70. Recombination Mosig G. and recombination-dependent DNA replication in bacteriophage T4. Annual review of genetics. 1998;32:379–413. doi: 10.1146/annurev.genet.32.1.379 9928485.

71. Mosig G, Gewin J, Luder A, Colowick N, Vo D. Two recombination-dependent DNA replication pathways of bacteriophage T4, and their roles in mutagenesis and horizontal gene transfer. Proceedings of the National Academy of Sciences of the United States of America. 2001;98(15):8306–11. doi: 10.1073/pnas.131007398 11459968; PubMed Central PMCID: PMC37436.

72. Wilkinson DE, Weller SK. The role of DNA recombination in herpes simplex virus DNA replication. IUBMB Life. 2003;55(8):451–8. doi: 10.1080/15216540310001612237 14609200.

73. Lee PH, Meng X, Kapler GM. Developmental Regulation of the Tetrahymena thermophila Origin Recognition Complex. PLoS genetics. 2015;11(1):e1004875. doi: 10.1371/journal.pgen.1004875 25569357; PubMed Central PMCID: PMC4287346.

74. Natsume T, Nishimura K, Minocherhomji S, Bhowmick R, Hickson ID, Kanemaki MT. Acute inactivation of the replicative helicase in human cells triggers MCM8-9-dependent DNA synthesis. Genes & development. 2017;31(8):816–29. doi: 10.1101/gad.297663.117 28487407; PubMed Central PMCID: PMC5435893.

75. Akiyoshi B, Gull K. Discovery of unconventional kinetochores in kinetoplastids. Cell. 2014;156(6):1247–58. doi: 10.1016/j.cell.2014.01.049 24582333; PubMed Central PMCID: PMC3978658.

76. Garcia-Silva MR, Sollelis L, MacPherson CR, Stanojcic S, Kuk N, Crobu L, et al. Identification of the centromeres of Leishmania major: revealing the hidden pieces. EMBO reports. 2017;18(11):1968–77. doi: 10.15252/embr.201744216 28935715; PubMed Central PMCID: PMC5666652.

77. Forsburg SL, Shen KF. Centromere Stability: The Replication Connection. Genes (Basel). 2017;8(1). doi: 10.3390/genes8010037 28106789; PubMed Central PMCID: PMC5295031.

78. Onaka AT, Toyofuku N, Inoue T, Okita AK, Sagawa M, Su J, et al. Rad51 and Rad54 promote noncrossover recombination between centromere repeats on the same chromatid to prevent isochromosome formation. Nucleic Acids Res. 2016;44(22):10744–57. doi: 10.1093/nar/gkw874 27697832; PubMed Central PMCID: PMC5159554.

79. McFarlane RJ, Humphrey TC. A role for recombination in centromere function. Trends in genetics: TIG. 2010;26(5):209–13. doi: 10.1016/j.tig.2010.02.005 20382440.

80. Santos R, Silva GLA, Santos EV, Duncan SM, Mottram JC, Damasceno JD, et al. A DiCre recombinase-based system for inducible expression in Leishmania major. Molecular and biochemical parasitology. 2017;216:45–8. doi: 10.1016/j.molbiopara.2017.06.006 28629935.

81. Beneke T, Madden R, Makin L, Valli J, Sunter J, Gluenz E. A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids. R Soc Open Sci. 2017;4(5):170095. doi: 10.1098/rsos.170095 28573017; PubMed Central PMCID: PMC5451818.

82. Damasceno JD, Obonaga R, Silva GLA, Reis-Cunha JL, Duncan SM, Bartholomeu DC, et al. Conditional genome engineering reveals canonical and divergent roles for the Hus1 component of the 9-1-1 complex in the maintenance of the plastic genome of Leishmania. Nucleic Acids Res. 2018;46(22):11835–46. doi: 10.1093/nar/gky1017 30380080; PubMed Central PMCID: PMC6294564.

83. Duncan SM, Myburgh E, Philipon C, Brown E, Meissner M, Brewer J, et al. Conditional gene deletion with DiCre demonstrates an essential role for CRK3 in Leishmania mexicana cell cycle regulation. Molecular microbiology. 2016;100(6):931–44. doi: 10.1111/mmi.13375 26991545; PubMed Central PMCID: PMC4913733.

84. Stortz JA, Serafim TD, Alsford S, Wilkes J, Fernandez-Cortes F, Hamilton G, et al. Genome-wide and protein kinase-focused RNAi screens reveal conserved and novel damage response pathways in Trypanosoma brucei. PLoS pathogens. 2017;13(7):e1006477. doi: 10.1371/journal.ppat.1006477 28742144; PubMed Central PMCID: PMC5542689.

85. Afgan E, Baker D, Batut B, van den Beek M, Bouvier D, Cech M, et al. The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update. Nucleic Acids Res. 2018;46(W1):W537–W44. doi: 10.1093/nar/gky379 29790989; PubMed Central PMCID: PMC6030816.

86. Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30(15):2114–20. Epub 2014/04/04. doi: 10.1093/bioinformatics/btu170 24695404; PubMed Central PMCID: PMC4103590.

87. Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009;25(14):1754–60. Epub 2009/05/20. doi: 10.1093/bioinformatics/btp324 19451168; PubMed Central PMCID: PMC2705234.

88. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009;25(16):2078–9. Epub 2009/06/10. doi: 10.1093/bioinformatics/btp352 19505943; PubMed Central PMCID: PMC2723002.

89. McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, et al. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 2010;20(9):1297–303. Epub 2010/07/21. doi: 10.1101/gr.107524.110 20644199; PubMed Central PMCID: PMC2928508.

90. Garrison E, Marth G. Haplotype-based variant detection from short-read sequencing. arXiv e-prints [Internet]. 2012 July 01, 2012. Available from:

91. Danecek P, Auton A, Abecasis G, Albers CA, Banks E, DePristo MA, et al. The variant call format and VCFtools. Bioinformatics. 2011;27(15):2156–8. Epub 2011/06/10. doi: 10.1093/bioinformatics/btr330 21653522; PubMed Central PMCID: PMC3137218.

92. Ramirez F, Ryan DP, Gruning B, Bhardwaj V, Kilpert F, Richter AS, et al. deepTools2: a next generation web server for deep-sequencing data analysis. Nucleic Acids Res. 2016;44(W1):W160–5. Epub 2016/04/16. doi: 10.1093/nar/gkw257 27079975; PubMed Central PMCID: PMC4987876.

93. Nik-Zainal S, Davies H, Staaf J, Ramakrishna M, Glodzik D, Zou X, et al. Landscape of somatic mutations in 560 breast cancer whole-genome sequences. Nature. 2016;534(7605):47–54. Epub 2016/05/03. doi: 10.1038/nature17676 27135926; PubMed Central PMCID: PMC4910866.

94. Hahne F, Ivanek R. Visualizing Genomic Data Using Gviz and Bioconductor. Methods Mol Biol. 2016;1418:335–51. Epub 2016/03/24. doi: 10.1007/978-1-4939-3578-9_16 27008022.

Článek vyšel v časopise

PLOS Genetics

2020 Číslo 7

Nejčtenější v tomto čísle
Zapomenuté heslo

Nemáte účet?  Registrujte se

Zapomenuté heslo

Zadejte e-mailovou adresu, se kterou jste vytvářel(a) účet, budou Vám na ni zaslány informace k nastavení nového hesla.


Nemáte účet?  Registrujte se