Mechanisms regulating where and when eukaryotic DNA replication initiates remain a fundamental mystery in molecular biology.
Genome-scale approaches are now being used to identify the location of replication origins and to evaluate replication timing.
Mapping replication origins in yeasts has been successful, but the current data sets from multicellular organisms are scarce and inconsistent.
Methods for mapping origins include trapping the earliest replicated DNA by replication fork arrest, mapping small nascent leading strands and trapping replication bubbles.
Studies of replication timing can involve prospective or retroactive synchronization of cells, followed by comparison of early- and late-S-phase DNA by microarray or sequencing.
Genome-wide studies of replication timing have shown that timing is regulated at the level of replication domains and that there are links between replication timing and chromatin structure.
Mechanisms regulating where and when eukaryotic DNA replication initiates remain a mystery. Recently, genome-scale methods have been brought to bear on this problem. The identification of replication origins and their associated proteins in yeasts is a well-integrated investigative tool, but corresponding data sets from multicellular organisms are scarce. By contrast, standardized protocols for evaluating replication timing have generated informative data sets for most eukaryotic systems. Here, I summarize the genome-scale methods that are most frequently used to analyse replication in eukaryotes, the kinds of questions each method can address and the technical hurdles that must be overcome to gain a complete understanding of the nature of eukaryotic replication origins.
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Masai, H., Matsumoto, S., You, Z., Yoshizawa-Sugata, N. & Oda, M. Eukaryotic chromosome DNA replication: where, when, and how? Annu. Rev. Biochem. 79, 89–130 (2010).
Woodward, A. M. et al. Excess Mcm2–7 license dormant origins of replication that can be used under conditions of replicative stress. J. Cell Biol. 173, 673–683 (2006).
Ibarra, A., Schwob, E. & Mendez, J. Excess MCM proteins protect human cells from replicative stress by licensing backup origins of replication. Proc. Natl Acad. Sci. USA 105, 8956–8961 (2008).
Doksani, Y., Bermejo, R., Fiorani, S., Haber, J. E. & Foiani, M. Replicon dynamics, dormant origin firing, and terminal fork integrity after double-strand break formation. Cell 137, 247–258 (2009).
Koren, A., Soifer, I. & Barkai, N. MRC1-dependent scaling of the budding yeast DNA replication timing program. Genome Res. 20, 781–790 (2010).
Raghuraman, M. K. & Brewer, B. J. Molecular analysis of the replication program in unicellular model organisms. Chromosome Res. 18, 19–34 (2010).
Krysan, P. J., Smith, J. G. & Calos, M. P. Autonomous replication in human cells of multimers of specific human and bacterial DNA sequences. Mol. Cell. Biol. 13, 2688–2696 (1993).
Aladjem, M. I. Replication in context: dynamic regulation of DNA replication patterns in metazoans. Nature Rev. Genet. 8, 588–600 (2007).
Hamlin, J. L. et al. A revisionist replicon model for higher eukaryotic genomes. J. Cell. Biochem. 105, 321–329 (2008).
Eaton, M. L., Galani, K., Kang, S., Bell, S. P. & MacAlpine, D. M. Conserved nucleosome positioning defines replication origins. Genes Dev. 24, 748–753 (2010). This paper combined genome-wide ChIP–seq analysis of ORC and nucleosome positions with in vitro nucleosome reconstitution to demonstrate that ORC binds to sequence-defined NFRs and directs local nucleosome positioning at budding yeast origins.
MacAlpine, H. K., Gordan, R., Powell, S. K., Hartemink, A. J. & MacAlpine, D. M. Drosophila ORC localizes to open chromatin and marks sites of cohesin complex loading. Genome Res. 20, 201–211 (2010). This paper used ChIP–chip to show that ORC localizes to NFRs in D. melanogaster cells. In combination with reference 10, it suggests that nucleosome organization may be a defining feature of all eukaryotic origins.
Deal, R. B., Henikoff, J. G. & Henikoff, S. Genome-wide kinetics of nucleosome turnover determined by metabolic labeling of histones. Science 328, 1161–1164 (2010).
Breier, A. M., Chatterji, S. & Cozzarelli, N. R. Prediction of Saccharomyces cerevisiae replication origins. Genome Biol. 5, R22 (2004).
Wyrick, J. J. et al. Genome-wide distribution of ORC and MCM proteins in S. cerevisiae: high-resolution mapping of replication origins. Science 294, 2357–2360 (2001).
Xu, W., Aparicio, J. G., Aparicio, O. M. & Tavare, 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 7, 276 (2006).
MacAlpine, D. M. & Bell, S. P. A genomic view of eukaryotic DNA replication. Chromosome Res. 13, 309–326 (2005).
Hayashi, M. et al. Genome-wide localization of pre-RC sites and identification of replication origins in fission yeast. EMBO J. 26, 1327–1339 (2007).
Nieduszynski, C. A., Knox, Y. & Donaldson, A. D. Genome-wide identification of replication origins in yeast by comparative genomics. Genes Dev. 20, 1874–1879 (2006).
Dai, J., Chuang, R. Y. & Kelly, T. J. DNA replication origins in the Schizosaccharomyces pombe genome. Proc. Natl Acad. Sci. USA 102, 337–342 (2005).
Cotobal, C., Segurado, M. & Antequera, F. Structural diversity and dynamics of genomic replication origins in Schizosaccharomyces pombe. EMBO J. 29, 934–942 (2010).
Chuang, R. Y. & Kelly, T. J. The fission yeast homologue of Orc4p binds to replication origin DNA via multiple AT-hooks. Proc. Natl Acad. Sci. USA 96, 2656–2661 (1999).
Lantermann, A. B. et al. Schizosaccharomyces pombe genome-wide nucleosome mapping reveals positioning mechanisms distinct from those of Saccharomyces cerevisiae. Nature Struct. Mol. Biol. 17, 251–257 (2010).
Gilbert, D. M. In search of the holy replicator. Nature Rev. Mol. Cell Biol. 5, 848–855 (2004).
Paixao, S. et al. Modular structure of the human lamin B2 replicator. Mol. Cell. Biol. 24, 2958–2967 (2004).
Altman, A. L. & Fanning, E. Defined sequence modules and an architectural element cooperate to promote initiation at an ectopic mammalian chromosomal replication origin. Mol. Cell. Biol. 24, 4138–4150 (2004).
Liu, G., Malott, M. & Leffak, M. Multiple functional elements comprise a mammalian chromosomal replicator. Mol. Cell. Biol. 23, 1832–1842 (2003).
Guan, Z. et al. Decreased replication origin activity in temporal transition regions. J. Cell Biol. 187, 623–635 (2009).
Lin, H. B., Dijkwel, P. A. & Hamlin, J. L. Promiscuous initiation on mammalian chromosomal DNA templates and its possible suppression by transcription. Exp. Cell Res. 308, 53–64 (2005).
Vashee, S. et al. Sequence-independent DNA binding and replication initiation by the human origin recognition complex. Genes Dev. 17, 1894–1908 (2003).
Remus, D., Beall, E. L. & Botchan, M. R. DNA topology, not DNA sequence, is a critical determinant for Drosophila ORC-DNA binding. EMBO J. 23, 897–907 (2004).
Gupta, S. et al. Predicting human nucleosome occupancy from primary sequence. PLoS Comput. Biol. 4, e1000134 (2008).
Schepers, A. & Papior, P. Why are we where we are? Understanding replication origins and initiation sites in eukaryotes using ChIP-approaches. Chromosome Res. 18, 63–77 (2010).
Friedman, K. L., Brewer, B. J. & Fangman, W. L. Replication profile of Saccharomyces cerevisiae chromosome VI. Genes Cells 2, 667–678 (1997).
Poloumienko, A., Dershowitz, A., De, J. & Newlon, C. S. Completion of replication map of Saccharomyces cerevisiae chromosome III. Mol. Biol. Cell 12, 3317–3327 (2001).
Heichinger, C., Penkett, C. J., Bahler, J. & Nurse, P. Genome-wide characterization of fission yeast DNA replication origins. EMBO J. 25, 5171–5179 (2006).
Wu, P. Y. & Nurse, P. Establishing the program of origin firing during S phase in fission yeast. Cell 136, 852–864 (2009). This report used genome-wide replication profiling of the earliest BrdU-labelled DNA synthesized in the presence of hydroxyurea to show that holding cells in mitosis increases the binding of ORC to certain origins in fission yeast and causes these origins to become earlier replicating.
Patel, P. K., Arcangioli, B., Baker, S. P., Bensimon, A. & Rhind, N. DNA replication origins fire stochastically in fission yeast. Mol. Biol. Cell 17, 308–316 (2006).
Norio, P. et al. Progressive activation of DNA replication initiation in large domains of the immunoglobulin heavy chain locus during B cell development. Mol. Cell 20, 575–587 (2005).
Norio, P. & Schildkraut, C. L. Plasticity of DNA replication initiation in epstein-barr virus episomes. PLoS Biol. 2, e152 (2004).
Lebofsky, R., Heilig, R., Sonnleitner, M., Weissenbach, J. & Bensimon, A. DNA replication origin interference increases the spacing between initiation events in human cells. Mol. Biol. Cell 17, 5337–5345 (2006).
Mesner, L. D., Crawford, E. L. & Hamlin, J. L. Isolating apparently pure libraries of replication origins from complex genomes. Mol. Cell 21, 719–726 (2006).
Anglana, M., Apiou, F., Bensimon, A. & Debatisse, M. Dynamics of DNA replication in mammalian somatic cells: nucleotide pool modulates origin choice and interorigin spacing. Cell 114, 385–394 (2003).
Yabuki, N., Terashima, H. & Kitada, K. Mapping of early firing origins on a replication profile of budding yeast. Genes Cells 7, 781–789 (2002).
Katou, Y. et al. S-phase checkpoint proteins Tof1 and Mrc1 form a stable replication-pausing complex. Nature 424, 1078–1083 (2003).
Viggiani, C. J., Knott, S. R. & Aparicio, O. M. Genome-wide analysis of DNA synthesis by BrdU immunoprecipitation on tiling microarrays (BrdU-IP-chip) in Saccharomyces cerevisiae. Cold Spring Harb. Protoc. 2010, pdb.prot5385 (2010).
Feng, W. et al. Genomic mapping of single-stranded DNA in hydroxyurea-challenged yeasts identifies origins of replication. Nature Cell Biol. 8, 148–155 (2006).
Sasaki, T. et al. The Chinese hamster dihydrofolate reductase replication origin decision point follows activation of transcription and suppresses initiation of replication within transcription units. Mol. Cell. Biol. 26, 1051–1062 (2006).
Alvino, G. M. et al. Replication in hydroxyurea: it's a matter of time. Mol. Cell. Biol. 27, 6396–6406 (2007).
Mickle, K. L. et al. Checkpoint independence of most DNA replication origins in fission yeast. BMC Mol. Biol. 8, 112 (2007).
Gilbert, D. M. Replication origin plasticity, Taylor-made: inhibition vs recruitment of origins under conditions of replication stress. Chromosoma 116, 341–347 (2007).
Courbet, S. et al. Replication fork movement sets chromatin loop size and origin choice in mammalian cells. Nature 455, 557–560 (2008).
Knott, S. R., Viggiani, C. J., Tavare, S. & Aparicio, O. M. 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. 23, 1077–1090 (2009). In this report, the authors examined the role of the budding yeast histone deacetylase Rpd3 in the timing of origin firing using genome-wide replication profiling of the earliest BrdU-labelled DNA synthesized in the presence of hydroxyurea.
Cadoret, J. C. & Prioleau, M. N. Genome-wide approaches to determining origin distribution. Chromosome Res. 18, 79–89 (2010).
Cadoret, J. C. et al. Genome-wide studies highlight indirect links between human replication origins and gene regulation. Proc. Natl Acad. Sci. USA 105, 15837–15842 (2008). A genome-scale analysis of sites of enrichment of small nascent single-stranded DNA within the human ENCODE regions using the Lexo method and hybridization to microarrays.
Gerbi, S. A. & Bielinsky, A. K. Replication initiation point mapping. Methods 13, 271–280 (1997).
Das-Bradoo, S. & Bielinsky, A. K. Replication initiation point mapping: approach and implications. Methods Mol. Biol. 521, 105–120 (2009).
Lucas, I. et al. High-throughput mapping of origins of replication in human cells. EMBO Rep. 8, 770–777 (2007).
Karnani, N., Taylor, C. M., Malhotra, A. & Dutta, A. 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 (2010). A second genome-scale analysis of sites of enrichment of small nascent single-stranded DNA within the human ENCODE regions using both the Lexo and BrdU-IP methods and hybridization to microarrays. Reasons for the low concordance with results in reference 54 are discussed although many features of the sites were in common.
Sequeira-Mendes, J. et al. Transcription initiation activity sets replication origin efficiency in mammalian cells. PLoS Genet. 5, e1000446 (2009). A genome-scale analysis of sites of enrichment of small nascent single-stranded DNA across a segment of the mouse genome in ES cells using hybridization to microarrays. Similarities in the features found for these origins and those in reference 54 are discussed.
Maric, C. & Prioleau, M. N. Interplay between DNA replication and gene expression: a harmonious coexistence. Curr. Opin. Cell Biol. 22, 277–283 (2010).
Mesner, L. D. & Hamlin, J. L. Isolation of restriction fragments containing origins of replication from complex genomes. Methods Mol. Biol. 521, 315–328 (2009).
Francis, L. I., Randell, J. C., Takara, T. J., Uchima, L. & Bell, S. P. Incorporation into the prereplicative complex activates the Mcm2–7 helicase for Cdc7-Dbf4 phosphorylation. Genes Dev. 23, 643–654 (2009).
Gelbart, M. E., Bachman, N., Delrow, J., Boeke, J. D. & Tsukiyama, T. Genome-wide identification of Isw2 chromatin-remodeling targets by localization of a catalytically inactive mutant. Genes Dev. 19, 942–954 (2005).
Romero, J. & Lee, H. Asymmetric bidirectional replication at the human DBF4 origin. Nature Struct. Mol. Biol. 15, 722–729 (2008).
Tuduri, S., Tourriere, H. & Pasero, P. Defining replication origin efficiency using DNA fiber assays. Chromosome Res. 18, 91–102 (2010).
Cohen, S. M. et al. BRG1 co-localizes with DNA replication factors and is required for efficient replication fork progression. Nucleic Acids Res. 22 Jun 2010 (doi:10.1093/nar/gkq559).
Sullivan, B. A. & Karpen, G. H. Centromeric chromatin exhibits a histone modification pattern that is distinct from both euchromatin and heterochromatin. Nature Struct. Mol. Biol. 11, 1076–1083 (2004).
Cipriany, B. R. et al. Single molecule epigenetic analysis in a nanofluidic channel. Anal. Chem. 82, 2480–2487 (2010).
Gilbert, D. M. Making sense of eukaryotic DNA replication origins. Science 294, 96–100 (2001).
Knott, S. R., Viggiani, C. J. & Aparicio, O. M. To promote and protect: coordinating DNA replication and transcription for genome stability. Epigenetics 4, 362–365 (2009).
Huvet, M. et al. Human gene organization driven by the coordination of replication and transcription. Genome Res. 17, 1278–1285 (2007).
Jorgensen, F. G. & Schierup, M. H. Increased rate of human mutations where DNA and RNA polymerases collide. Trends Genet. 25, 523–527 (2009).
Bermejo, R. et al. Genome-organizing factors Top2 and Hmo1 prevent chromosome fragility at sites of S phase transcription. Cell 138, 870–884 (2009).
Tuduri, S. et al. Topoisomerase I suppresses genomic instability by preventing interference between replication and transcription. Nature Cell Biol. 11, 1315–1324 (2009).
Necsulea, A., Guillet, C., Cadoret, J. C., Prioleau, M. N. & Duret, L. The relationship between DNA replication and human genome organization. Mol. Biol. Evol. 26, 729–741 (2009).
Azvolinsky, A., Giresi, P. G., Lieb, J. D. & Zakian, V. A. Highly transcribed RNA polymerase II genes are impediments to replication fork progression in Saccharomyces cerevisiae. Mol. Cell 34, 722–734 (2009).
Liachko, I. et al. A comprehensive genome-wide map of autonomously replicating sequences in a naive genome. PLoS Genet. 6, e1000946 (2010).
Aladjem, M. I. et al. Replication initiation patterns in the β-globin loci of totipotent and differentiated murine cells: evidence for multiple initiation regions. Mol. Cell. Biol. 22, 442–452 (2002).
Hiratani, I. & Gilbert, D. M. in The Cell Biology of Stem Cells (eds Meshorer, E. & Plath, K.) 41–58 (Landes and Springer, Austin, Texas, 2010).
Karnani, N., Taylor, C. M. & Dutta, A. Microarray analysis of DNA replication timing. Methods Mol. Biol. 556, 191–203 (2009).
Farkash-Amar, S. & Simon, I. Genome-wide analysis of the replication program in mammals. Chromosome Res. 18, 115–125 (2009).
Gilbert, D. M. & Cohen, S. N. Bovine papilloma virus plasmids replicate randomly in mouse fibroblasts throughout S-phase of the cell cycle. Cell 50, 59–68 (1987).
Gilbert, D. M. Temporal order of replication of Xenopus laevis 5S ribosomal RNA genes in somatic cells. Proc. Natl Acad. Sci. USA 83, 2924–2928 (1986).
Hansen, R., Canfield, T., Lamb, M., Gartler, S. & Laird, C. Association of fragile X syndrome with delayed replication of the FMR1 gene. Cell 73, 1403–1409 (1993).
Schubeler, D. et al. Genome-wide DNA replication profile for Drosophila melanogaster: a link between transcription and replication timing. Nature Genet. 32, 438–442 (2002).
Schwaiger, M. et al. Chromatin state marks cell-type- and gender-specific replication of the Drosophila genome. Genes Dev. 23, 589–601 (2009). The first comprehensive genome-wide mapping of replication timing in two D. melanogaster tissue culture cell lines derived from different tissues. The study revealed developmental and gender-specific differences.
Schwaiger, M., Kohler, H., Oakeley, E. J., Stadler, M. B. & Schubeler, D. Heterochromatin protein 1 (HP1) modulates replication timing of the Drosophila genome. Genome Res. 20, 771–780 (2010). A genome-wide analysis of replication timing in D. melanogaster cultured cells lacking heterochromatin protein 1 (HP1). Surprisingly, the study revealed roles for HP1 both in very late replication of centromeric DNA and in early replication of certain euchromatic regions with high levels of repeats.
Hansen, R. S. et al. Sequencing newly replicated DNA reveals widespread plasticity in human replication timing. Proc. Natl Acad. Sci. USA 107, 139–144 (2010). A genome-wide replication timing analysis in four human cell lines using the BrdU-IP method followed by sequencing. This study revealed many differences in and links to the accessibility of chromatin by DNase I.
Hiratani, I. et al. Genome-wide dynamics of replication timing revealed by in vitro models of mouse embryogenesis. Genome Res. 20, 155–169 (2010). A genome-wide analysis of replication timing in 22 cell lines representing 10 stages of early mouse development. It revealed widespread changes in replication timing, including a set of early to late changes during epiblast maturation that harboured genes which became difficult to reactivate when late replicating.
Ryba, T. et al. Evolutionarily conserved replication timing profiles predict long-range chromatin interactions and distinguish closely related cell types. Genome Res. 20, 761–770 (2010). A genome-wide replication timing analysis of eight human cell types, including four human ES cell lines, and a comparison to corresponding mouse profiles. The study revealed overall cell-type-specific evolutionary conservation of replication timing and a much closer alignment of human ES cells to mouse epiblast-derived stem cells than to mouse ES cells, as well as a surprisingly close alliance of replication timing to chromatin spatial proximity. This report also showed the equivalence of microarray to sequencing methods in the same cell line.
Desprat, R. et al. Predictable dynamic program of timing of DNA replication in human cells. Genome Res. 19, 2288–2299 (2009). A replication timing analysis of 3% of the genome in three human cell lines using the S/G1 microarray hybridization method, followed by genome-wide analysis by S/G1 sequencing. Equivalent results were obtained by both methods, and this group also identified a close linkage between early replication and proximity to expressed genes.
Yokochi, T. et al. G9a selectively represses a class of late-replicating genes at the nuclear periphery. Proc. Natl Acad. Sci. USA 106, 19363–19368 (2009). Genome-wide replication timing analysis is used as a tool to show that loss of the G9a histone methyltransferase in mouse ES cells results in the de-repression of a set of genes that are almost exclusively late replicating. However, loss of this enzyme has no detectable effect on replication timing of the genome, except for peri-centromeric regions.
Hiratani, I. et al. Global reorganization of replication domains during embryonic stem cell differentiation. PLoS Biol. 6, e245 (2008). The first genome-wide analysis of replication timing during the course of directed differentiation in three mouse ES cell lines. It revealed widespread consolidation of replication domains, which resulted in the production of fewer segments of discordant replication during neural differentiation. Furthermore, the study highlighted links to transcriptional contol, promoter classes and chromatin marks, and also compared the S/G1 and BrdU-IP profiling methods.
Farkash-Amar, S. et al. Global organization of replication time zones of the mouse genome. Genome Res. 18, 1562–1570 (2008). The authors used an elegant synchronization scheme to create a high-resolution genome-wide map of regions that replicate within fine cell cycle windows.
Woodfine, K. et al. Replication timing of human chromosome 6. Cell Cycle 4, 172–176 (2005).
Woodfine, K. et al. Replication timing of the human genome. Hum. Mol. Genet. 13, 191–202 (2004).
Lee, T. J. et al. Arabidopsis thaliana chromosome 4 replicates in two phases that correlate with chromatin state. PLoS Genet. 6, e1000982 (2010). The first genome-scale analysis of replication timing across a segment of the A. thaliana genome using the BrdU-IP method. Many parallels to animal cells were found.
Gilbert, D. M. & Cohen, S. N. Position effects on the timing of replication of chromosomally integrated simian virus 40 molecules in Chinese hamster cells. Mol. Cell. Biol. 10, 4345–4355 (1990).
Karnani, N., Taylor, C., Malhotra, A. & Dutta, A. Pan-S replication patterns and chromosomal domains defined by genome-tiling arrays of ENCODE genomic areas. Genome Res. 17, 865–876 (2007).
Lu, J., Li, F., Murphy, C. S., Davidson, M. W. & Gilbert, D. M. G2 phase chromatin lacks determinants of replication timing. J. Cell Biol. 189, 967–980 (2010).
Hiratani, I., Takebayashi, S., Lu, J. & Gilbert, D. M. Replication timing and transcriptional control: beyond cause and effect — part II. Curr. Opin. Genet. Dev. 19, 142–149 (2009).
Hiratani, I. & Gilbert, D. M. Replication timing as an epigenetic mark. Epigenetics 4, 93–97 (2009).
Pope, B. D., Hiratani, I. & Gilbert, D. M. Domain-wide regulation of DNA replication timing during mammalian development. Chromosome Res. 18, 127–136 (2010).
Mendez, J. Temporal regulation of DNA replication in mammalian cells. Crit. Rev. Biochem. Mol. Biol. 44, 343–351 (2009).
McCune, H. J. et al. The temporal program of chromosome replication: genomewide replication in clb5 δ Saccharomyces cerevisiae. Genetics 180, 1833–1847 (2008). This report used density transfer to isolate DNA replicated at specific times during S phase, followed by array hybridization. The study showed that, in Clb5-deleted budding yeast that can only initiate at early origins, large blocks of chromosomes became considerably delayed in their replication, providing evidence for clustering of temporally related origins in budding yeast.
MacAlpine, D. M., Rodriguez, H. K. & Bell, S. P. Coordination of replication and transcription along a Drosophila chromosome. Genes Dev. 18, 3094–3105 (2004).
Yaffe, E. et al. Comparative analysis of DNA replication timing reveals conserved large-scale chromosomal architecture. PLoS Genet. 6, e1001011 (2010). Genome-wide replication timing profiles in mouse and human fibroblast and lymphoblast cell lines using the S/G1 method revealed high cell-type-specific conservation of replication timing, consistent with reference 90. These authors further showed that evolutionary break points are usually located between regions of similar replication time that are in close proximity.
Jorgensen, H. F. et al. The impact of chromatin modifiers on the timing of locus replication in mouse embryonic stem cells. Genome Biol. 8, R169 (2007).
Li, J., Santoro, R., Koberna, K. & Grummt, I. The chromatin remodeling complex NoRC controls replication timing of rRNA genes. EMBO J. 24, 120–127 (2004).
Wu, R., Singh, P. B. & Gilbert, D. M. Uncoupling global and fine-tuning replication timing determinants for mouse pericentric heterochromatin. J. Cell Biol. 174, 185–194 (2006).
Hayashi, M. T., Takahashi, T. S., Nakagawa, T., Nakayama, J. I. & Masukata, H. The heterochromatin protein Swi6/HP1 activates replication origins at the pericentromeric region and silent mating-type locus. Nature Cell Biol. 11, 357–362 (2009).
Duan, Z. et al. A three-dimensional model of the yeast genome. Nature 465, 363–367 (2010).
Azuara, V. et al. Heritable gene silencing in lymphocytes delays chromatid resolution without affecting the timing of DNA replication. Nature Cell Biol. 5, 668–674 (2003).
Lande-Diner, L., Zhang, J. & Cedar, H. Shifts in replication timing actively affect histone acetylation during nucleosome reassembly. Mol. Cell 34, 767–774 (2009).
Pink, C. J. & Hurst, L. D. Timing of replication is a determinant of neutral substitution rates but does not explain slow Y chromosome evolution in rodents. Mol. Biol. Evol. 27, 1077–1086 (2010).
Chen, C. L. et al. Impact of replication timing on non-CpG and CpG substitution rates in mammalian genomes. Genome Res. 20, 447–457 (2010).
Hyrien, O. & Goldar, A. Mathematical modelling of eukaryotic DNA replication. Chromosome Res. 18, 147–161 (2010).
Huberman, J. A. & Riggs, A. D. Autoradiography of chromosomal DNA fibers from Chinese hamster cells. Proc. Natl Acad. Sci. USA 55, 599–606 (1966).
Takebayashi, S. I., Manders, E. M., Kimura, H., Taguchi, H. & Okumura, K. Mapping sites where replication initiates in mammalian cells using DNA fibers. Exp. Cell Res. 271, 263–268 (2001).
Guan, J. & Lee, L. J. Generating highly ordered DNA nanostrand arrays. Proc. Natl Acad. Sci. USA 102, 18321–18325 (2005).
Sekula, S. et al. Multiplexed lipid dip-pen nanolithography on subcellular scales for the templating of functional proteins and cell culture. Small 4, 1785–1793 (2008).
I thank J. Huberman, D. Macalpine, O. Aparicio, A. Beilinsky, N. Rhind, M.-N. Prioleau, A. Dutta, H. Masukata, A. Schepers, J. Hamlin, K. Plath, B. Papp, L. Mesner, M. Mechali, K. Ekwall and members of my laboratory for helpful discussions during the preparation of this article.
The author declares no competing financial interests.
- Pre-replication complex
(pre-RC). A complex of proteins that forms at the origin of replication during the initiation step of DNA replication. All pre-RC proteins are essential for DNA replication. The pre-RC is typically thought to consist of origin recognition complex (ORC), DNA replication factor Cdt1, cell division cycle protein 6 (Cdc6) and mini-chromosome maintenance (MCM) complex.
- Dormant origin
Cells have a large excess of replication origins over what they need to complete DNA replication. Origins that are in the vicinity of a recently initiated origin normally will be replicated passively when the replication fork passes through them. However, if DNA damage or low-nucleotide pools slow the replication forks, these origins can fire to complete duplication of nearby DNA in a timely fashion.
- Replication origin
A site where replication is initiated during S phase. It is bound by the origin recognition complex.
- Replication timing programme
All eukaryotic cells replicate segments of their genomes in a defined temporal sequence. This process is referred to as replication timing. The temporal order in which segments of DNA are replicated is specific to specific cell types, and that temporal order is its replication timing programme.
- Origin recognition complex
A complex of six subunits that binds to the origins of DNA replication in an ATP-dependent manner before initiation to recruit additional protein members of the pre-replication complex.
- Chromatin immunoprecipitation
A technique that is used to identify the location of DNA-binding proteins and epigenetic marks in the genome. Genomic sequences containing the mark of interest are enriched by binding soluble DNA chromatin extracts (complexes of DNA and protein) to an antibody that recognizes the mark.
- Mini-chromosome maintenance complex
An oligomeric complex that is suggested to be the helicase involved in replication.
- Phased nucleosomes
Nucleosomes that are evenly spaced. This usually occurs when a nucleosome is positioned by a DNA sequence or chromatin protein, which restricts the possible locations of its nearest neighbours.
The percentage of replication cycles in which any given origin is used as an initiation site.
- Replication fork
The branch-point structure that forms at the site of active DNA synthesis, where helicases break the hydrogen bonds tethering the two DNA strands and unwind the DNA.
- Primer extension
Any configuration in which a partially single-stranded nucleic acid is annealed with a 5′ overhang to a smaller complementary strand. The 3′ hydroxyl of the annealed complementary strand can serve as a primer that can be extended by DNA polymerase along the remaining single-stranded portion of the larger template molecule.
- DNase I hypersensitive site
A region of the genome that is readily degraded by the enzyme DNase I owing to decreased nucleosome occupancy (an 'open' chromatin structure).
- Replication bubble
The structure formed where two replication forks, derived from the same replication origins, are moving bidirectionally away from the site of initiation. The intervening DNA consists of two newly synthesized strands.
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Gilbert, D. Evaluating genome-scale approaches to eukaryotic DNA replication. Nat Rev Genet 11, 673–684 (2010). https://doi.org/10.1038/nrg2830
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