A new class of disordered elements controls DNA replication through initiator self-assembly

Abstract

The initiation of DNA replication in metazoans occurs at thousands of chromosomal sites known as origins. At each origin, the Origin Recognition Complex (ORC), Cdc6, and Cdt1 co-assemble to load the Mcm2-7 replicative helicase onto chromatin. Current replication models envisage a linear arrangement of isolated origins functioning autonomously; the extent of inter-origin organization and communication is unknown. Here, we report that the replication initiation machinery of D. melanogaster unexpectedly undergoes liquid-liquid phase separation (LLPS) upon binding DNA in vitro. We find that ORC, Cdc6, and Cdt1 contain intrinsically disordered regions (IDRs) that drive LLPS and constitute a new class of phase separating elements. Initiator IDRs are shown to regulate multiple functions, including chromosome recruitment, initiator-specific co-assembly, and Mcm2-7 loading. These data help explain how CDK activity controls replication initiation and suggest that replication programs are subject to higher-order levels of inter-origin organization.

References 122

1. 1.

   Arias EE, Walter JC. PCNA functions as a molecular platform to trigger Cdt1 destruction and prevent re-replication. Nature Cell Biology. 2006;8:84-90.

   Not in our library10.1038/ncb1346

2. 2.

   Aumiller WM, Keating CD. Phosphorylation-mediated RNA/peptide complex coacervation as a model for intracellular liquid organelles. Nature Chemistry. 2016;8:129-137.

   Not in our library10.1038/nchem.2414

3. 3.

   Austin RJ, Orr-Weaver TL, Bell SP. Drosophila ORC specifically binds to ACE3, an origin of DNA replication control element. Genes & Development. 1999;13:2639-2649.

   Not in our library10.1101/gad.13.20.2639

4. 4.

   Balasov M, Huijbregts RP, Chesnokov I. Role of the Orc6 protein in origin recognition complex-dependent DNA binding and replication in Drosophila Melanogaster. Molecular and Cellular Biology. 2007;27:3143-3153.

   Not in our library10.1128/mcb.02382-06

5. 5.

   Baldinger T, Gossen M. Binding of Drosophila ORC proteins to anaphase chromosomes requires cessation of mitotic cyclin-dependent kinase activity. Molecular and Cellular Biology. 2009;29:140-149.

   Not in our library10.1128/mcb.00981-08

6. 6.

   Bell SP, Stillman B. ATP-dependent recognition of eukaryotic origins of DNA replication by a multiprotein complex. Nature. 1992;357:128-134.

   Not in our library10.1038/357128a0

7. 7.

   Bleichert F, Botchan MR, Berger JM. Mechanisms for initiating cellular DNA replication. Science. 2017;355:eaah6317.

   Not in our library10.1126/science.aah6317

8. 8.

   Bleichert F, Leitner A, Aebersold R, Botchan MR, Berger JM. Conformational control and DNA-binding mechanism of the metazoan origin recognition complex. PNAS. 2018;115:E5906-E5915.

   Not in our library10.1073/pnas.1806315115

9. 9.

   Boeynaems S, Alberti S, Fawzi NL, Mittag T, Polymenidou M, Rousseau F, Schymkowitz J, Shorter J, Wolozin B, Van Den Bosch L, Tompa P, Fuxreiter M. Protein phase separation: a new phase in cell biology. Trends in Cell Biology. 2018;28:420-435.

   Not in our library10.1016/j.tcb.2018.02.004

10. 10.

    Bowers JL, Randell JC, Chen S, Bell SP. ATP hydrolysis by ORC catalyzes reiterative Mcm2-7 assembly at a defined origin of replication. Molecular Cell. 2004;16:967-978.

    Not in our library10.1016/j.molcel.2004.11.038

11. 11.

    Brangwynne CP, Eckmann CR, Courson DS, Rybarska A, Hoege C, Gharakhani J, Jülicher F, Hyman AA. Germline P granules are liquid droplets that localize by controlled dissolution/condensation. Science. 2009;324:1729-1732.

    Not in our library10.1126/science.1172046

12. 12.

    Brüne D, Andrade-Navarro MA, Mier P. Proteome-wide comparison between the amino acid composition of domains and linkers. BMC Research Notes. 2018;11:117.

    Not in our library10.1186/s13104-018-3221-0

13. 13.

    Cayrou C, Coulombe P, Vigneron A, Stanojcic S, Ganier O, Peiffer I, Rivals E, Puy A, Laurent-Chabalier S, Desprat R, Méchali M. Genome-scale analysis of metazoan replication origins reveals their organization in specific but flexible sites defined by conserved features. Genome Research. 2011;21:1438-1449.

    Not in our library10.1101/gr.121830.111

14. 14.

    Cayrou C, Coulombe P, Puy A, Rialle S, Kaplan N, Segal E, Méchali M. New insights into replication origin characteristics in metazoans. Cell Cycle. 2012;11:658-667.

    Not in our library10.4161/cc.11.4.19097

15. 15.

    Cayrou C, Ballester B, Peiffer I, Fenouil R, Coulombe P, Andrau JC, van Helden J, Méchali M. The chromatin environment shapes DNA replication origin organization and defines origin classes. Genome Research. 2015;25:1873-1885.

    Not in our library10.1101/gr.192799.115

16. 16.
    4D Visualization of replication foci in mammalian cells corresponding to individual replicons · 2016

    No notice
17. 17.

    Chen BC, Legant WR, Wang K, Shao L, Milkie DE, Davidson MW, Janetopoulos C, Wu XS, Hammer JA, Liu Z, English BP, Mimori-Kiyosue Y, Romero DP, Ritter AT, Lippincott-Schwartz J, Fritz-Laylin L, Mullins RD, Mitchell DM, Bembenek JN, Reymann AC, Böhme R, Grill SW, Wang JT, Seydoux G, Tulu US, Kiehart DP, Betzig E. Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution. Science. 2014;346:1257998.

    Not in our library10.1126/science.1257998

18. 18.

    Chesnokov I, Remus D, Botchan M. Functional analysis of mutant and wild-type Drosophila origin recognition complex. PNAS. 2001;98:11997-12002.

    Not in our library10.1073/pnas.211342798

19. 19.

    Chong PA, Vernon RM, Forman-Kay JD. RGG/RG motif regions in RNA binding and phase separation. Journal of Molecular Biology. 2018a;430:4650-4665.

    Not in our library10.1016/j.jmb.2018.06.014

20. 20.

    Chong S, Dugast-Darzacq C, Liu Z, Dong P, Dailey GM, Cattoglio C, Heckert A, Banala S, Lavis L, Darzacq X, Tjian R. Imaging dynamic and selective low-complexity domain interactions that control gene transcription. Science. 2018b;361:eaar2555.

    Not in our library10.1126/science.aar2555

21. 21.
    The fission yeast homologue of Orc4p binds to replication origin DNA via multiple AT-hooks · 1999

    Not checked
22. 22.

    Cook PR. The organization of replication and transcription. Science. 1999;284:1790-1795.

    Not in our library10.1126/science.284.5421.1790

23. 23.

    Cremer T, Cremer C. Chromosome territories, nuclear architecture and gene regulation in mammalian cells. Nature Reviews Genetics. 2001;2:292-301.

    Not in our library10.1038/35066075

24. 24.

    Davey NE, Van Roey K, Weatheritt RJ, Toedt G, Uyar B, Altenberg B, Budd A, Diella F, Dinkel H, Gibson TJ. Attributes of short linear motifs. Mol. BioSyst. 2012;8:268-281.

    Not in our library10.1039/c1mb05231d

25. 25.

    Drosophila 12 Genomes Consortium, Clark AG, Eisen MB, Smith DR, Bergman CM, Oliver B, Markow TA, Kaufman TC, Kellis M, Gelbart W, Iyer VN, Pollard DA, Sackton TB, Larracuente AM, Singh ND, Abad JP, Abt DN, Adryan B, Aguade M, Akashi H, Anderson WW, Aquadro CF, Ardell DH, Arguello R, Artieri CG, Barbash DA, Barker D, Barsanti P, Batterham P, Batzoglou S, Begun D, Bhutkar A, Blanco E, Bosak SA, Bradley RK, Brand AD, Brent MR, Brooks AN, Brown RH, Butlin RK, Caggese C, Calvi BR, Bernardo de Carvalho A, Caspi A, Castrezana S, Celniker SE, Chang JL, Chapple C, Chatterji S, Chinwalla A, Civetta A, Clifton SW, Comeron JM, Costello JC, Coyne JA, Daub J, David RG, Delcher AL, Delehaunty K, Do CB, Ebling H, Edwards K, Eickbush T, Evans JD, Filipski A, Findeiss S, Freyhult E, Fulton L, Fulton R, Garcia AC, Gardiner A, Garfield DA, Garvin BE, Gibson G, Gilbert D, Gnerre S, Godfrey J, Good R, Gotea V, Gravely B, Greenberg AJ, Griffiths-Jones S, Gross S, Guigo R, Gustafson EA, Haerty W, Hahn MW, Halligan DL, Halpern AL, Halter GM, Han MV, Heger A, Hillier L, Hinrichs AS, Holmes I, Hoskins RA, Hubisz MJ, Hultmark D, Huntley MA, Jaffe DB, Jagadeeshan S, Jeck WR, Johnson J, Jones CD, Jordan WC, Karpen GH, Kataoka E, Keightley PD, Kheradpour P, Kirkness EF, Koerich LB, Kristiansen K, Kudrna D, Kulathinal RJ, Kumar S, Kwok R, Lander E, Langley CH, Lapoint R, Lazzaro BP, Lee SJ, Levesque L, Li R, Lin CF, Lin MF, Lindblad-Toh K, Llopart A, Long M, Low L, Lozovsky E, Lu J, Luo M, Machado CA, Makalowski W, Marzo M, Matsuda M, Matzkin L, McAllister B, McBride CS, McKernan B, McKernan K, Mendez-Lago M, Minx P, Mollenhauer MU, Montooth K, Mount SM, Mu X, Myers E, Negre B, Newfeld S, Nielsen R, Noor MA, O'Grady P, Pachter L, Papaceit M, Parisi MJ, Parisi M, Parts L, Pedersen JS, Pesole G, Phillippy AM, Ponting CP, Pop M, Porcelli D, Powell JR, Prohaska S, Pruitt K, Puig M, Quesneville H, Ram KR, Rand D, Rasmussen MD, Reed LK, Reenan R, Reily A, Remington KA, Rieger TT, Ritchie MG, Robin C, Rogers YH, Rohde C, Rozas J, Rubenfield MJ, Ruiz A, Russo S, Salzberg SL, Sanchez-Gracia A, Saranga DJ, Sato H, Schaeffer SW, Schatz MC, Schlenke T, Schwartz R, Segarra C, Singh RS, Sirot L, Sirota M, Sisneros NB, Smith CD, Smith TF, Spieth J, Stage DE, Stark A, Stephan W, Strausberg RL, Strempel S, Sturgill D, Sutton G, Sutton GG, Tao W, Teichmann S, Tobari YN, Tomimura Y, Tsolas JM, Valente VL, Venter E, Venter JC, Vicario S, Vieira FG, Vilella AJ, Villasante A, Walenz B, Wang J, Wasserman M, Watts T, Wilson D, Wilson RK, Wing RA, Wolfner MF, Wong A, Wong GK, Wu CI, Wu G, Yamamoto D, Yang HP, Yang SP, Yorke JA, Yoshida K, Zdobnov E, Zhang P, Zhang Y, Zimin AV, Baldwin J, Abdouelleil A, Abdulkadir J, Abebe A, Abera B, Abreu J, Acer SC, Aftuck L, Alexander A, An P, Anderson E, Anderson S, Arachi H, Azer M, Bachantsang P, Barry A, Bayul T, Berlin A, Bessette D, Bloom T, Blye J, Boguslavskiy L, Bonnet C, Boukhgalter B, Bourzgui I, Brown A, Cahill P, Channer S, Cheshatsang Y, Chuda L, Citroen M, Collymore A, Cooke P, Costello M, D'Aco K, Daza R, De Haan G, DeGray S, DeMaso C, Dhargay N, Dooley K, Dooley E, Doricent M, Dorje P, Dorjee K, Dupes A, Elong R, Falk J, Farina A, Faro S, Ferguson D, Fisher S, Foley CD, Franke A, Friedrich D, Gadbois L, Gearin G, Gearin CR, Giannoukos G, Goode T, Graham J, Grandbois E, Grewal S, Gyaltsen K, Hafez N, Hagos B, Hall J, Henson C, Hollinger A, Honan T, Huard MD, Hughes L, Hurhula B, Husby ME, Kamat A, Kanga B, Kashin S, Khazanovich D, Kisner P, Lance K, Lara M, Lee W, Lennon N, Letendre F, LeVine R, Lipovsky A, Liu X, Liu J, Liu S, Lokyitsang T, Lokyitsang Y, Lubonja R, Lui A, MacDonald P, Magnisalis V, Maru K, Matthews C, McCusker W, McDonough S, Mehta T, Meldrim J, Meneus L, Mihai O, Mihalev A, Mihova T, Mittelman R, Mlenga V, Montmayeur A, Mulrain L, Navidi A, Naylor J, Negash T, Nguyen T, Nguyen N, Nicol R, Norbu C, Norbu N, Novod N, O'Neill B, Osman S, Markiewicz E, Oyono OL, Patti C, Phunkhang P, Pierre F, Priest M, Raghuraman S

    Not in our library10.1038/nature06341

26. 26.

    Duzdevich D, Warner MD, Ticau S, Ivica NA, Bell SP, Greene EC. The dynamics of eukaryotic replication initiation: origin specificity, licensing, and firing at the single-molecule level. Molecular Cell. 2015;58:483-494.

    Not in our library10.1016/j.molcel.2015.03.017

27. 27.

    Elbaum-Garfinkle S, Kim Y, Szczepaniak K, Chen CC, Eckmann CR, Myong S, Brangwynne CP. The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics. PNAS. 2015;112:7189-7194.

    Not in our library10.1073/pnas.1504822112

28. 28.

    Evrin C, Clarke P, Zech J, Lurz R, Sun J, Uhle S, Li H, Stillman B, Speck C. A double-hexameric MCM2-7 complex is loaded onto origin DNA during licensing of eukaryotic DNA replication. PNAS. 2009;106:20240-20245.

    Not in our library10.1073/pnas.0911500106

29. 29.

    Evrin C, Fernandez-Cid A, Zech J, Herrera MC, Riera A, Clarke P, Brill S, Lurz R, Speck C. In the absence of ATPase activity, pre-RC formation is blocked prior to MCM2-7 hexamer dimerization. Nucleic Acids Research. 2013;41:3162-3172.

    Not in our library10.1093/nar/gkt043

30. 30.
    Identification and Characterization of Saccharomyces cerevisiae Cdc6 DNA-binding Properties · 2000

    Not checked
31. 31.

    Feric M, Vaidya N, Harmon TS, Mitrea DM, Zhu L, Richardson TM, Kriwacki RW, Pappu RV, Brangwynne CP. Coexisting liquid phases underlie nucleolar subcompartments. Cell. 2016;165:1686-1697.

    Not in our library10.1016/j.cell.2016.04.047

32. 32.

    Findeisen M, El-Denary M, Kapitza T, Graf R, Strausfeld U. Cyclin A-dependent kinase activity affects chromatin binding of ORC, Cdc6, and MCM in egg extracts of xenopus laevis. European Journal of Biochemistry. 1999;264:415-426.

    Not in our library10.1046/j.1432-1327.1999.00613.x

33. 33.

    Frigola J, He J, Kinkelin K, Pye VE, Renault L, Douglas ME, Remus D, Cherepanov P, Costa A, Diffley JFX. Cdt1 stabilizes an open MCM ring for helicase loading. Nature Communications. 2017;8:15720.

    Not in our library10.1038/ncomms15720

34. 34.

    Gilbert N, Gilchrist S, Bickmore WA. Chromatin organization in the mammalian nucleus. International Review of Cytology. 2005;242:283-336.

    Not in our library10.1016/s0074-7696(04)42007-5

35. 35.

    Giordano-Coltart J, Ying CY, Gautier J, Hurwitz J. Studies of the properties of human origin recognition complex and its walker A motif mutants. PNAS. 2005;102:69-74.

    Not in our library10.1073/pnas.0408690102

36. 36.

    Heinzel SS, Krysan PJ, Tran CT, Calos MP. Autonomous DNA replication in human cells is affected by the size and the source of the DNA. Molecular and Cellular Biology. 1991;11:2263-2272.

    Not in our library10.1128/mcb.11.4.2263

37. 37.

    Hemerly AS, Prasanth SG, Siddiqui K, Stillman B. Orc1 controls centriole and centrosome copy number in human cells. Science. 2009;323:789-793.

    Not in our library10.1126/science.1166745

38. 38.

    Hennig S, Kong G, Mannen T, Sadowska A, Kobelke S, Blythe A, Knott GJ, Iyer KS, Ho D, Newcombe EA, Hosoki K, Goshima N, Kawaguchi T, Hatters D, Trinkle-Mulcahy L, Hirose T, Bond CS, Fox AH. Prion-like domains in RNA binding proteins are essential for building subnuclear paraspeckles. The Journal of Cell Biology. 2015;210:529-539.

    Not in our library10.1083/jcb.201504117

39. 39.

    Hossain M, Bhalla K, Stillman B. Cyclin binding cy motifs have multiple activities in the initiation of DNA replication. bioRxiv. 2019.

    Not in our library10.1101/681668

40. 40.

    Houchens CR, Lu W, Chuang RY, Frattini MG, Fuller A, Simancek P, Kelly TJ. Multiple mechanisms contribute to Schizosaccharomyces pombe origin recognition complex-DNA interactions. Journal of Biological Chemistry. 2008;283:30216-30224.

    Not in our library10.1074/jbc.m802649200

Cited by papers we hold 2

• Structural mechanism for replication origin binding and remodeling by a metazoan origin recognition complex and its co-loader Cdc6 · 2020
  Not checked
• Biological phase separation: cell biology meets biophysics · 2020
  No notice

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