Best Practices for Mapping Replication Origins in Eukaryotic Chromosomes

Emilie Besnard1, Romain Desprat2, Michael Ryan3, Malik Kahli1, Mirit I. Aladjem4, Jean‐Marc Lemaitre4

1 Laboratory of Genome Plasticity and Aging, Institute of Functional Genomics, CNRS UMR5203, INSERM U661, UMI, Montpellier, 2 Stem Cell core Facility, Institute of Regenerative Medicine and Biotherapies, Saint‐Eloi Hospital, Montpellier, 3 InSilico Inc, Falls Church, Virginia, 4 Corresponding authors: Mirit I. Aladjem ( and Jean‐Marc Lemaitre (
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 22.18
DOI:  10.1002/0471143030.cb2218s64
Online Posting Date:  September, 2014
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Understanding the regulatory principles ensuring complete DNA replication in each cell division is critical for deciphering the mechanisms that maintain genomic stability. Recent advances in genome sequencing technology facilitated complete mapping of DNA replication sites and helped move the field from observing replication patterns at a handful of single loci to analyzing replication patterns genome‐wide. These advances address issues, such as the relationship between replication initiation events, transcription, and chromatin modifications, and identify potential replication origin consensus sequences. This unit summarizes the technological and fundamental aspects of replication profiling and briefly discusses novel insights emerging from mining large datasets, published in the last 3 years, and also describes DNA replication dynamics on a whole‐genome scale. Curr. Protoc. Cell Biol. 64:22.18.1‐22.18.13. © 2014 by John Wiley & Sons, Inc.

Keywords: DNA replication; origins; replication timing; next‐generation sequencing (NGS); bioinformatics

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Table of Contents

  • Introduction
  • Questions and Unresolved Paradoxes in Understanding Eukaryotic DNA Replication
  • Techniques Used in Replication Profiling
  • Quality Control and Standardization Issues
  • Integrating Different Sources of Genomic Data
  • Insights from Recent Whole‐Genome Replication Profiling Studies
  • Remaining Issues and Future Directions
  • Literature Cited
  • Figures
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Literature Cited

Literature Cited
  Aladjem, M.I. 2007. Replication in context: Dynamic regulation of DNA replication patterns in metazoans. Nat. Rev. Genet. 8:588‐600.
  Aladjem, M.I., Groudine, M., Brody, L.L., Dieken, E.S., Fournier, R.E., Wahl, G.M., and Epner, E.M. 1995. Participation of the human beta‐globin locus control region in initiation of DNA replication. Science 270:815‐819.
  Aladjem, M.I., Rodewald, L.W., Kolman, J.L., and Wahl, G.M. 1998. Genetic dissection of a mammalian replicator in the human beta‐globin locus. Science 281:1005‐1009.
  Audit, B., Zaghloul, L., Baker, A., Arneodo, A., Chen, C.L., d'Aubenton‐Carafa, Y., and Thermes, C. 2012. Megabase replication domains along the human genome: Relation to chromatin structure and genome organisation. Subcell. Biochem. 61:57‐80.
  Baker, A., Audit, B., Chen, C.L., Moindrot, B., Leleu, A., Guilbaud, G., Rappailles, A., Vaillant, C., Goldar, A., Mongelard, F., d'Aubenton‐Carafa, Y., Hyrien, O., Thermes, C., and Arneodo, A. 2012. Replication fork polarity gradients revealed by megabase‐sized U‐shaped replication timing domains in human cell lines. PLoS Comput. Biol. 8:e1002443.
  Besnard, E., Babled, A., Lapasset, L., Milhavet, O., Parrinello, H., Dantec, C., Marin, J.M., and Lemaitre, J.M. 2012. Unraveling cell type‐specific and reprogrammable human replication origin signatures associated with G‐quadruplex consensus motifs. Nat. Struct. Mol. Biol. 19:837‐844.
  Bielinsky, A.K. and Gerbi, S.A. 1998. Discrete start sites for DNA synthesis in the yeast ARS1 origin. Science 279:95‐98.
  Blahnik, K.R., Dou, L., O'Geen, H., McPhillips, T., Xu, X., Cao, A.R., Iyengar, S., Nicolet, C.M., Ludascher, B., Korf, I., and Farnham, P.J. 2010. Sole‐Search: An integrated analysis program for peak detection and functional annotation using ChIP‐seq data. Nucleic Acids Res. 38:e13.
  Brewer, B.J. and Fangman, W.L. 1987. The localization of replication origins on ARS plasmids in S. cerevisiae. Cell 51:463‐471.
  Burhans, W.C., Selegue, J.E., and Heintz, N.H. 1986. Isolation of the origin of replication associated with the amplified Chinese hamster dihydrofolate reductase domain. Proc. Natl. Acad. Sci. U.S.A. 83:7790‐7794.
  Burhans, W.C., Vassilev, L.T., Caddle, M.S., Heintz, N.H., and DePamphilis, M.L. 1990. Identification of an origin of bidirectional DNA replication in mammalian chromosomes. Cell 62:955‐965.
  Cadoret, J.C., Meisch, F., Hassan‐Zadeh, V., Luyten, I., Guillet, C., Duret, L., Quesneville, H., and Prioleau, M.N. 2008. Genome‐wide studies highlight indirect links between human replication origins and gene regulation. Proc. Natl. Acad. Sci. U.S.A. 105:15837‐15842.
  Cayrou, C., Coulombe, P., Vigneron, A., Stanojcic, S., Ganier, O., Peiffer, I., Rivals, E., Puy, A., Laurent‐Chabalier, S., Desprat, R., and Mechali, M. 2011. Genome‐scale analysis of metazoan replication origins reveals their organization in specific but flexible sites defined by conserved features. Genome Res. 21:1438‐1449.
  Chang, F., May, C.D., Hoggard, T., Miller, J., Fox, C.A., and Weinreich, M. 2011. High‐resolution analysis of four efficient yeast replication origins reveals new insights into the ORC and putative MCM binding elements. Nucleic Acids Res. 39:6523‐6535.
  Chen, C.L., Duquenne, L., Audit, B., Guilbaud, G., Rappailles, A., Baker, A., Huvet, M., d'Aubenton‐Carafa, Y., Hyrien, O., Arneodo, A., and Thermes, C. 2011. Replication‐associated mutational asymmetry in the human genome. Mol. Biol. Evol. 28:2327‐2337.
  Dellino, G.I., Cittaro, D., Piccioni, R., Luzi, L., Banfi, S., Segalla, S., Cesaroni, M., Mendoza‐Maldonado, R., Giacca, M., and Pelicci, P.G. 2013. Genome‐wide mapping of human DNA‐replication origins: Levels of transcription at ORC1 sites regulate origin selection and replication timing. Genome Res. 23:1‐11.
  Demczuk, A., Gauthier, M.G., Veras, I., Kosiyatrakul, S., Schildkraut, C.L., Busslinger, M., Bechhoefer, J., and Norio, P. 2012. Regulation of DNA replication within the immunoglobulin heavy‐chain locus during B cell commitment. PLoS Biol. 10:e1001360.
  DePamphilis, M.L. 1993. Origins of DNA replication in metazoan chromosomes. J. Biol. Chem. 268:1‐4.
  Desprat, R., Thierry‐Mieg, D., Lailler, N., Lajugie, J., Schildkraut, C., Thierry‐Mieg, J., and Bouhassira, E.E. 2009. Predictable dynamic program of timing of DNA replication in human cells. Genome Res. 19:2288‐2299.
  Dorn, E.S. and Cook, J.G. 2011. Nucleosomes in the neighborhood: New roles for chromatin modifications in replication origin control. Epigenetics 6:552‐559.
  Eaton, M.L., Galani, K., Kang, S., Bell, S.P., and MacAlpine, D.M. 2010. Conserved nucleosome positioning defines replication origins. Genes Dev. 24:748‐753.
  Edwards, M.C., Tutter, A.V., Cvetic, C., Gilbert, C.H., Prokhorova, T.A., and Walter, J.C. 2002. MCM2‐7 complexes bind chromatin in a distributed pattern surrounding the origin recognition complex in Xenopus egg extracts. J. Biol. Chem. 277:33049‐33057.
  Farkash‐Amar, S., Lipson, D., Polten, A., Goren, A., Helmstetter, C., Yakhini, Z., and Simon, I. 2008. Global organization of replication time zones of the mouse genome. Genome Res. 18:1562‐1570.
  Fu, H., Maunakea, A.K., Martin, M.M., Huang, L., Zhang, Y., Ryan, M., Kim, R., Lin, C.M., Zhao, K., and Aladjem, M.I. 2013. Methylation of histone H3 on lysine 79 associates with a group of replication origins and helps limit DNA replication once per cell cycle. PLoS Genetics 9:e1003542.
  Ge, X.Q., Jackson, D.A., and Blow, J.J. 2007. Dormant origins licensed by excess Mcm2‐7 are required for human cells to survive replicative stress. Genes Dev. 21:3331‐3341.
  Ghosh, M., Liu, G., Randall, G., Bevington, J., and Leffak, M. 2004. Transcription factor binding and induced transcription alter chromosomal c‐myc replicator activity. Mol. Cell Biol. 24:10193‐10207.
  Gilbert, D.M. 2007. Replication origin plasticity, Taylor‐made: Inhibition vs. recruitment of origins under conditions of replication stress. Chromosoma 116:341‐347.
  Gilbert, D.M. 2012. Replication origins run (ultra) deep. Nat. Struct. Mol. Biol. 19:740‐742.
  Guan, Z., Hughes, C.M., Kosiyatrakul, S., Norio, P., Sen, R., Fiering, S., Allis, C.D., Bouhassira, E.E., and Schildkraut, C.L. 2009. Decreased replication origin activity in temporal transition regions. J. Cell Biol. 187:623‐635.
  Guilbaud, G., Rappailles, A., Baker, A., Chen, C.L., Arneodo, A., Goldar, A., d'Aubenton‐Carafa, Y., Thermes, C., Audit, B., and Hyrien, O. 2011. Evidence for sequential and increasing activation of replication origins along replication timing gradients in the human genome. PLoS Comput. Biol. 7:e1002322.
  Hamlin, J.L., Mesner, L.D., Lar, O., Torres, R., Chodaparambil, S.V., and Wang, L. 2008. A revisionist replicon model for higher eukaryotic genomes. J. Cell Biochem. 105:321‐329.
  Handeli, S., Klar, A., Meuth, M., and Cedar, H. 1989. Mapping replication units in animal cells. Cell 57:909‐920.
  Harismendy, O., Ng, P.C., Strausberg, R.L., Wang, X., Stockwell, T.B., Beeson, K.Y., Schork, N.J., Murray, S.S., Topol, E.J., Levy, S., and Frazer, K.A. 2009. Evaluation of next generation sequencing platforms for population targeted sequencing studies. Genome Biol. 10:R32.
  Herrick, J. and Bensimon, A. 2009. Introduction to molecular combing: Genomics, DNA replication, and cancer. Methods Mol. Biol. 521:71‐101.
  Hiratani, I., Takebayashi, S., Lu, J., and Gilbert, D.M. 2009. Replication timing and transcriptional control: Beyond cause and effect—part II. Curr. Opin. Genet. Dev. 19:142‐149.
  Huvet, M., Nicolay, S., Touchon, M., Audit, B., d'Aubenton‐Carafa, Y., Arneodo, A., and Thermes, C. 2007. Human gene organization driven by the coordination of replication and transcription. Genome Res. 17:1278‐1285.
  Hyrien, O., Maric, C., and Mechali, M. 1995. Transition in specification of embryonic metazoan DNA replication origins. Science 270:994‐997.
  Hyrien, O., Marheineke, K., and Goldar, A. 2003. Paradoxes of eukaryotic DNA replication: MCM proteins and the random completion problem. BioEssays 25:116‐125.
  Ibarra, A., Schwob, E., and Mendez, J. 2008. Excess MCM proteins protect human cells from replicative stress by licensing backup origins of replication. Proc. Natl. Acad. Sci. U.S.A. 105:8956‐8961.
  Karnani, N., Taylor, C.M., Malhotra, A., and Dutta, A. 2010. Genomic study of replication initiation in human chromosomes reveals the influence of transcription regulation and chromatin structure on origin selection. Mol. Biol. Cell 21:393‐404.
  Kuo, A.J., Song, J., Cheung, P., Ishibe‐Murakami, S., Yamazoe, S., Chen, J.K., Patel, D.J., and Gozani, O. 2012. The BAH domain of ORC1 links H4K20me2 to DNA replication licensing and Meier‐Gorlin syndrome. Nature 484:115‐119.
  Lebofsky, R. and Bensimon, A. 2005. DNA replication origin plasticity and perturbed fork progression in human inverted repeats. Mol. Cell Biol. 25:6789‐6797.
  Letessier, A., Millot, G.A., Koundrioukoff, S., Lachages, A.M., Vogt, N., Hansen, R.S., Malfoy, B., Brison, O., and Debatisse, M. 2011. Cell‐type‐specific replication initiation programs set fragility of the FRA3B fragile site. Nature 470:120‐123.
  Lipford, J.R. and Bell, S.P. 2001. Nucleosomes positioned by ORC facilitate the initiation of DNA replication. Mol. Cell 7:21‐30.
  Little, R.D., Platt, T.H., and Schildkraut, C.L. 1993. Initiation and termination of DNA replication in human rRNA genes. Mol. Cell Biol. 13:6600‐6613.
  Lubelsky, Y., Sasaki, T., Kuipers, M.A., Lucas, I., Le Beau, M.M., Carignon, S., Debatisse, M., Prinz, J.A., Dennis, J.H., and Gilbert, D.M. 2011. Pre‐replication complex proteins assemble at regions of low nucleosome occupancy within the Chinese hamster dihydrofolate reductase initiation zone. Nucleic Acids Res. 39:3141‐3155.
  Martin, M.M., Ryan, M., Kim, R., Zakas, A.L., Fu, H., Lin, C.M., Reinhold, W.C., Davis, S.R., Bilke, S., Liu, H., Doroshow, J.H., Reimers, M.A., Valenzuela, M.S., Pommier, Y., Meltzer, P.S., and Aladjem, M.I. 2011. Genome‐wide depletion of replication initiation events in highly transcribed regions. Genome Res. 21:1822‐1832.
  McGuffee, S.R., Smith, D.J., and Whitehouse, I. 2013. Quantitative, genome‐wide analysis of eukaryotic replication initiation and termination. Mol. Cell 50:123‐135.
  Mechali, M. 2010. Eukaryotic DNA replication origins: Many choices for appropriate answers. Nat. Rev. Mol. Cell Biol. 11:728‐738.
  Mesner, L.D., Valsakumar, V., Karnani, N., Dutta, A., Hamlin, J.L., and Bekiranov, S. 2011. Bubble‐chip analysis of human origin distributions demonstrates on a genomic scale significant clustering into zones and significant association with transcription. Genome Res. 21:377‐389.
  Mesner, L.D., Valsakumar, V., Cieslik, M., Pickin, R., Hamlin, J.L., and Bekiranov, S. 2013. Bubble‐seq analysis of the human genome reveals distinct chromatin‐mediated mechanisms for regulating early‐ and late‐firing origins. Genome Res. 23:1774‐1788.
  Nordman, J., Li, S., Eng, T., Macalpine, D., and Orr‐Weaver, T.L. 2011. Developmental control of the DNA replication and transcription programs. Genome Res. 21:175‐181.
  Norio, P., Kosiyatrakul, S., Yang, Q., Guan, Z., Brown, N.M., Thomas, S., Riblet, R., and Schildkraut, C.L. 2005. Progressive activation of DNA replication initiation in large domains of the immunoglobulin heavy chain locus during B cell development. Mol. Cell 20:575‐587.
  Paces, J., Zíka, R., Paces, V., Pavlícek, A., Clay, O., and Bernardi, G. 2004. Representing GC variation along eukaryotic chromosomes. Gene 333:135‐141.
  Petermann, E., Woodcock, M., and Helleday, T. 2010. Chk1 promotes replication fork progression by controlling replication initiation. Proc. Natl. Acad. Sci. U.S.A. 107:16090‐16095.
  Raghuraman, M.K., Winzeler, E.A., Collingwood, D., Hunt, S., Wodicka, L., Conway, A., Lockhart, D.J., Davis, R.W., Brewer, B.J., and Fangman, W.L. 2001. Replication dynamics of the yeast genome. Science 294:115‐121.
  Ryba, T., Hiratani, I., Lu, J., Itoh, M., Kulik, M., Zhang, J., Schulz, T.C., Robins, A.J., Dalton, S., and Gilbert, D.M. 2010. Evolutionarily conserved replication timing profiles predict long‐range chromatin interactions and distinguish closely related cell types. Genome Res. 20:761‐770.
  Schepers, A. and Papior, P. 2010. Why are we where we are? Understanding replication origins and initiation sites in eukaryotes using ChIP‐approaches. Chromosome Res. 18:63‐77.
  Sequeira‐Mendes, J., Diaz‐Uriarte, R., Apedaile, A., Huntley, D., Brockdorff, N., and Gomez, M. 2009. Transcription initiation activity sets replication origin efficiency in mammalian cells. PLoS Genetics 5:e1000446.
  Smith, D.J. and Whitehouse, I. 2012. Intrinsic coupling of lagging‐strand synthesis to chromatin assembly. Nature 483:434‐438.
  Tardat, M., Brustel, J., Kirsh, O., Lefevbre, C., Callanan, M., Sardet, C., and Julien, E. 2010. The histone H4 Lys 20 methyltransferase PR‐Set7 regulates replication origins in mammalian cells. Nat. Cell Biol. 12:1086‐1093.
  Touchon, M., Nicolay, S., Audit, B., Brodie of Brodie, E.B., d'Aubenton‐Carafa, Y., Arneodo, A., and Thermes, C. 2005. Replication‐associated strand asymmetries in mammalian genomes: Toward detection of replication origins. Proc. Natl. Acad. Sci. U.S.A. 102:9836‐9841.
  Valenzuela, M.S., Chen, Y., Davis, S., Yang, F., Walker, R.L., Bilke, S., Lueders, J., Martin, M.M., Aladjem, M.I., Massion, P.P., and Meltzer, P.S. 2011. Preferential localization of human origins of DNA replication at the 5'‐ends of expressed genes and at evolutionarily conserved DNA sequences. PloS One 6:e17308.
  Valton, A.L., Hassan‐Zadeh, V., Lema, I., Boggetto, N., Alberti, P., Saintomé, C., Riou, J.F., and Prioleau, M.N. 2014. G4 motifs affect origin positioning and efficiency in two vertebrate replicators. EMBO J. 33:732‐746.
  Vashee, S., Cvetic, C., Lu, W., Simancek, P., Kelly, T.J., and Walter, J.C. 2003. Sequence‐independent DNA binding and replication initiation by the human origin recognition complex. Genes Dev. 17:1894‐1908.
  Vaughn, J.P., Dijkwel, P.A., and Hamlin, J.L. 1990. Replication initiates in a broad zone in the amplified CHO dihydrofolate reductase domain. Cell 61:1075‐1087.
  Yin, S., Deng, W., Hu, L., and Kong, X. 2009. The impact of nucleosome positioning on the organization of replication origins in eukaryotes. Biochem. Biophys. Res. Comm. 385:363‐368.
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