The capacity of origins to load MCM establishes replication timing patterns

Autoři: Livio Dukaj aff001;  Nicholas Rhind aff001
Působiště autorů: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School Worcester, Massachusetts, United States of America aff001
Vyšlo v časopise: The capacity of origins to load MCM establishes replication timing patterns. PLoS Genet 17(3): e1009467. doi:10.1371/journal.pgen.1009467
Kategorie: Research Article


Loading of the MCM replicative helicase at origins of replication is a highly regulated process that precedes DNA replication in all eukaryotes. The stoichiometry of MCM loaded at origins has been proposed to be a key determinant of when those origins initiate replication during S phase. Nevertheless, the genome-wide regulation of MCM loading stoichiometry and its direct effect on replication timing remain unclear. In order to investigate why some origins load more MCM than others, we perturbed MCM levels in budding yeast cells and, for the first time, directly measured MCM levels and replication timing in the same experiment. Reduction of MCM levels through degradation of Mcm4, one of the six obligate components of the MCM complex, slowed progression through S phase and increased sensitivity to replication stress. Reduction of MCM levels also led to differential loading at origins during G1, revealing origins that are sensitive to reductions in MCM and others that are not. Sensitive origins loaded less MCM under normal conditions and correlated with a weak ability to recruit the origin recognition complex (ORC). Moreover, reduction of MCM loading at specific origins of replication led to a delay in their replication during S phase. In contrast, overexpression of MCM had no effects on cell cycle progression, relative MCM levels at origins, or replication timing, suggesting that, under optimal growth conditions, cellular MCM levels are not limiting for MCM loading. Our results support a model in which the loading capacity of origins is the primary determinant of MCM stoichiometry in wild-type cells, but that stoichiometry is controlled by origins’ ability to recruit ORC and compete for MCM when MCM becomes limiting.

Klíčová slova:

Auxins – DNA replication – Genomic signal processing – Genomics – Helicases – Hyperexpression techniques – Stoichiometry – Synthesis phase


1. Bell SP, Labib K. Chromosome Duplication in Saccharomyces cerevisiae. Genetics. 2016;203: 1027–1067. doi: 10.1534/genetics.115.186452 27384026

2. Bleichert F. Mechanisms of replication origin licensing: a structural perspective. Curr Opin Struct Biol. 2019;59: 195–204. doi: 10.1016/ 31630057

3. Boos D, Ferreira P. Origin Firing Regulations to Control Genome Replication Timing. Genes (Basel). 2019;10: doi: 10.3390/genes10030199 30845782

4. Gilbert DM, Takebayashi SI, Ryba T, Lu J, Pope BD, Wilson KA et al. Space and time in the nucleus: developmental control of replication timing and chromosome architecture. Cold Spring Harb Symp Quant Biol. 2010;75: 143–153. doi: 10.1101/sqb.2010.75.011 21139067

5. Goren A, Cedar H. Replicating by the clock. Nat Rev Mol Cell Biol. 2003;4: 25–32. doi: 10.1038/nrm1008 12511866

6. Lang GI, Murray AW. Mutation rates across budding yeast chromosome VI are correlated with replication timing. Genome Biol Evol. 2011;3: 799–811. doi: 10.1093/gbe/evr054 21666225

7. Müller CA, Nieduszynski CA. DNA replication timing influences gene expression level. J Cell Biol. 2017;216: 1907–1914. doi: 10.1083/jcb.201701061 28539386

8. Rhind N, Gilbert DM. DNA replication timing. Cold Spring Harb Perspect Biol. 2013;5: a010132. doi: 10.1101/cshperspect.a010132 23838440

9. Nguyen VQ, Co C, Li JJ. Cyclin-dependent kinases prevent DNA re-replication through multiple mechanisms. Nature. 2001;411: 1068–1073. doi: 10.1038/35082600 11429609

10. Tanaka S, Diffley JF. Interdependent nuclear accumulation of budding yeast Cdt1 and Mcm2-7 during G1 phase. Nat Cell Biol. 2002;4: 198–207. doi: 10.1038/ncb757 11836525

11. Eaton ML, Galani K, Kang S, Bell SP, MacAlpine DM. Conserved nucleosome positioning defines replication origins. Genes Dev. 2010;24: 748–753. doi: 10.1101/gad.1913210 20351051

12. Xu W, Aparicio JG, Aparicio OM, Tavaré S. Genome-wide mapping of ORC and Mcm2p binding sites on tiling arrays and identification of essential ARS consensus sequences in S. cerevisiae. BMC Genomics. 2006;7: 276. doi: 10.1186/1471-2164-7-276 17067396

13. Ticau S, Friedman LJ, Ivica NA, Gelles J, Bell SP. Single-Molecule Studies of Origin Licensing Reveal Mechanisms Ensuring Bidirectional Helicase Loading. Cell. 2015 doi: 10.1016/j.cell.2015.03.012 25892223

14. Labib K. How do Cdc7 and cyclin-dependent kinases trigger the initiation of chromosome replication in eukaryotic cells. Genes Dev. 2010;24: 1208–1219. doi: 10.1101/gad.1933010 20551170

15. Mantiero D, Mackenzie A, Donaldson A, Zegerman P. Limiting replication initiation factors execute the temporal programme of origin firing in budding yeast. EMBO J. 2011;30: 4805–4814. doi: 10.1038/emboj.2011.404 22081107

16. Hiraga S, Alvino GM, Chang F, Lian HY, Sridhar A, Kubota T et al. Rif1 controls DNA replication by directing Protein Phosphatase 1 to reverse Cdc7-mediated phosphorylation of the MCM complex. Genes Dev. 2014;28: 372–383. doi: 10.1101/gad.231258.113 24532715

17. Hayashi MT, Takahashi TS, Nakagawa T, Nakayama J, Masukata H. The heterochromatin protein Swi6/HP1 activates replication origins at the pericentromeric region and silent mating-type locus. Nat Cell Biol. 2009;11: 357–362. doi: 10.1038/ncb1845 19182789

18. De Piccoli G, Katou Y, Itoh T, Nakato R, Shirahige K, Labib K. Replisome stability at defective DNA replication forks is independent of S phase checkpoint kinases. Mol Cell. 2012;45: 696–704. doi: 10.1016/j.molcel.2012.01.007 22325992

19. Belsky JA, MacAlpine HK, Lubelsky Y, Hartemink AJ, MacAlpine DM. Genome-wide chromatin footprinting reveals changes in replication origin architecture induced by pre-RC assembly. Genes Dev. 2015;29: 212–224. doi: 10.1101/gad.247924.114 25593310

20. Das SP, Borrman T, Liu VW, Yang SC, Bechhoefer J, Rhind N. Replication timing is regulated by the number of MCMs loaded at origins. Genome Res. 2015;25: 1886–1892. doi: 10.1101/gr.195305.115 26359232

21. Foss EJ, Sripathy S, Gatbonton-Schwager T, Kwak H, Thiesen AH, Lao U et al. Chromosomal Mcm2-7 distribution is the primary driver of the genome replication program in species from yeast to humans. bioRxiv. 2020737742. doi: 10.1101/737742

22. Yang SC, Rhind N, Bechhoefer J. Modeling genome-wide replication kinetics reveals a mechanism for regulation of replication timing. Mol Syst Biol. 2010;6: 404. doi: 10.1038/msb.2010.61 20739926

23. Rhind N, Yang SC, Bechhoefer J. Reconciling stochastic origin firing with defined replication timing. Chromosome Res. 2010;18: 35–43. doi: 10.1007/s10577-009-9093-3 20205352

24. Stillman B. Origin recognition and the chromosome cycle. FEBS Letters. 2005;579: 877–884. doi: 10.1016/j.febslet.2004.12.011 15680967

25. McCune HJ, Danielson LS, Alvino GM, Collingwood D, Delrow JJ, Fangman WL et al. The temporal program of chromosome replication: genomewide replication in clb5{Delta} Saccharomyces cerevisiae. Genetics. 2008;180: 1833–1847. doi: 10.1534/genetics.108.094359 18832352

26. de Moura AP, Retkute R, Hawkins M, Nieduszynski CA. Mathematical modelling of whole chromosome replication. Nucleic Acids Res. 2010;38: 5623–5633. doi: 10.1093/nar/gkq343 20457753

27. Nishimura K, Fukagawa T, Takisawa H, Kakimoto T, Kanemaki M. An auxin-based degron system for the rapid depletion of proteins in nonplant cells. Nat Methods. 2009;6: 917–922. doi: 10.1038/nmeth.1401 19915560

28. Nishimura K, Kanemaki MT. Rapid Depletion of Budding Yeast Proteins via the Fusion of an Auxin-Inducible Degron (AID). Curr Protoc Cell Biol. 2014;64: 20.9.1–16. doi: 10.1002/0471143030.cb2009s64 25181302

29. Labib K, Kearsey SE, Diffley JF. MCM2-7 proteins are essential components of prereplicative complexes that accumulate cooperatively in the nucleus during G1-phase and are required to establish, but not maintain, the S-phase checkpoint. Mol Biol Cell. 2001;12: 3658–3667. doi: 10.1091/mbc.12.11.3658 11694596

30. Wal M, Pugh BF. Genome-wide mapping of nucleosome positions in yeast using high-resolution MNase ChIP-Seq. Methods Enzymol. 2012;513: 233–250. doi: 10.1016/B978-0-12-391938-0.00010-0 22929772

31. Hoggard T, Shor E, Müller CA, Nieduszynski CA, Fox CA. A Link between ORC-origin binding mechanisms and origin activation time revealed in budding yeast. PLoS Genet. 2013;9: e1003798. doi: 10.1371/journal.pgen.1003798 24068963

32. McGuffee SR, Smith DJ, Whitehouse I. Quantitative, genome-wide analysis of eukaryotic replication initiation and termination. Mol Cell. 2013;50: 123–135. doi: 10.1016/j.molcel.2013.03.004 23562327

33. Miller TCR, Locke J, Greiwe JF, Diffley JFX, Costa A. Mechanism of head-to-head MCM double-hexamer formation revealed by cryo-EM. Nature. 2019;575: 704–710. doi: 10.1038/s41586-019-1768-0 31748745

34. Henikoff JG, Belsky JA, Krassovsky K, MacAlpine DM, Henikoff S. Epigenome characterization at single base-pair resolution. Proc Natl Acad Sci U S A. 2011;108: 18318–18323. doi: 10.1073/pnas.1110731108 22025700

35. Coster G, Diffley JFX. Bidirectional eukaryotic DNA replication is established by quasi-symmetrical helicase loading. Science. 2017;357: 314–318. doi: 10.1126/science.aan0063 28729513

36. Remus D, Beuron F, Tolun G, Griffith JD, Morris EP, Diffley JF. Concerted loading of Mcm2-7 double hexamers around DNA during DNA replication origin licensing. Cell. 2009;139: 719–730. doi: 10.1016/j.cell.2009.10.015 19896182

37. Müller CA, Hawkins M, Retkute R, Malla S, Wilson R, Blythe MJ et al. The dynamics of genome replication using deep sequencing. Nucleic Acids Res. 2014;42: e3. doi: 10.1093/nar/gkt878 24089142

38. Knott SR, Viggiani CJ, Tavaré S, Aparicio OM. Genome-wide replication profiles indicate an expansive role for Rpd3L in regulating replication initiation timing or efficiency, and reveal genomic loci of Rpd3 function in Saccharomyces cerevisiae. Genes Dev. 2009;23: 1077–1090. doi: 10.1101/gad.1784309 19417103

39. Lian HY, Robertson ED, Hiraga S, Alvino GM, Collingwood D, McCune HJ et al. The effect of Ku on telomere replication time is mediated by telomere length but is independent of histone tail acetylation. Mol Biol Cell. 2011;22: 1753–1765. doi: 10.1091/mbc.E10-06-0549 21441303

40. Knott SR, Peace JM, Ostrow AZ, Gan Y, Rex AE, Viggiani CJ et al. Forkhead transcription factors establish origin timing and long-range clustering in S. cerevisiae. Cell. 2012;148: 99–111. doi: 10.1016/j.cell.2011.12.012 22265405

41. Natsume T, Müller CA, Katou Y, Retkute R, Gierliński M, Araki H et al. Kinetochores coordinate pericentromeric cohesion and early DNA replication by Cdc7-Dbf4 kinase recruitment. Mol Cell. 2013;50: 661–674. doi: 10.1016/j.molcel.2013.05.011 23746350

42. Das SP, Rhind N. How and why multiple MCMs are loaded at origins of DNA replication. Bioessays. 2016;38: 613–617. doi: 10.1002/bies.201600012 27174869

43. Ghaemmaghami S, Huh WK, Bower K, Howson RW, Belle A, Dephoure N et al. Global analysis of protein expression in yeast. Nature. 2003;425: 737–741. doi: 10.1038/nature02046 14562106

44. Donovan S, Harwood J, Drury LS, Diffley JF. Cdc6p-dependent loading of Mcm proteins onto pre-replicative chromatin in budding yeast. Proc Natl Acad Sci U S A. 1997;94: 5611–5616. doi: 10.1073/pnas.94.11.5611 9159120

45. Lei M, Kawasaki Y, Tye BK. Physical interactions among Mcm proteins and effects of Mcm dosage on DNA replication in Saccharomyces cerevisiae. Mol Cell Biol. 1996;16: 5081–5090. doi: 10.1128/mcb.16.9.5081 8756666

46. Ho B, Baryshnikova A, Brown GW. Unification of Protein Abundance Datasets Yields a Quantitative Saccharomyces cerevisiae Proteome. Cell Syst. 2018;6: 192–205.e3. doi: 10.1016/j.cels.2017.12.004 29361465

47. Bowers JL, Randell JC, Chen S, Bell SP. ATP hydrolysis by ORC catalyzes reiterative Mcm2-7 assembly at a defined origin of replication. Mol Cell. 2004;16: 967–978. doi: 10.1016/j.molcel.2004.11.038 15610739

48. Edwards MC, Tutter AV, Cvetic C, Gilbert CH, Prokhorova TA, Walter JC. MCM2-7 complexes bind chromatin in a distributed pattern surrounding the origin recognition complex in Xenopus egg extracts. J Biol Chem. 2002;277: 33049–33057. doi: 10.1074/jbc.M204438200 12087101

49. Woodward AM, Göhler T, Luciani MG, Oehlmann M, Ge X, Gartner A et al. Excess Mcm2-7 license dormant origins of replication that can be used under conditions of replicative stress. J Cell Biol. 2006;173: 673–683. doi: 10.1083/jcb.200602108 16754955

50. Ge XQ, Jackson DA, Blow JJ. Dormant origins licensed by excess Mcm2-7 are required for human cells to survive replicative stress. Genes Dev. 2007;21: 3331–3341. doi: 10.1101/gad.457807 18079179

51. Ibarra A, Schwob E, Méndez J. Excess MCM proteins protect human cells from replicative stress by licensing backup origins of replication. Proc Natl Acad Sci U S A. 2008;105: 8956–8961. doi: 10.1073/pnas.0803978105 18579778

52. Li N, Lam WH, Zhai Y, Cheng J, Cheng E, Zhao Y et al. Structure of the origin recognition complex bound to DNA replication origin. Nature. 2018;559: 217–222. doi: 10.1038/s41586-018-0293-x 29973722

53. Maine GT, Sinha P, Tye BK. Mutants of S. cerevisiae defective in the maintenance of minichromosomes. Genetics. 1984;106: 365–385. 6323245

54. Zou L, Stillman B. Assembly of a complex containing Cdc45p, replication protein A, and Mcm2p at replication origins controlled by S-phase cyclin-dependent kinases and Cdc7p-Dbf4p kinase. Mol Cell Biol. 2000;20: 3086–3096. doi: 10.1128/mcb.20.9.3086-3096.2000 10757793

55. Lynch KL, Alvino GM, Kwan EX, Brewer BJ, Raghuraman MK. The effects of manipulating levels of replication initiation factors on origin firing efficiency in yeast. PLoS Genet. 2019;15: e1008430. doi: 10.1371/journal.pgen.1008430 31584938

56. Dion MF, Kaplan T, Kim M, Buratowski S, Friedman N, Rando OJ. Dynamics of replication-independent histone turnover in budding yeast. Science. 2007;315: 1405–1408. doi: 10.1126/science.1134053 17347438

57. Sonneville R, Querenet M, Craig A, Gartner A, Blow JJ. The dynamics of replication licensing in live Caenorhabditis elegans embryos. J Cell Biol. 2012;196: 233–246. doi: 10.1083/jcb.201110080 22249291

58. Tanaka S, Nakato R, Katou Y, Shirahige K, Araki H. Origin association of Sld3, Sld7, and Cdc45 proteins is a key step for determination of origin-firing timing. Curr Biol. 2011;21: 2055–2063. doi: 10.1016/j.cub.2011.11.038 22169533

59. Müller CA, Nieduszynski CA. Conservation of replication timing reveals global and local regulation of replication origin activity. Genome Res. 2012;22: 1953–1962. doi: 10.1101/gr.139477.112 22767388

60. Nieduszynski CA, Knox Y, Donaldson AD. Genome-wide identification of replication origins in yeast by comparative genomics. Genes Dev. 2006;20: 1874–1879. doi: 10.1101/gad.385306 16847347

61. Green MR (2012) Molecular cloning: a laboratory manual. Fourth edition. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press.

62. Chen S, Bell SP. CDK prevents Mcm2-7 helicase loading by inhibiting Cdt1 interaction with Orc6. Genes Dev. 2011;25: 363–372. doi: 10.1101/gad.2011511 21289063

63. Siow CC, Nieduszynska SR, Müller CA, Nieduszynski CA. OriDB, the DNA replication origin database updated and extended. Nucleic Acids Res. 2012;40: D682–6. doi: 10.1093/nar/gkr1091 22121216

64. Batrakou DG, Müller CA, Wilson RHC, Nieduszynski CA. DNA copy-number measurement of genome replication dynamics by high-throughput sequencing: the sort-seq, sync-seq and MFA-seq family. Nat Protoc. 2020;15: 1255–1284. doi: 10.1038/s41596-019-0287-7 32051615

65. Huh WK, Falvo JV, Gerke LC, Carroll AS, Howson RW, Weissman JS et al. Global analysis of protein localization in budding yeast. Nature. 2003;425: 686–691. doi: 10.1038/nature02026 14562095

Článek vyšel v časopise

PLOS Genetics

2021 Číslo 3
Nejčtenější tento týden
Nejčtenější v tomto čísle

Zvyšte si kvalifikaci online z pohodlí domova

Důležitost adherence při depresivním onemocnění
nový kurz
Autoři: MUDr. Eliška Bartečková, Ph.D.

Koncepce osteologické péče pro gynekology a praktické lékaře
Autoři: MUDr. František Šenk

Sekvenční léčba schizofrenie
Autoři: MUDr. Jana Hořínková, Ph.D.

Hypertenze a hypercholesterolémie – synergický efekt léčby
Autoři: prof. MUDr. Hana Rosolová, DrSc.

Multidisciplinární zkušenosti u pacientů s diabetem
Autoři: Prof. MUDr. Martin Haluzík, DrSc., prof. MUDr. Vojtěch Melenovský, CSc., prof. MUDr. Vladimír Tesař, DrSc.

Všechny kurzy
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