Editing Origin of replication (section)

== Eukaryotic ==
[[file:Origins of DNA replication Figure 4.jpg|thumb|300px|Origin organization and recognition in eukaryotes. Specific DNA elements and epigenetic features involved in ORC recruitment and origin function are summarized for ''S. cerevisiae'', ''S. pombe'', and [[metazoan]] origins. A schematic of the ORC architecture is also shown, highlighting the arrangement of the AAA+ and winged-helix domains into a pentameric ring that encircles origin DNA. Ancillary domains of several ORC subunits involved in targeting ORC to origins are included. Other regions in ORC subunits may also be involved in initiator recruitment, either by directly or indirectly associating with partner proteins. A few examples are listed. Note that the BAH domain in ''S. cerevisiae'' Orc1 binds nucleosomes<ref name="#18158899">{{cite journal | vauthors = Onishi M, Liou GG, Buchberger JR, Walz T, Moazed D | title = Role of the conserved Sir3-BAH domain in nucleosome binding and silent chromatin assembly | journal = Molecular Cell | volume = 28 | issue = 6 | pages = 1015–28 | date = December 2007 | pmid = 18158899 | doi = 10.1016/j.molcel.2007.12.004 | doi-access = free }}</ref> but does not recognize H4K20me2.<ref name="#22398447" /><br/>BAH – bromo-adjacent homology domain, WH – winged-helix domain, TFIIB – transcription factor II B-like domain in Orc6, G4 – G quadruplex, OGRE – origin G-rich repeated element. ORC gene names are indicated by a single number; e.g. 3 refers to [[ORC3]].]]

Origin organization, specification, and activation in [[Eukaryote|eukaryotes]] are more complex than in [[Bacteria|bacterial]] or [[Archaea|archaeal]] domains and significantly deviate from the paradigm established for prokaryotic replication initiation. The large [[Genome size|genome sizes]] of eukaryotic cells, which range from 12 Mbp in ''S. cerevisiae'' to more than 100 Gbp in some plants, necessitates that DNA replication starts at several hundred (in budding yeast) to tens of thousands (in humans) origins to complete DNA replication of all chromosomes during each cell cycle.<ref name=":3" /><ref name=":10" /> With the exception of ''S. cerevisiae'' and related ''Saccharomycotina'' species, eukaryotic origins do not contain consensus DNA sequence elements but their location is influenced by contextual cues such as local DNA topology, DNA structural features, and chromatin environment.<ref name="#15459665"/><ref name=":9" /><ref name=":11" /> 

Eukaryotic origin function relies on a conserved initiator protein complex to load replicative helicases onto DNA during the late M and [[G1 phase|G1]] phases of the cell cycle, a step known as '''origin licensing'''.<ref name="#28209641">{{cite journal | vauthors = Bleichert F, Botchan MR, Berger JM | title = Mechanisms for initiating cellular DNA replication | journal = Science | volume = 355 | issue = 6327 | pages = eaah6317 | date = February 2017 | pmid = 28209641 | doi = 10.1126/science.aah6317 | doi-access = free }}</ref> In contrast to their bacterial counterparts, replicative helicases in eukaryotes are loaded onto origin duplex DNA in an inactive, double-hexameric form and only a subset of them (10-20% in mammalian cells) is activated during any given [[S phase]], events that are referred to as '''origin firing'''.<ref name="#21282109">{{cite journal | vauthors = Gambus A, Khoudoli GA, Jones RC, Blow JJ | title = MCM2-7 form double hexamers at licensed origins in Xenopus egg extract | journal = The Journal of Biological Chemistry | volume = 286 | issue = 13 | pages = 11855–64 | date = April 2011 | pmid = 21282109 | pmc = 3064236 | doi = 10.1074/jbc.M110.199521 | doi-access = free }}</ref><ref name="#19896182">{{cite journal | vauthors = Remus D, Beuron F, Tolun G, Griffith JD, Morris EP, Diffley JF | title = Concerted loading of Mcm2-7 double hexamers around DNA during DNA replication origin licensing | journal = Cell | volume = 139 | issue = 4 | pages = 719–30 | date = November 2009 | pmid = 19896182 | pmc = 2804858 | doi = 10.1016/j.cell.2009.10.015 }}</ref><ref name="#19910535">{{cite journal | vauthors = Evrin C, Clarke P, Zech J, Lurz R, Sun J, Uhle S, Li H, Stillman B, Speck C | display-authors = 6 | title = A double-hexameric MCM2-7 complex is loaded onto origin DNA during licensing of eukaryotic DNA replication | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 106 | issue = 48 | pages = 20240–5 | date = December 2009 | pmid = 19910535 | pmc = 2787165 | doi = 10.1073/pnas.0911500106 | bibcode = 2009PNAS..10620240E | doi-access = free }}</ref>

The location of active eukaryotic origins is therefore determined on at least two different levels, origin licensing to mark all potential origins, and origin firing to select a subset that permits assembly of the replication machinery and initiation of DNA synthesis. The extra licensed origins serve as backup and are activated only upon slowing or stalling of nearby replication forks, ensuring that DNA replication can be completed when cells encounter replication stress.<ref name="#18079179">{{cite journal | vauthors = Ge XQ, Jackson DA, Blow JJ | title = Dormant origins licensed by excess Mcm2-7 are required for human cells to survive replicative stress | journal = Genes & Development | volume = 21 | issue = 24 | pages = 3331–41 | date = December 2007 | pmid = 18079179 | pmc = 2113033 | doi = 10.1101/gad.457807 }}</ref><ref name="#18579778">{{cite journal | vauthors = Ibarra A, Schwob E, Méndez J | title = Excess MCM proteins protect human cells from replicative stress by licensing backup origins of replication | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 105 | issue = 26 | pages = 8956–61 | date = July 2008 | pmid = 18579778 | pmc = 2449346 | doi = 10.1073/pnas.0803978105 | bibcode = 2008PNAS..105.8956I | doi-access = free }}</ref> In the absence of stress, firing of extra origins is suppressed by a replication-associated signaling mechanism.<ref>{{cite journal | vauthors = Moiseeva TN, Yin Y, Calderon MJ, Qian C, Schamus-Haynes S, Sugitani N, Osmanbeyoglu HU, Rothenberg E, Watkins SC, Bakkenist CJ | display-authors = 6 | title = An ATR and CHK1 kinase signaling mechanism that limits origin firing during unperturbed DNA replication | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 116 | issue = 27 | pages = 13374–13383 | date = July 2019 | pmid = 31209037 | pmc = 6613105 | doi = 10.1073/pnas.1903418116 | bibcode = 2019PNAS..11613374M | doi-access = free }}</ref><ref>{{cite journal | vauthors = Moiseeva TN, Bakkenist CJ | title = Dormant origin signaling during unperturbed replication | journal = DNA Repair | volume = 81 | pages = 102655 | date = September 2019 | pmid = 31311769 | pmc = 6764875 | doi = 10.1016/j.dnarep.2019.102655 }}</ref> Together, the excess of licensed origins and the tight cell cycle control of origin licensing and firing embody two important strategies to prevent under- and overreplication and to maintain the integrity of eukaryotic genomes.<ref name="Ekundayo et al" />

Early studies in ''S. cerevisiae'' indicated that replication origins in eukaryotes might be recognized in a DNA-sequence-specific manner analogously to those in prokaryotes. In budding yeast, the search for genetic replicators lead to the identification of autonomously replicating sequences (ARS) that support efficient DNA replication initiation of extrachromosomal DNA.<ref name="#388229">{{cite journal | vauthors = Stinchcomb DT, Struhl K, Davis RW | title = Isolation and characterisation of a yeast chromosomal replicator | journal = Nature | volume = 282 | issue = 5734 | pages = 39–43 | date = November 1979 | pmid = 388229 | doi = 10.1038/282039a0 | bibcode = 1979Natur.282...39S | s2cid = 4326901 }}</ref><ref name="#3311385">{{cite journal | vauthors = Huberman JA, Spotila LD, Nawotka KA, el-Assouli SM, Davis LR | title = The in vivo replication origin of the yeast 2 microns plasmid | journal = Cell | volume = 51 | issue = 3 | pages = 473–81 | date = November 1987 | pmid = 3311385 | doi = 10.1016/0092-8674(87)90643-x | s2cid = 54385402 }}</ref><ref name="#2822257">{{cite journal | vauthors = Brewer BJ, Fangman WL | title = The localization of replication origins on ARS plasmids in S. cerevisiae | journal = Cell | volume = 51 | issue = 3 | pages = 463–71 | date = November 1987 | pmid = 2822257 | doi = 10.1016/0092-8674(87)90642-8 | s2cid = 20152681 }}</ref> These ARS regions are approximately 100-200 bp long and exhibit a multipartite organization, containing A, B1, B2, and sometimes B3 elements that together are essential for origin function.<ref name="#1536007">{{cite journal | vauthors = Marahrens Y, Stillman B | title = A yeast chromosomal origin of DNA replication defined by multiple functional elements | journal = Science | volume = 255 | issue = 5046 | pages = 817–23 | date = February 1992 | pmid = 1536007 | doi = 10.1126/science.1536007 | bibcode = 1992Sci...255..817M }}</ref><ref name="#7935478">{{cite journal | vauthors = Rao H, Marahrens Y, Stillman B | title = Functional conservation of multiple elements in yeast chromosomal replicators | journal = Molecular and Cellular Biology | volume = 14 | issue = 11 | pages = 7643–51 | date = November 1994 | pmid = 7935478 | pmc = 359300 | doi = 10.1128/mcb.14.11.7643-7651.1994 }}</ref> The A element encompasses the conserved 11 bp ARS consensus sequence (ACS),<ref name="#6345070">{{cite journal | vauthors = Broach JR, Li YY, Feldman J, Jayaram M, Abraham J, Nasmyth KA, Hicks JB | title = Localization and sequence analysis of yeast origins of DNA replication | journal = Cold Spring Harbor Symposia on Quantitative Biology | volume = 47 Pt 2 | pages = 1165–73 | pmid = 6345070 | doi = 10.1101/sqb.1983.047.01.132 | year = 1983 }}</ref><ref name="#6392851">{{cite journal | vauthors = Celniker SE, Sweder K, Srienc F, Bailey JE, Campbell JL | title = Deletion mutations affecting autonomously replicating sequence ARS1 of Saccharomyces cerevisiae | journal = Molecular and Cellular Biology | volume = 4 | issue = 11 | pages = 2455–66 | date = November 1984 | pmid = 6392851 | pmc = 369077 | doi = 10.1128/mcb.4.11.2455-2466.1984 }}</ref> which, in conjunction with the B1 element, constitutes the primary binding site for the heterohexameric [[origin recognition complex]] (ORC), the eukaryotic replication initiator.<ref name="#7892251">{{cite journal | vauthors = Rao H, Stillman B | title = The origin recognition complex interacts with a bipartite DNA binding site within yeast replicators | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 92 | issue = 6 | pages = 2224–8 | date = March 1995 | pmid = 7892251 | pmc = 42456 | doi = 10.1073/pnas.92.6.2224 | bibcode = 1995PNAS...92.2224R | doi-access = free }}</ref><ref name="#7781615">{{cite journal | vauthors = Rowley A, Cocker JH, Harwood J, Diffley JF | title = Initiation complex assembly at budding yeast replication origins begins with the recognition of a bipartite sequence by limiting amounts of the initiator, ORC | journal = The EMBO Journal | volume = 14 | issue = 11 | pages = 2631–41 | date = June 1995 | pmid = 7781615 | pmc = 398377 | doi = 10.1002/j.1460-2075.1995.tb07261.x }}</ref><ref name="#1579162">{{cite journal | vauthors = Bell SP, Stillman B | title = ATP-dependent recognition of eukaryotic origins of DNA replication by a multiprotein complex | journal = Nature | volume = 357 | issue = 6374 | pages = 128–34 | date = May 1992 | pmid = 1579162 | doi = 10.1038/357128a0 | bibcode = 1992Natur.357..128B | s2cid = 4346767 }}</ref><ref name="#29973722">{{cite journal | vauthors = Li N, Lam WH, Zhai Y, Cheng J, Cheng E, Zhao Y, Gao N, Tye BK | display-authors = 6 | title = Structure of the origin recognition complex bound to DNA replication origin | journal = Nature | volume = 559 | issue = 7713 | pages = 217–222 | date = July 2018 | pmid = 29973722 | doi = 10.1038/s41586-018-0293-x | bibcode = 2018Natur.559..217L | s2cid = 49577101 }}</ref> Within ORC, five subunits are predicated on conserved AAA+ ATPase and winged-helix folds and co-assemble into a pentameric ring that encircles DNA.<ref name="#29973722" /><ref name="#25762138">{{cite journal | vauthors = Bleichert F, Botchan MR, Berger JM | title = Crystal structure of the eukaryotic origin recognition complex | journal = Nature | volume = 519 | issue = 7543 | pages = 321–6 | date = March 2015 | pmid = 25762138 | pmc = 4368505 | doi = 10.1038/nature14239 | bibcode = 2015Natur.519..321B }}</ref><ref name="#23851460">{{cite journal | vauthors = Sun J, Evrin C, Samel SA, Fernández-Cid A, Riera A, Kawakami H, Stillman B, Speck C, Li H | display-authors = 6 | title = Cryo-EM structure of a helicase loading intermediate containing ORC-Cdc6-Cdt1-MCM2-7 bound to DNA | journal = Nature Structural & Molecular Biology | volume = 20 | issue = 8 | pages = 944–51 | date = August 2013 | pmid = 23851460 | pmc = 3735830 | doi = 10.1038/nsmb.2629 }}</ref> In budding yeast ORC, DNA binding elements in the ATPase and winged-helix domains, as well as adjacent basic patch regions in some of the ORC subunits, are positioned in the central pore of the ORC ring such that they aid the DNA-sequence-specific recognition of the ACS in an ATP-dependent manner.<ref name="#29973722" /><ref name="#26456755">{{cite journal | vauthors = Kawakami H, Ohashi E, Kanamoto S, Tsurimoto T, Katayama T | title = Specific binding of eukaryotic ORC to DNA replication origins depends on highly conserved basic residues | journal = Scientific Reports | volume = 5 | pages = 14929 | date = October 2015 | pmid = 26456755 | pmc = 4601075 | doi = 10.1038/srep14929 | bibcode = 2015NatSR...514929K }}</ref> By contrast, the roles of the B2 and B3 elements are less clear. The B2 region is similar to the ACS in sequence and has been suggested to function as a second ORC binding site under certain conditions, or as a binding site for the replicative helicase core.<ref name="#3284655">{{cite journal | vauthors = Palzkill TG, Newlon CS | title = A yeast replication origin consists of multiple copies of a small conserved sequence | journal = Cell | volume = 53 | issue = 3 | pages = 441–50 | date = May 1988 | pmid = 3284655 | doi = 10.1016/0092-8674(88)90164-x | s2cid = 7534654 }}</ref><ref name="#11756674">{{cite journal | vauthors = Wilmes GM, Bell SP | title = The B2 element of the Saccharomyces cerevisiae ARS1 origin of replication requires specific sequences to facilitate pre-RC formation | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 99 | issue = 1 | pages = 101–6 | date = January 2002 | pmid = 11756674 | pmc = 117521 | doi = 10.1073/pnas.012578499 | bibcode = 2002PNAS...99..101W | doi-access = free }}</ref><ref name="#28729513">{{cite journal | vauthors = Coster G, Diffley JF | title = Bidirectional eukaryotic DNA replication is established by quasi-symmetrical helicase loading | journal = Science | volume = 357 | issue = 6348 | pages = 314–318 | date = July 2017 | pmid = 28729513 | pmc = 5608077 | doi = 10.1126/science.aan0063 | bibcode = 2017Sci...357..314C }}</ref><ref name="#10757793">{{cite journal | vauthors = Zou L, Stillman B | title = 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 | journal = Molecular and Cellular Biology | volume = 20 | issue = 9 | pages = 3086–96 | date = May 2000 | pmid = 10757793 | pmc = 85601 | doi = 10.1128/mcb.20.9.3086-3096.2000 }}</ref><ref name="#11172708">{{cite journal | vauthors = Lipford JR, Bell SP | title = Nucleosomes positioned by ORC facilitate the initiation of DNA replication | journal = Molecular Cell | volume = 7 | issue = 1 | pages = 21–30 | date = January 2001 | pmid = 11172708 | doi = 10.1016/s1097-2765(01)00151-4 | doi-access = free }}</ref> Conversely, the B3 element recruits the transcription factor Abf1, albeit B3 is not found at all budding yeast origins and Abf1 binding does not appear to be strictly essential for origin function.<ref name="Ekundayo et al"/><ref name="#1536007" /><ref name="#1579168">{{cite journal | vauthors = Diffley JF, Cocker JH | title = Protein-DNA interactions at a yeast replication origin | journal = Nature | volume = 357 | issue = 6374 | pages = 169–72 | date = May 1992 | pmid = 1579168 | doi = 10.1038/357169a0 | bibcode = 1992Natur.357..169D | s2cid = 4354585 }}</ref><ref name="#3281162">{{cite journal | vauthors = Diffley JF, Stillman B | title = Purification of a yeast protein that binds to origins of DNA replication and a transcriptional silencer | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 85 | issue = 7 | pages = 2120–4 | date = April 1988 | pmid = 3281162 | pmc = 279940 | doi = 10.1073/pnas.85.7.2120 | bibcode = 1988PNAS...85.2120D | doi-access = free }}</ref>

Origin recognition in eukaryotes other than ''S. cerevisiae'' or its close relatives does not conform to the sequence-specific read-out of conserved origin DNA elements. Pursuits to isolate specific chromosomal replicator sequences more generally in eukaryotic species, either genetically or by genome-wide mapping of initiator binding or replication start sites, have failed to identify clear consensus sequences at origins.<ref name="#27436900">{{cite journal | vauthors = Miotto B, Ji Z, Struhl K | title = Selectivity of ORC binding sites and the relation to replication timing, fragile sites, and deletions in cancers | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 113 | issue = 33 | pages = E4810-9 | date = August 2016 | pmid = 27436900 | pmc = 4995967 | doi = 10.1073/pnas.1609060113 | bibcode = 2016PNAS..113E4810M | doi-access = free }}</ref><ref name="#19996087">{{cite journal | vauthors = MacAlpine HK, Gordân R, Powell SK, Hartemink AJ, MacAlpine DM | title = Drosophila ORC localizes to open chromatin and marks sites of cohesin complex loading | journal = Genome Research | volume = 20 | issue = 2 | pages = 201–11 | date = February 2010 | pmid = 19996087 | pmc = 2813476 | doi = 10.1101/gr.097873.109 }}</ref><ref name="#21177973">{{cite journal | vauthors = Eaton ML, Prinz JA, MacAlpine HK, Tretyakov G, Kharchenko PV, MacAlpine DM | title = Chromatin signatures of the Drosophila replication program | journal = Genome Research | volume = 21 | issue = 2 | pages = 164–74 | date = February 2011 | pmid = 21177973 | pmc = 3032920 | doi = 10.1101/gr.116038.110 }}</ref><ref name="#23187890">{{cite journal | vauthors = Dellino GI, Cittaro D, Piccioni R, Luzi L, Banfi S, Segalla S, Cesaroni M, Mendoza-Maldonado R, Giacca M, Pelicci PG | display-authors = 6 | title = Genome-wide mapping of human DNA-replication origins: levels of transcription at ORC1 sites regulate origin selection and replication timing | journal = Genome Research | volume = 23 | issue = 1 | pages = 1–11 | date = January 2013 | pmid = 23187890 | pmc = 3530669 | doi = 10.1101/gr.142331.112 }}</ref><ref name="#26560631">{{cite journal | vauthors = Cayrou C, Ballester B, Peiffer I, Fenouil R, Coulombe P, Andrau JC, van Helden J, Méchali M | display-authors = 6 | title = The chromatin environment shapes DNA replication origin organization and defines origin classes | journal = Genome Research | volume = 25 | issue = 12 | pages = 1873–85 | date = December 2015 | pmid = 26560631 | pmc = 4665008 | doi = 10.1101/gr.192799.115 }}</ref><ref name="#21750104">{{cite journal | vauthors = Cayrou C, Coulombe P, Vigneron A, Stanojcic S, Ganier O, Peiffer I, Rivals E, Puy A, Laurent-Chabalier S, Desprat R, Méchali M | display-authors = 6 | title = Genome-scale analysis of metazoan replication origins reveals their organization in specific but flexible sites defined by conserved features | journal = Genome Research | volume = 21 | issue = 9 | pages = 1438–49 | date = September 2011 | pmid = 21750104 | pmc = 3166829 | doi = 10.1101/gr.121830.111 }}</ref><ref name="#21148149">{{cite journal | vauthors = Lubelsky Y, Sasaki T, Kuipers MA, Lucas I, Le Beau MM, Carignon S, Debatisse M, Prinz JA, Dennis JH, Gilbert DM | display-authors = 6 | title = Pre-replication complex proteins assemble at regions of low nucleosome occupancy within the Chinese hamster dihydrofolate reductase initiation zone | journal = Nucleic Acids Research | volume = 39 | issue = 8 | pages = 3141–55 | date = April 2011 | pmid = 21148149 | pmc = 3082903 | doi = 10.1093/nar/gkq1276 }}</ref><ref name="#17304213">{{cite journal | vauthors = Hayashi M, Katou Y, Itoh T, Tazumi A, Tazumi M, Yamada Y, Takahashi T, Nakagawa T, Shirahige K, Masukata H | display-authors = 6 | title = Genome-wide localization of pre-RC sites and identification of replication origins in fission yeast | journal = The EMBO Journal | volume = 26 | issue = 5 | pages = 1327–39 | date = March 2007 | pmid = 17304213 | pmc = 1817633 | doi = 10.1038/sj.emboj.7601585 }}</ref><ref name="#21813623">{{cite journal | vauthors = Martin MM, Ryan M, Kim R, Zakas AL, Fu H, Lin CM, Reinhold WC, Davis SR, Bilke S, Liu H, Doroshow JH, Reimers MA, Valenzuela MS, Pommier Y, Meltzer PS, Aladjem MI | display-authors = 6 | title = Genome-wide depletion of replication initiation events in highly transcribed regions | journal = Genome Research | volume = 21 | issue = 11 | pages = 1822–32 | date = November 2011 | pmid = 21813623 | pmc = 3205567 | doi = 10.1101/gr.124644.111 }}</ref><ref name="#28009254">{{cite journal | vauthors = Pourkarimi E, Bellush JM, Whitehouse I | title = C. elegans | journal = eLife | volume = 5 | date = December 2016 | pmid = 28009254 | pmc = 5222557 | doi = 10.7554/eLife.21728 | doi-access = free }}</ref><ref name="#28112731">{{cite journal | vauthors = Rodríguez-Martínez M, Pinzón N, Ghommidh C, Beyne E, Seitz H, Cayrou C, Méchali M | title = The gastrula transition reorganizes replication-origin selection in Caenorhabditis elegans | journal = Nature Structural & Molecular Biology | volume = 24 | issue = 3 | pages = 290–299 | date = March 2017 | pmid = 28112731 | doi = 10.1038/nsmb.3363 | s2cid = 7445974 }}</ref><ref name="#22751019">{{cite journal | vauthors = Besnard E, Babled A, Lapasset L, Milhavet O, Parrinello H, Dantec C, Marin JM, Lemaitre JM | display-authors = 6 | title = Unraveling cell type-specific and reprogrammable human replication origin signatures associated with G-quadruplex consensus motifs | journal = Nature Structural & Molecular Biology | volume = 19 | issue = 8 | pages = 837–44 | date = August 2012 | pmid = 22751019 | doi = 10.1038/nsmb.2339 | s2cid = 20710237 }}</ref> Thus, sequence-specific DNA-initiator interactions in budding yeast signify a specialized mode for origin recognition in this system rather than an archetypal mode for origin specification across the eukaryotic domain. Nonetheless, DNA replication does initiate at discrete sites that are not randomly distributed across eukaryotic genomes, arguing that alternative means determine the chromosomal location of origins in these systems. These mechanisms involve a complex interplay between DNA accessibility, nucleotide sequence skew (both AT-richness and CpG islands have been linked to origins), [[Nucleosome]] positioning, [[Epigenetics|epigenetic]] features, DNA topology and certain DNA structural features (e.g., G4 motifs), as well as regulatory proteins and transcriptional interference.<ref name=":15" /><ref name=":16" /><ref name=":8" /><ref name=":9" /><ref name=":11" /><ref name="#9545253">{{cite journal | vauthors = Delgado S, Gómez M, Bird A, Antequera F | title = Initiation of DNA replication at CpG islands in mammalian chromosomes | journal = The EMBO Journal | volume = 17 | issue = 8 | pages = 2426–35 | date = April 1998 | pmid = 9545253 | pmc = 1170585 | doi = 10.1093/emboj/17.8.2426 }}</ref><ref name="#19360092">{{cite journal | vauthors = Sequeira-Mendes J, Díaz-Uriarte R, Apedaile A, Huntley D, Brockdorff N, Gómez M | title = Transcription initiation activity sets replication origin efficiency in mammalian cells | journal = PLOS Genetics | volume = 5 | issue = 4 | pages = e1000446 | date = April 2009 | pmid = 19360092 | pmc = 2661365 | doi = 10.1371/journal.pgen.1000446 | doi-access = free }}</ref><ref name="#21750104"/><ref name="#30718387">{{cite journal | vauthors = Kelly T, Callegari AJ | title = Dynamics of DNA replication in a eukaryotic cell | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 116 | issue = 11 | pages = 4973–4982 | date = March 2019 | pmid = 30718387 | pmc = 6421431 | doi = 10.1073/pnas.1818680116 | bibcode = 2019PNAS..116.4973K | doi-access = free }}</ref> Importantly, origin properties vary not only between different origins in an organism and among species, but some can also change during development and cell differentiation. The chorion locus in ''Drosophila'' follicle cells constitutes a well-established example for spatial and developmental control of initiation events. This region undergoes DNA-replication-dependent gene amplification at a defined stage during oogenesis and relies on the timely and specific activation of chorion origins, which in turn is regulated by origin-specific cis-elements and several protein factors, including the Myb complex, E2F1, and E2F2.<ref name="#10541550">{{cite journal | vauthors = Austin RJ, Orr-Weaver TL, Bell SP | title = Drosophila ORC specifically binds to ACE3, an origin of DNA replication control element | journal = Genes & Development | volume = 13 | issue = 20 | pages = 2639–49 | date = October 1999 | pmid = 10541550 | pmc = 317108 | doi = 10.1101/gad.13.20.2639 }}</ref><ref name="#12490953">{{cite journal | vauthors = Beall EL, Manak JR, Zhou S, Bell M, Lipsick JS, Botchan MR |author5-link=Joseph S. Lipsick | title = Role for a Drosophila Myb-containing protein complex in site-specific DNA replication | journal = Nature | volume = 420 | issue = 6917 | pages = 833–7 | pmid = 12490953 | doi = 10.1038/nature01228 | year = 2002 | bibcode = 2002Natur.420..833B | s2cid = 4425307 }}</ref><ref name="#15256498">{{cite journal | vauthors = Beall EL, Bell M, Georlette D, Botchan MR | title = Dm-myb mutant lethality in Drosophila is dependent upon mip130: positive and negative regulation of DNA replication | journal = Genes & Development | volume = 18 | issue = 14 | pages = 1667–80 | date = July 2004 | pmid = 15256498 | pmc = 478189 | doi = 10.1101/gad.1206604 }}</ref><ref name="#15545624">{{cite journal | vauthors = Lewis PW, Beall EL, Fleischer TC, Georlette D, Link AJ, Botchan MR | title = Identification of a Drosophila Myb-E2F2/RBF transcriptional repressor complex | journal = Genes & Development | volume = 18 | issue = 23 | pages = 2929–40 | date = December 2004 | pmid = 15545624 | pmc = 534653 | doi = 10.1101/gad.1255204 }}</ref><ref name="#11231579">{{cite journal | vauthors = Bosco G, Du W, Orr-Weaver TL | title = DNA replication control through interaction of E2F-RB and the origin recognition complex | journal = Nature Cell Biology | volume = 3 | issue = 3 | pages = 289–95 | date = March 2001 | pmid = 11231579 | doi = 10.1038/35060086 | s2cid = 24942902 }}</ref> This combinatorial specification and multifactorial regulation of metazoan origins has complicated the identification of unifying features that determine the location of replication start sites across eukaryotes more generally.<ref name="Ekundayo et al"/>

To facilitate replication initiation and origin recognition, ORC assemblies from various species have evolved specialized auxiliary domains that are thought to aid initiator targeting to chromosomal origins or chromosomes in general. For example, the [[ORC4|Orc4]] subunit in ''S. pombe'' ORC contains several AT-hooks that preferentially bind AT-rich DNA,<ref name="#10077566">{{cite journal | vauthors = Chuang RY, Kelly TJ | title = The fission yeast homologue of Orc4p binds to replication origin DNA via multiple AT-hooks | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 96 | issue = 6 | pages = 2656–61 | date = March 1999 | pmid = 10077566 | pmc = 15824 | doi = 10.1073/pnas.96.6.2656 | bibcode = 1999PNAS...96.2656C | doi-access = free }}</ref> while in metazoan (animal) ORC the TFIIB-like domain of [[ORC6|Orc6]] is thought to perform a similar function.<ref name="#17283052">{{cite journal | vauthors = Balasov M, Huijbregts RP, Chesnokov I | title = Role of the Orc6 protein in origin recognition complex-dependent DNA binding and replication in Drosophila melanogaster | journal = Molecular and Cellular Biology | volume = 27 | issue = 8 | pages = 3143–53 | date = April 2007 | pmid = 17283052 | pmc = 1899928 | doi = 10.1128/MCB.02382-06 }}</ref> Metazoan [[ORC1|Orc1]] proteins also harbor a bromo-adjacent homology (BAH) domain that interacts with H4K20me2-nucleosomes.<ref name="#22398447">{{cite journal | vauthors = Kuo AJ, Song J, Cheung P, Ishibe-Murakami S, Yamazoe S, Chen JK, Patel DJ, Gozani O | display-authors = 6 | title = The BAH domain of ORC1 links H4K20me2 to DNA replication licensing and Meier-Gorlin syndrome | journal = Nature | volume = 484 | issue = 7392 | pages = 115–9 | date = March 2012 | pmid = 22398447 | pmc = 3321094 | doi = 10.1038/nature10956 | bibcode = 2012Natur.484..115K }}</ref> Particularly in mammalian cells, [[H4K20me|H4K20 methylation]] has been reported to be required for efficient replication initiation, and the Orc1's BAH domain facilitates ORC association with chromosomes and Epstein-Barr virus origin-dependent replication.<ref name="#20953199">{{cite journal | vauthors = Tardat M, Brustel J, Kirsh O, Lefevbre C, Callanan M, Sardet C, Julien E | title = The histone H4 Lys 20 methyltransferase PR-Set7 regulates replication origins in mammalian cells | journal = Nature Cell Biology | volume = 12 | issue = 11 | pages = 1086–93 | date = November 2010 | pmid = 20953199 | doi = 10.1038/ncb2113 | s2cid = 6710289 }}</ref><ref name="#23152447">{{cite journal | vauthors = Beck DB, Burton A, Oda H, Ziegler-Birling C, Torres-Padilla ME, Reinberg D | title = The role of PR-Set7 in replication licensing depends on Suv4-20h | journal = Genes & Development | volume = 26 | issue = 23 | pages = 2580–9 | date = December 2012 | pmid = 23152447 | pmc = 3521623 | doi = 10.1101/gad.195636.112 }}</ref><ref name="#28778956">{{cite journal | vauthors = Brustel J, Kirstein N, Izard F, Grimaud C, Prorok P, Cayrou C, Schotta G, Abdelsamie AF, Déjardin J, Méchali M, Baldacci G, Sardet C, Cadoret JC, Schepers A, Julien E | display-authors = 6 | title = Histone H4K20 tri-methylation at late-firing origins ensures timely heterochromatin replication | journal = The EMBO Journal | volume = 36 | issue = 18 | pages = 2726–2741 | date = September 2017 | pmid = 28778956 | pmc = 5599798 | doi = 10.15252/embj.201796541 }}</ref><ref name="#30209253">{{cite journal | vauthors = Shoaib M, Walter D, Gillespie PJ, Izard F, Fahrenkrog B, Lleres D, Lerdrup M, Johansen JV, Hansen K, Julien E, Blow JJ, Sørensen CS | display-authors = 6 | title = Histone H4K20 methylation mediated chromatin compaction threshold ensures genome integrity by limiting DNA replication licensing | journal = Nature Communications | volume = 9 | issue = 1 | pages = 3704 | date = September 2018 | pmid = 30209253 | pmc = 6135857 | doi = 10.1038/s41467-018-06066-8 | bibcode = 2018NatCo...9.3704S }}</ref><ref name="#17066079">{{cite journal | vauthors = Noguchi K, Vassilev A, Ghosh S, Yates JL, DePamphilis ML | title = The BAH domain facilitates the ability of human Orc1 protein to activate replication origins in vivo | journal = The EMBO Journal | volume = 25 | issue = 22 | pages = 5372–82 | date = November 2006 | pmid = 17066079 | pmc = 1636626 | doi = 10.1038/sj.emboj.7601396 }}</ref> Therefore, it is intriguing to speculate that both observations are mechanistically linked at least in a subset of metazoa, but this possibility needs to be further explored in future studies. In addition to the recognition of certain DNA or epigenetic features, ORC also associates directly or indirectly with several partner proteins that could aid initiator recruitment, including LRWD1, PHIP (or DCAF14), HMGA1a, among others.<ref name=":7" /><ref name="#22645314">{{cite journal | vauthors = Shen Z, Chakraborty A, Jain A, Giri S, Ha T, Prasanth KV, Prasanth SG | title = Dynamic association of ORCA with prereplicative complex components regulates DNA replication initiation | journal = Molecular and Cellular Biology | volume = 32 | issue = 15 | pages = 3107–20 | date = August 2012 | pmid = 22645314 | pmc = 3434513 | doi = 10.1128/MCB.00362-12 }}</ref><ref name="#27924004">{{cite journal | vauthors = Wang Y, Khan A, Marks AB, Smith OK, Giri S, Lin YC, Creager R, MacAlpine DM, Prasanth KV, Aladjem MI, Prasanth SG | display-authors = 6 | title = Temporal association of ORCA/LRWD1 to late-firing origins during G1 dictates heterochromatin replication and organization | journal = Nucleic Acids Research | volume = 45 | issue = 5 | pages = 2490–2502 | date = March 2017 | pmid = 27924004 | pmc = 5389698 | doi = 10.1093/nar/gkw1211 }}</ref><ref name="#21029866">{{cite journal | vauthors = Bartke T, Vermeulen M, Xhemalce B, Robson SC, Mann M, Kouzarides T | title = Nucleosome-interacting proteins regulated by DNA and histone methylation | journal = Cell | volume = 143 | issue = 3 | pages = 470–84 | date = October 2010 | pmid = 21029866 | pmc = 3640253 | doi = 10.1016/j.cell.2010.10.012 }}</ref><ref name="#20850016">{{cite journal | vauthors = Vermeulen M, Eberl HC, Matarese F, Marks H, Denissov S, Butter F, Lee KK, Olsen JV, Hyman AA, Stunnenberg HG, Mann M | display-authors = 6 | title = Quantitative interaction proteomics and genome-wide profiling of epigenetic histone marks and their readers | journal = Cell | volume = 142 | issue = 6 | pages = 967–80 | date = September 2010 | pmid = 20850016 | doi = 10.1016/j.cell.2010.08.020 | s2cid = 7926456 | doi-access = free | hdl = 2066/84114 | hdl-access = free }}</ref><ref name="#26496610">{{cite journal | vauthors = Hein MY, Hubner NC, Poser I, Cox J, Nagaraj N, Toyoda Y, Gak IA, Weisswange I, Mansfeld J, Buchholz F, Hyman AA, Mann M | display-authors = 6 | title = A human interactome in three quantitative dimensions organized by stoichiometries and abundances | journal = Cell | volume = 163 | issue = 3 | pages = 712–23 | date = October 2015 | pmid = 26496610 | doi = 10.1016/j.cell.2015.09.053 | doi-access = free }}</ref><ref name="#18234858">{{cite journal | vauthors = Thomae AW, Pich D, Brocher J, Spindler MP, Berens C, Hock R, Hammerschmidt W, Schepers A | display-authors = 6 | title = Interaction between HMGA1a and the origin recognition complex creates site-specific replication origins | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 105 | issue = 5 | pages = 1692–7 | date = February 2008 | pmid = 18234858 | pmc = 2234206 | doi = 10.1073/pnas.0707260105 | bibcode = 2008PNAS..105.1692T | doi-access = free }}</ref><ref name="#27272143">{{cite journal | vauthors = Zhang Y, Huang L, Fu H, Smith OK, Lin CM, Utani K, Rao M, Reinhold WC, Redon CE, Ryan M, Kim R, You Y, Hanna H, Boisclair Y, Long Q, Aladjem MI | display-authors = 6 | title = A replicator-specific binding protein essential for site-specific initiation of DNA replication in mammalian cells | journal = Nature Communications | volume = 7 | pages = 11748 | date = June 2016 | pmid = 27272143 | pmc = 4899857 | doi = 10.1038/ncomms11748 | bibcode = 2016NatCo...711748Z }}</ref> Interestingly, ''Drosophila'' ORC, like its budding yeast counterpart, bends DNA and negative supercoiling has been reported to enhance DNA binding of this complex, suggesting that DNA shape and malleability might influence the location of ORC binding sites across metazoan genomes.<ref name=":6" /><ref name="#29973722" /><ref name="#29899147">{{cite journal | vauthors = Bleichert F, Leitner A, Aebersold R, Botchan MR, Berger JM | title = Conformational control and DNA-binding mechanism of the metazoan origin recognition complex | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 115 | issue = 26 | pages = E5906–E5915 | date = June 2018 | pmid = 29899147 | pmc = 6042147 | doi = 10.1073/pnas.1806315115 | bibcode = 2018PNAS..115E5906B | doi-access = free }}</ref><ref name="#18824234">{{cite journal | vauthors = Clarey MG, Botchan M, Nogales E | title = Single particle EM studies of the Drosophila melanogaster origin recognition complex and evidence for DNA wrapping | journal = Journal of Structural Biology | volume = 164 | issue = 3 | pages = 241–9 | date = December 2008 | pmid = 18824234 | pmc = 2640233 | doi = 10.1016/j.jsb.2008.08.006 }}</ref><ref name="#9372948">{{cite journal | vauthors = Lee DG, Bell SP | title = Architecture of the yeast origin recognition complex bound to origins of DNA replication | journal = Molecular and Cellular Biology | volume = 17 | issue = 12 | pages = 7159–68 | date = December 1997 | pmid = 9372948 | pmc = 232573 | doi = 10.1128/mcb.17.12.7159 }}</ref> A molecular understanding for how ORC's DNA binding regions might support the read out of structural properties of the DNA duplex in metazoans rather than of specific DNA sequences as in ''S. cerevisiae'' awaits high-resolution structural information of DNA-bound metazoan initiator assemblies. Likewise, whether and how different epigenetic factors contribute to initiator recruitment in metazoan systems is poorly defined and is an important question that needs to be addressed in more detail.<ref name="Ekundayo et al"/>

Once recruited to origins, ORC and its co-factors [[Cdc6]] and [[DNA replication factor CDT1|Cdt1]] drive the deposition of the [[Minichromosome maintenance|minichromosome maintenance 2-7]] (Mcm2-7) complex onto DNA.<ref name="#28209641" /><ref name="#28717046">{{cite journal | vauthors = Riera A, Barbon M, Noguchi Y, Reuter LM, Schneider S, Speck C | title = From structure to mechanism-understanding initiation of DNA replication | journal = Genes & Development | volume = 31 | issue = 11 | pages = 1073–1088 | date = June 2017 | pmid = 28717046 | pmc = 5538431 | doi = 10.1101/gad.298232.117 }}</ref> Like the archaeal replicative helicase core, Mcm2-7 is loaded as a head-to-head double hexamer onto DNA to license origins.<ref name="#21282109" /><ref name="#19896182" /><ref name="#19910535" /> In S-phase, Dbf4-dependent kinase (DDK) and [[Cyclin-dependent kinase]] (CDK) phosphorylate several Mcm2-7 subunits and additional initiation factors to promote the recruitment of the helicase co-activators Cdc45 and GINS, DNA melting, and ultimately bidirectional replisome assembly at a subset of the licensed origins.<ref name=":4" /><ref name="#25308420">{{cite journal | vauthors = Tognetti S, Riera A, Speck C | title = Switch on the engine: how the eukaryotic replicative helicase MCM2-7 becomes activated | journal = Chromosoma | volume = 124 | issue = 1 | pages = 13–26 | date = March 2015 | pmid = 25308420 | doi = 10.1007/s00412-014-0489-2 | hdl = 10044/1/27085 | s2cid = 175510 | hdl-access = free }}</ref> In both yeast and metazoans, origins are free or depleted of nucleosomes, a property that is crucial for Mcm2-7 loading, indicating that chromatin state at origins regulates not only initiator recruitment but also helicase loading.<ref name="#21148149" /><ref name="#20824081">{{cite journal | vauthors = Berbenetz NM, Nislow C, Brown GW | title = Diversity of eukaryotic DNA replication origins revealed by genome-wide analysis of chromatin structure | journal = PLOS Genetics | volume = 6 | issue = 9 | pages = e1001092 | date = September 2010 | pmid = 20824081 | pmc = 2932696 | doi = 10.1371/journal.pgen.1001092 | doi-access = free }}</ref><ref name="#20351051">{{cite journal | vauthors = Eaton ML, Galani K, Kang S, Bell SP, MacAlpine DM | title = Conserved nucleosome positioning defines replication origins | journal = Genes & Development | volume = 24 | issue = 8 | pages = 748–53 | date = April 2010 | pmid = 20351051 | pmc = 2854390 | doi = 10.1101/gad.1913210 }}</ref><ref name="#28322723">{{cite journal | vauthors = Azmi IF, Watanabe S, Maloney MF, Kang S, Belsky JA, MacAlpine DM, Peterson CL, Bell SP | display-authors = 6 | title = Nucleosomes influence multiple steps during replication initiation | journal = eLife | volume = 6 | date = March 2017 | pmid = 28322723 | pmc = 5400510 | doi = 10.7554/eLife.22512 | doi-access = free }}</ref><ref name="#20129055">{{cite journal | vauthors = Miotto B, Struhl K | title = HBO1 histone acetylase activity is essential for DNA replication licensing and inhibited by Geminin | journal = Molecular Cell | volume = 37 | issue = 1 | pages = 57–66 | date = January 2010 | pmid = 20129055 | pmc = 2818871 | doi = 10.1016/j.molcel.2009.12.012 }}</ref><ref name="#26227968">{{cite journal | vauthors = Liu J, Zimmer K, Rusch DB, Paranjape N, Podicheti R, Tang H, Calvi BR | title = DNA sequence templates adjacent nucleosome and ORC sites at gene amplification origins in Drosophila | journal = Nucleic Acids Research | volume = 43 | issue = 18 | pages = 8746–61 | date = October 2015 | pmid = 26227968 | pmc = 4605296 | doi = 10.1093/nar/gkv766 }}</ref> A permissive chromatin environment is further important for origin activation and has been implicated in regulating both origin efficiency and the timing of origin firing. Euchromatic origins typically contain active chromatin marks, replicate early, and are more efficient than late-replicating, [[Heterochromatin|heterochromatic]] origins, which conversely are characterized by repressive marks.<ref name=":3" /><ref name="#28322723" /><ref name="#29357061">{{cite book | vauthors = Zhao PA, Rivera-Mulia JC, Gilbert DM | title = DNA Replication | chapter = Replication Domains: Genome Compartmentalization into Functional Replication Units | series = Advances in Experimental Medicine and Biology | volume = 1042 | pages = 229–257 | pmid = 29357061 | doi = 10.1007/978-981-10-6955-0_11 | year = 2017 | isbn = 978-981-10-6954-3 }}</ref> Not surprisingly, several [[Chromatin remodeling|chromatin remodelers]] and [[Chromatin remodeling|chromatin-modifying enzymes]] have been found to associate with origins and certain initiation factors,<ref name="#29357053">{{cite book | vauthors = Sugimoto N, Fujita M | title = DNA Replication | chapter = Molecular Mechanism for Chromatin Regulation During MCM Loading in Mammalian Cells | series = Advances in Experimental Medicine and Biology | volume = 1042 | pages = 61–78 | pmid = 29357053 | doi = 10.1007/978-981-10-6955-0_3 | year = 2017 | isbn = 978-981-10-6954-3 }}</ref><ref name="#23751185">{{cite journal | vauthors = MacAlpine DM, Almouzni G | title = Chromatin and DNA replication | journal = Cold Spring Harbor Perspectives in Biology | volume = 5 | issue = 8 | pages = a010207 | date = August 2013 | pmid = 23751185 | pmc = 3721285 | doi = 10.1101/cshperspect.a010207 }}</ref> but how their activities impact different replication initiation events remains largely obscure. Remarkably, cis-acting “early replication control elements” (ECREs) have recently also been identified to help regulate replication timing and to influence 3D genome architecture in mammalian cells.<ref name="#30595451">{{cite journal | vauthors = Sima J, Chakraborty A, Dileep V, Michalski M, Klein KN, Holcomb NP, Turner JL, Paulsen MT, Rivera-Mulia JC, Trevilla-Garcia C, Bartlett DA, Zhao PA, Washburn BK, Nora EP, Kraft K, Mundlos S, Bruneau BG, Ljungman M, Fraser P, Ay F, Gilbert DM | display-authors = 6 | title = Identifying cis Elements for Spatiotemporal Control of Mammalian DNA Replication | journal = Cell | volume = 176 | issue = 4 | pages = 816–830.e18 | date = February 2019 | pmid = 30595451 | pmc = 6546437 | doi = 10.1016/j.cell.2018.11.036 }}</ref> Understanding the molecular and biochemical mechanisms that orchestrate this complex interplay between 3D genome organization, local and higher-order chromatin structure, and replication initiation is an exciting topic for further studies.<ref name="Ekundayo et al"/>

Why have metazoan replication origins diverged from the DNA sequence-specific recognition paradigm that determines replication start sites in prokaryotes and budding yeast? Observations that metazoan origins often co-localize with promoter regions in ''Drosophila'' and mammalian cells and that replication-transcription conflicts due to collisions of the underlying molecular machineries can lead to DNA damage suggest that proper coordination of transcription and replication is important for maintaining genome stability.<ref name="#19996087" /><ref name="#23187890" /><ref name="#21750104" /><ref name="#21813623" /><ref name="#18838675">{{cite journal | vauthors = Cadoret JC, Meisch F, Hassan-Zadeh V, Luyten I, Guillet C, Duret L, Quesneville H, Prioleau MN | display-authors = 6 | title = Genome-wide studies highlight indirect links between human replication origins and gene regulation | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 105 | issue = 41 | pages = 15837–42 | date = October 2008 | pmid = 18838675 | pmc = 2572913 | doi = 10.1073/pnas.0805208105 | bibcode = 2008PNAS..10515837C | doi-access = free }}</ref><ref name="#8638128"/><ref name="#27362223">{{cite journal | vauthors = Sankar TS, Wastuwidyaningtyas BD, Dong Y, Lewis SA, Wang JD | title = The nature of mutations induced by replication–transcription collisions | journal = Nature | volume = 535 | issue = 7610 | pages = 178–81 | date = July 2016 | pmid = 27362223 | pmc = 4945378 | doi = 10.1038/nature18316 | bibcode = 2016Natur.535..178S }}</ref><ref name="#19560424">{{cite journal | vauthors = Azvolinsky A, Giresi PG, Lieb JD, Zakian VA | title = Highly transcribed RNA polymerase II genes are impediments to replication fork progression in Saccharomyces cerevisiae | journal = Molecular Cell | volume = 34 | issue = 6 | pages = 722–34 | date = June 2009 | pmid = 19560424 | doi = 10.1016/j.molcel.2009.05.022 | pmc = 2728070 }}</ref> Recent findings also point to a more direct role of transcription in influencing the location of origins, either by inhibiting Mcm2-7 loading or by repositioning of loaded Mcm2-7 on chromosomes.<ref name="#26656162">{{cite journal | vauthors = Gros J, Kumar C, Lynch G, Yadav T, Whitehouse I, Remus D | title = Post-licensing Specification of Eukaryotic Replication Origins by Facilitated Mcm2-7 Sliding along DNA | journal = Molecular Cell | volume = 60 | issue = 5 | pages = 797–807 | date = December 2015 | pmid = 26656162 | pmc = 4680849 | doi = 10.1016/j.molcel.2015.10.022 }}</ref><ref name="#30718387"/> Sequence-independent (but not necessarily random) initiator binding to DNA additionally allows for flexibility in specifying helicase loading sites and, together with transcriptional interference and the variability in activation efficiencies of licensed origins, likely determines origin location and contributes to the co-regulation of DNA replication and transcriptional programs during development and cell fate transitions. Computational modeling of initiation events in ''S. pombe'', as well as the identification of cell-type specific and developmentally-regulated origins in metazoans, are in agreement with this notion.<ref name="#21177973" /><ref name="#28112731" /><ref name="#21258320">{{cite journal | vauthors = Letessier A, Millot GA, Koundrioukoff S, Lachagès AM, Vogt N, Hansen RS, Malfoy B, Brison O, Debatisse M | display-authors = 6 | title = Cell-type-specific replication initiation programs set fragility of the FRA3B fragile site | journal = Nature | volume = 470 | issue = 7332 | pages = 120–3 | date = February 2011 | pmid = 21258320 | doi = 10.1038/nature09745 | bibcode = 2011Natur.470..120L | s2cid = 4302940 }}</ref><ref name="#27168766">{{cite journal | vauthors = Smith OK, Kim R, Fu H, Martin MM, Lin CM, Utani K, Zhang Y, Marks AB, Lalande M, Chamberlain S, Libbrecht MW, Bouhassira EE, Ryan MC, Noble WS, Aladjem MI | display-authors = 6 | title = Distinct epigenetic features of differentiation-regulated replication origins | journal = Epigenetics & Chromatin | volume = 9 | pages = 18 | pmid = 27168766 | pmc = 4862150 | doi = 10.1186/s13072-016-0067-3 | year = 2016 | doi-access = free }}</ref><ref name="#22090375">{{cite journal | vauthors = Sher N, Bell GW, Li S, Nordman J, Eng T, Eaton ML, Macalpine DM, Orr-Weaver TL | display-authors = 6 | title = Developmental control of gene copy number by repression of replication initiation and fork progression | journal = Genome Research | volume = 22 | issue = 1 | pages = 64–75 | date = January 2012 | pmid = 22090375 | pmc = 3246207 | doi = 10.1101/gr.126003.111 }}</ref><ref name="#25921534">{{cite journal | vauthors = Comoglio F, Schlumpf T, Schmid V, Rohs R, Beisel C, Paro R | title = High-resolution profiling of Drosophila replication start sites reveals a DNA shape and chromatin signature of metazoan origins | journal = Cell Reports | volume = 11 | issue = 5 | pages = 821–34 | date = May 2015 | pmid = 25921534 | pmc = 4562395 | doi = 10.1016/j.celrep.2015.03.070 }}</ref><ref name="#9499407">{{cite journal | vauthors = Calvi BR, Lilly MA, Spradling AC | title = Cell cycle control of chorion gene amplification | journal = Genes & Development | volume = 12 | issue = 5 | pages = 734–44 | date = March 1998 | pmid = 9499407 | pmc = 316579 | doi = 10.1101/gad.12.5.734 }}</ref><ref name="#30718387"/> However, a large degree of flexibility in origin choice also exists among different cells within a single population,<ref name="#21750104" /><ref name="#22751019" /><ref name="#27168766" /> albeit the molecular mechanisms that lead to the heterogeneity in origin usage remain ill-defined. Mapping origins in single cells in metazoan systems and correlating these initiation events with single-cell gene expression and chromatin status will be important to elucidate whether origin choice is purely stochastic or controlled in a defined manner.<ref name="Ekundayo et al"/>