Think global, act local - How to regulate S phase from individual replication origins

Current Opinion in Genetics and Development

Volumn 10, Issue 2, 2000, Pages 178-186

  Pasero, Philippe ^a   Schwob, Etienne ^a  

^a CNRS   (France)

Author keywords

[No Author keywords available]

Indexed keywords

CYCLIN DEPENDENT KINASE; DNA BINDING PROTEIN; INITIATION FACTOR;

CELL CYCLE S PHASE; CHROMOSOME; DNA REPLICATION ORIGIN; DROSOPHILA; EUKARYOTE; NONHUMAN; PRIORITY JOURNAL; REVIEW; YEAST;

ARACHNIDA; DROSOPHILA; EUKARYOTA; HEXAPODA; SCHIZOSACCHAROMYCES POMBE;

EID: 0034069075     PISSN: 0959437X     EISSN: None     Source Type: Journal    
DOI: 10.1016/S0959-437X(00)00067-8     Document Type: Review

References (81)

1. Donaldson A.D., Blow J.J. The regulation of replication origin activation. Curr Opin Genet Dev. 9:1999;62-68.
2. Tye B.K. MCM proteins in DNA replication. Annu Rev Biochem. 68:1999;649-686.
3. You Z., Komamura Y., Ishimi Y. Biochemical analysis of the intrinsic Mcm4-Mcm6-Mcm7 DNA helicase activity. Mol Cell Biol. 19:1999;8003-8015.
4. Kelman Z., Lee J.K., Hurwitz J. The single minichromosome maintenance protein of methanobacterium thermoautotrophicum DeltaH contains DNA helicase activity. Proc Natl Acad Sci USA. 96:1999;14783-14788.
5. ••], this report indicates that Orc4 binds strongly to DNA and mediates the interaction of S. pombe ORC with replication origins.
6. Chesnokov I., Gossen M., Remus D., Botchan M. Assembly of functionally active Drosophila origin recognition complex from recombinant proteins. Genes Dev. 13:1999;1289-1296. A report of the cloning of the genes encoding the six Drosophila ORC subunits and the reconstitution of a functional complex from recombinant proteins restoring initiation in ORC-depleted Drosophila egg extracts. Interestingly, this complex is able to partially complement initiation in ORC-depleted Xenopus egg extracts, indicating that ORC function is highly conserved among eukaryotes.
7. Springer J., Kneissl M., Putter V., Grummt F. Identification and characterization of MmORC4 and MmORC5, two subunits of the mouse origin of replication recognition complex. Chromosoma. 108:1999;243-249.
8. Feger G. Identification and complete cDNA sequence of the missing Drosophila MCMs: DmMCM3, DmMCM6 and DmMCM7. Gene. 227:1999;149-155.
9. Ogawa Y., Takahashi T., Masukata H. Association of fission yeast Orp1 and Mcm6 proteins with chromosomal replication origins. Mol Cell Biol. 19:1999;7228-7236. The first evidence that ORC binds origin DNA in vivo in an organism other than S. cerevisiae. Using a chromatin immunoprecipitation assay, Orp1 is shown to interact specifically with essential regions of S. pombe ars2004 and ars3002. The loading of Mcm6 on these origins is also reported. In contrast to Orp1, this interaction is cell-cycle regulated. Mcm6 loading on origins depends on ORC and Cdc18 functions, as previously reported for budding yeast and Xenopus.
10. 1 progression, provided that the nuclear envelope has been permeabilized to allow Cdc6 entry.
11. Perkins G., Diffley J.F. Nucleotide-dependent prereplicative complex assembly by Cdc6p, a homolog of eukaryotic and prokaryotic clamp-loaders. Mol Cell. 2:1998;23-32.
12. Wang B., Feng L., Hu Y., Huang S.H., Reynolds C.P., Wu L., Jong A.Y. The essential role of Saccharomyces cerevisiae CDC6 nucleotide-binding site in cell growth, DNA synthesis, and Orc1 association. J Biol Chem. 274:1999;8291-8298.
13. Weinreich M., Liang C., Stillman B. The cdc6p nucleotide-binding motif is required for loading mcm proteins onto chromatin. Proc Natl Acad Sci USA. 96:1999;441-446.
14. DeRyckere D., Smith C.L., Martin G.S. The role of nucleotide binding and hydrolysis in the function of the fission yeast cdc18(+) gene product. Genetics. 151:1999;1445-1457.
15. 1, they interfere with endogenous Cdc6 function, suggesting that both ATP binding and hydrolysis activities are required for initiation.
16. Sanchez M., Calzada A., Bueno A. Functionally homologous DNA replication genes in fission and budding yeast. J Cell Sci. 112:1999;2381-2390.
17. •], that Him1 is important for S phase and phosphorylated. Point mutations in the internal BRCT-like motif cause HU-sensitivity and cut phenotypes. Conserved S/T residues in this motif could be targets of the Cds1 kinase.
18. James S.W., Bullock K.A., Gygax S.E., Kraynack B.A., Matura R.A., MacLeod J.A., McNeal K.K., Prasauckas K.A., Scacheri P.C., Shenefiel H.L.et al. nimO, an Aspergillus gene related to budding yeast Dbf4, is required for DNA synthesis and mitotic checkpoint control. J Cell Sci. 112:1999;1313-1324. NimO, the Aspergillus homologue of Dbf4, is required for S-phase entry as well as for S completion after release from HU. As for some dbf4 alleles, temperature-sensitive nimO18 mutants undergo mitotic catastrophe without DNA replication. Interallelic complementation between distinct nimO mutants suggests that the Cdc7 kinase forms oligomers.
19. Landis G., Tower J. The Drosophila chiffon gene is required for chorion gene amplification, and is related to the yeast Dbf4 regulator of DNA replication and cell cycle. Development. 126:1999;4281-4293.
20. Faul T., Staib C., Nanda I., Schmid M., Grummt F. Identification and characterization of mouse homologue to yeast Cdc7 protein and chromosomal localization of the cognate mouse gene Cdc7l. Chromosoma. 108:1999;26-31.
21. Roberts B.T., Ying C.Y., Gautier J., Maller J.L. DNA replication in vertebrates requires a homolog of the Cdc7 protein kinase. Proc Natl Acad Sci USA. 96:1999;2800-2804. The authors show that anti-Cdc7 antibodies block DNA synthesis when added to Xenopus egg extracts or when micro-injected into one-cell embryos. The latter continue cell division, albeit at a reduced rate, and form a blastula apparently devoid of nuclear DNA. Cdc7 interacts with Mcm3 in interphase, but not in mitosis, consistent with a role of Cdc7 at origins.
22. 1 (after CycE and Cdc6) and remains high throughout S phase. ASK protein is phosphorylated and forms bright speckles in the nucleus. Cdc7 kinase activity is detected throughout S, depends on ASK and correlates with its levels. Micro-injection of anti-ASK antibodies in fibroblasts blocks or greatly delays BrdU incorporation, indicating that Cdc7 kinase is also needed for DNA replication in mammalian cells.
23. ••] but also show that Mcm2 is the preferential Cdc7 substrate within the six subunit MCM complex. Metabolic labeling of HeLa cells and comparison of in vivo and in vitro two-dimensional-phosphopeptide maps indicate that Mcm2 is multiply phosphorylated and a likely in vivo target of Cdc7.
24. Johnston L.H., Masai H., Sugino A. First the CDKs, now the DDKs. Trends Cell Biol. 9:1999;249-252.
25. Bielinsky A.K., Gerbi S.A. Chromosomal ARS1 has a single leading strand start site. Mol Cell. 3:1999;477-486. Detailed study of initiation at the chromosomal origin ARS1 using the recently developed Replication Initiation Point mapping assay and a yeast strain defective for DNA ligase I. Transition between continuous and discontinuous replication is restricted to a single site, located immediately upstream to the ORC binding site. Interestingly, initiation at the episomal ARS1 is less strictly positioned but is also more efficient, suggesting that differences in chromatin structure could affect the unwinding kinetics of ARS1.
26. Okuno Y., Satoh H., Sekiguchi M., Masukata H. Clustered adenine/thymine stretches are essential for function of a fission yeast replication origin. Mol Cell Biol. 19:1999;6699-6709. A functional analysis of ars2004, a highly active chromosomal origin on S. pombe chromosome II. Two regions containing stretches of poly(dA/dT) are shown to be essential for origin activity. Interestingly, these regions can be replaced with synthetic (A/T)40 sequences without affecting initiation. Together with the presence of replication enhancer elements, the number of these A/T stretches and the extent of their clustering seem to be major determinants of origin activity in S. pombe.
27. Kim S.M., Huberman J.A. Multiple orientation-dependent, synergistically interacting, similar domains in the ribosomal DNA replication origin of the fission yeast, Schizosaccharomyces pombe. Mol Cell Biol. 18:1998;7294-7303. A high-resolution deletion and linker-scanning analysis of ars3001, the rDNA ARS of S. pombe, is reported. This relatively small origin (570 bp), contains three essential domains with stretches of poly(dA/dT). These regions can partially substitute for each other, suggesting that similar proteins bind these domains synergistically.
28. Vernis L., Chasles M., Pasero P., Lepingle A., Gaillardin C., Fournier P. Short DNA fragments without sequence similarity are initiation sites for replication in the chromosome of the yeast Yarrowia lipolytica. Mol Biol Cell. 10:1999;757-769.
29. Aladjem M.I., Rodewald L.W., Kolman J.L., Wahl G.M. Genetic dissection of a mammalian replicator in the human beta-globin locus. Science. 281:1998;1005-1009.
30. DePamphilis M.L. Replication origins in metazoan chromosomes: fact or fiction? Bioessays. 21:1999;5-16.
31. 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. 96:1999;2656-2661. This paper reports the characterization of Orp4, the S. pombe homologue of Orc4. The amino-terminal part Orp4 contains nine AT-hook motifs, which are also found in HMG-I(Y) chromosomal proteins and mediate binding to poly(dA/dT) tracts. The isolated amino-terminal domain is able to interact with sequences in S. pombe ars1 and most likely targets the ORC complex to replication origins.
32. Pak D.T., Pflumm M., Chesnokov I., Huang D.W., Kellum R., Marr J., Romanowski P., Botchan M.R. Association of the origin recognition complex with heterochromatin and HP1 in higher eukaryotes. Cell. 91:1997;311-323.
33. Okuhara K., Ohta K., Seo H., Shioda M., Yamada T., Tanaka Y., Dohmae N., Seyama Y., Shibata T., Murofushi H. A DNA unwinding factor involved in DNA replication in cell-free extracts of Xenopus eggs. Curr Biol. 9:1999;341-350.
34. Austin R.J., Orr-Weaver T.L., Bell S.P. Drosophila ORC specifically binds to ACE3, an origin of DNA replication control element. Genes Dev. 13:1999;2639-2649. In vivo crosslinking and chromatin immunoprecipitation techniques are here used to show that Orc2 binds ACE3, the element controlling amplification at the chorion gene locus. Purified Drosophila ORC binds an 80 bp ACE3 sequence in vitro in an ATP-dependent manner, providing the first direct evidence for a sequence-specific DNA binding of ORC in metazoans.
35. Royzman I., Austin R.J., Bosco G., Bell S.P., Orr-Weaver T.L. ORC localization in Drosophila follicle cells and the effects of mutations in dE2F and dDP. Genes Dev. 13:1999;827-840. Amplification of specific regions in Drosophila follicle cells is preceded by a redistribution of Orc2 to amplifying loci, once genomic replication ceases. This relocalization is impaired in E2F DNA-binding mutants but occurs earlier in E2F mutants lacking the retinoblastoma protein binding domain. E2F may regulate Orc2 localization via one of its transcriptional targets. However, a E2F mutant predicted to lack transcriptional activity exhibits normal Orc2 localization, suggesting that E2F may target Orc2 to the amplifying loci directly.
36. Diffley J.F. Once and only once upon a time: specifying and regulating origins of DNA replication in eukaryotic cells. Genes Dev. 10:1996;2819-2830.
37. 1 when Cdc18 is recruited to origins.
38. 1 through the end of S phase and this fluctuation is regulated by E2F. Orp1 levels vary widely according to developmental stages. Overexpression of Orc1 alters replication patterns in different cell types, suggesting that Orc1 abundance could regulate origin activity.
39. Rowles A., Tada S., Blow J.J. Changes in association of the Xenopus origin recognition complex with chromatin on licensing of replication origins. J Cell Sci. 112:1999;2011-2018.
40. Françon P., Maiorano D., Méchali M. Initiation of DNA replication in eukaryotes: questioning the origin. FEBS Lett. 452:1999;87-91.
41. Duncker B.P., Pasero P., Braguglia D., Heun P., Weinreich M., Gasser S.M. Cyclin B-cdk1 kinase stimulates ORC- and Cdc6-independent steps of semiconservative plasmid replication in yeast nuclear extracts. Mol Cell Biol. 19:1999;1226-1241. Semiconservative replication of plasmid DNA in yeast nuclear extracts can be achieved in the absence of ORC, Cdc6 and Cdc7 activity but DNA synthesis is impaired in extracts presenting a low level of Cdk activity. These results suggest that, in addition to pre-RC components, Cdks modulate the efficiency of the replication machinery itself.
42. Sanchez M., Calzada A., Bueno A. The Cdc6 protein is ubiquitinated in vivo for proteolysis in Saccharomyces cerevisiae. J Biol Chem. 274:1999;9092-9097.
43. Elsasser S., Chi Y., Yang P., Campbell J.L. Phosphorylation controls timing of cdc6p destruction: a biochemical analysis. Mol Biol Cell. 10:1999;3263-3277.
44. Drury L.S., Perkins G., Diffley J.F.X. The Cdc4/34/53 pathway targets cdc6p for proteolysis in budding yeast. EMBO J. 16:1997;5966-5976.
45. 1 nuclei lacking Cdc6p. Thus Cln-Cdk1 may remove the excess of unbound Mcms, whereas Clb-Cdk1 excludes the chromatin-bound fraction after it is displaced by DNA replication. This additional level of regulation suggests that the 'window of opportunity' for pre-RC assembly is even shorter than initially proposed.
46. 2/M. Therefore, the exclusion of Mcms from the nucleus is only one of the multiple mechanisms preventing rereplication in S. cerevisiae.
47. Saha P., Chen J., Thome K.C., Lawlis S.J., Hou Z.H., Hendricks M., Parvin J.D., Dutta A. Human CDC6/Cdc18 associates with Orc1 and cyclin-cdk and is selectively eliminated from the nucleus at the onset of S phase. Mol Cell Biol. 18:1998;2758-2767.
48. Jiang W., Wells N.J., Hunter T. Multistep regulation of DNA replication by Cdk phosphorylation of HsCdc6. Proc Natl Acad Sci USA. 96:1999;6193-6198.
49. Petersen B.O., Lukas J., Sorensen C.S., Bartek J., Helin K. Phosphorylation of mammalian CDC6 by Cyclin A/CDK2 regulates its subcellular localization. EMBO J. 18:1999;396-410. Together with [47], this paper shows that phosphorylation of human Cdc6 by Cdks regulates its subnuclear localization. Phosphorylation of Cdc6 is not required for entry into S phase but it drives the relocalization of the protein from the nucleus to the cytosol. A mutant that mimics phosphorylated Cdc6 is always cytoplasmic whereas a non-phosphorylatable mutant is always nuclear. Overexpression of the latter is not sufficient to induce rereplication.
50. 1 and in M by different mechanisms. Dfp1 is autophosphorylated by Hsk1, and hyperphosphorylated during S phase and in HU. The latter depends on Cds1 (Rad53).
51. 1 independently of each other, but that Cdc7 can perform its function for DNA replication only after S-CDKs have been activated. This suggests that S-CDKs must modify the pre-RC, perhaps by loading Cdc45, before Cdc7 can act on it. Accordingly, Cdc45 is shown to be phosphorylated by Cdc7 in vitro.
52. 1 leads to Cdc7 activation, suggesting that Cdc7 kinase activity is regulated solely by Dbf4 availability and independent of CDKs.
53. Cheng L., Collyer T., Hardy C.F. Cell cycle regulation of DNA replication initiator factor Dbf4p. Mol Cell Biol. 19:1999;4270-4278.
54. 1/S.
55. Brown G.W., Kelly T.J. Purification of Hsk1, a minichromosome maintenance protein kinase from fission yeast. J Biol Chem. 273:1998;22083-22090.
56. ••], that Dbf4 binds and dissociates from chromatin later than Mcm2. Dbf4 is hyperphosphorylated and Cdc7 kinase less active in HU-treated cells. Dbf4 accumulates in αF-treated cdc16 and cdc23 cells, suggesting that it is targeted for degradation by the APC. Deletion of CDC7 in bob1 cells causes HU sensitivity but no loss of viability, suggesting that Cdc7 may be required for recovery from arrest. Purified Dbf4/Cdc7 phosphorylates Mcm2, 3, 4, 6 and 7 as well as DNA Pol α-primase (p180 subunit) in vitro.
57. 1 in a Orc2-dependent but Cdc6-independent manner and is released during S, later than Mcm2. Targeting Dbf4-Cdc7 at origins may restrict and provide local regulation of origin firing. Accordingly, Dbf4 is stabilized and displaced from chromatin in HU in a Rad53-dependent manner, providing a mechanism for the block of late-origin firing in these cells.
58. Santocanale C., Diffley J.F. A Mec1- and Rad53-dependent checkpoint controls late-firing origins of DNA replication. Nature. 395:1998;615-618.
59. Shirahige K., Hori Y., Shiraishi K., Yamashita M., Takahashi K., Obuse C., Tsurimoto T., Yoshikawa H. Regulation of DNA-replication origins during cell-cycle progression. Nature. 395:1998;618-621.
60. 1, suggesting that it interferes with pre-RC assembly.
61. 1. Activation of this checkpoint requires entry into S. Thus, primary lesions signal through Mec1/Rad53 independently of Rad9/Rad24, unless damage is processed by the NER pathway. Interestingly, cells arrest early in S not only due to inhibition of late origins but probably also because fork progression is impaired.
62. Larner J.M., Lee H., Little R.D., Dijkwel P.A., Schildkraut C.L., Hamlin J.L. Radiation down-regulates replication origin activity throughout the S phase in mammalian cells. Nucleic Acids Res. 27:1999;803-809. A two-dimensional gel analysis of early- and late-firing rDNA origins in irradiated human cells is used to show that initiation is downregulated at all origins which have not yet fired when radiation doses are delivered. This intra-S phase checkpoint is defective in cells from AT patients deficient for ATM (ataxia telangiectasia mutated), the homologue of S. cerevisiae MEC1.
63. Santocanale C., Sharma K., Diffley J.F. Activation of dormant origins of DNA replication in budding yeast. Genes Dev. 13:1999;2360-2364. The authors show that inactive ARS301 is a dormant but potentially active origin that is normally replicated passively in late S phase. (See main text for details.).
64. ••], this paper shows that more origins are formed than are actually used, and that origin usage is influenced both by the distance and activation time of adjacent origins.
65. Tanaka T., Nasmyth K. Association of RPA with chromosomal replication origins requires an mcm protein, and is regulated by rad53, and cyclin- and Dbf4-dependent kinases. EMBO J. 17:1998;5182-5191.
66. Dimitrova D.S., Todorov I.T., Melendy T., Gilbert D.M. Mcm2, but Not RPA, is a component of the mammalian early G1-phase prereplication complex. J Cell Biol. 146:1999;709-722. The recruitment of Mcm2 and RPA (replication protein A) to early- and late-replicating domains pre-labeled in vivo with pulses of halogenated nucleotides is reported here. Mcm2 is loaded at the same time on early and late replication foci and is rapidly displaced from active replication foci. In contrast, detergent-resistant RPA foci are only detected in S phase.
67. 1 cells, raising the possibility that differential recruitment of Cdc45 onto origins might determine their time of activation.
68. Raghuraman M.K., Brewer B.J., Fangman W.L. Cell cycle-dependent establishment of a late replication program. Science. 276:1997;806-809.
69. Ofir R., Wong A.C., McDermid H.E., Skorecki K.L., Selig S. Position effect of human telomeric repeats on replication timing. Proc Natl Acad Sci USA. 96:1999;11434-11439.
70. Smith Z.E., Higgs D.R. The pattern of replication at a human telomeric region (16p13.3): its relationship to chromosome structure and gene expression. Hum Mol Genet. 8:1999;1373-1386. The authors use the fluorescence in situ hybridization doublet method to assess replication timing along a 325 kb region encompassing the human 16p telomere. This region contains loci that are replicated in early, mid and late S phase. As in [69], a natural 142 kb deletion which now places the α-globin locus telomere-proximal delays its replication. Removal of the α-globin control region (HS-40) abolishes transcription but does not change replication timing. However, chromatin remains open after deletion of HS-40, suggesting that chromatin structure rather than transcription is likely to regulate replication timing by restricting or facilitating access to multiple, redundant origins.
71. Stevenson J.B., Gottschling D.E. Telomeric chromatin modulates replication timing near chromosome ends. Genes Dev. 13:1999;146-151. Density transfer experiments are used to measure replication time near yeast telomeres. Deletion of SIR3 disrupts telomeric silencing and causes subtelomeric DNA on the right arm of chromosome V to replicate earlier. However, Sir3 is not responsible for the late activation of Ori501 and Ori1412, which are further away from telomeres. (See main text for details.).
72. van Brabant A.J., Fangman W.L., Brewer B.J. Active role of a human genomic insert in replication of a yeast artificial chromosome. Mol Cell Biol. 19:1999;4231-4240. This paper describes the replication pattern of a YAC (yeast artificial chromosome) containing 240 kb human DNA. Eight replication origins that surprisingly display the same characteristics than yeast origins in terms of ARS consensus, spacing as well as variable timing and firing efficiencies, are mapped. This suggests that human DNA contains fortuitous sequences that can serve as true origins in yeast. The presence of such sequences will favorably influence YAC stability.
73. Sasaki T., Sawado T., Yamaguchi M., Shinomiya T. Specification of regions of DNA replication initiation during embryogenesis in the 65-kilobase DNApolα-dE2F locus of Drosophila melanogaster. Mol Cell Biol. 19:1999;547-555. The authors compare origin usage within a 65 kb region between the DNA Polα and dE2F genes at different stages of Drosophila embryogenesis. Every fragment shows origin activity and passive replication during the rapid syncitial divisions, consistent with the presence of numerous origins and random initiation at this stage. In 5-hr embryos, when cell cycles lengthen and become asynchronous, origin activity is restricted to four regions. In differentiated cultured cells, only two of these four regions (oriDα and oriDβ) still function as origins. The presence of two oris upstream of dE2F in 5-hr embryos correlates with the existence of two E2F transcripts at this stage. Thus, origin specification occurs gradually during development and differentiation, and may be influenced by nearby transcription.
74. Ehrenhofer-Murray A.E., Kamakaka R.T., Rine J. A role for the replication proteins PCNA, RF-C, polymerase epsilon and cdc45 in transcriptional silencing in Saccharomyces cerevisiae. Genetics. 153:1999;1171-1182.
75. Smith J.S., Caputo E., Boeke J.D. A genetic screen for ribosomal DNA silencing defects identifies multiple DNA replication and chromatin-modulating factors. Mol Cell Biol. 19:1999;3184-3197.
76. ••], how the pattern of DNA replication can influence the developmental fate of sister cells.
77. Shibahara K., Stillman B. Replication-dependent marking of DNA by PCNA facilitates CAF-1-coupled inheritance of chromatin. Cell. 96:1999;575-585. CAF-1 is a conserved heterotrimeric complex that assembles chromatin on newly replicated DNA. Chromatin assembly can be uncoupled from DNA replication, suggesting the presence of a stable mark on replicated DNA. This paper shows that PCNA (proliferating cell nuclear antigen A), a DNA polymerase clamp, marks DNA and that RF-C has the ability to unload PCNA in an ATP-dependent manner. The asymmetric distribution of PCNA on leading and lagging strands may serve to
78. Simon I., Tenzen T., Reubinoff B.E., Hillman D., McCarrey J.R., Cedar H. Asynchronous replication of imprinted genes is established in the gametes and maintained during development. Nature. 401:1999;929-932. Paternal and maternal alleles of imprinted genes (Snrpn, Igf2 and Igf2r) replicate asynchronously. A differential replication pattern is already present in primordial germ cells, erased before meiosis, and then reset in a parent-specific fashion in late gametogenesis. The early establishment and persistence of asynchronous replication throughout development suggests that it may serve as a primary epigenetic marker for distinguishing between parental alleles.
79. Berezney R., Dubey D.D., Huberman J.A. Heterogeneity of eukaryotic replicons, replicon clusters and replication foci. Chromosoma. 2000;. in press.
80. Gomez M., Antequera F. Organization of DNA replication origins in the fission yeast genome. EMBO J. 18:1999;5683-5690. The identification of several new S. pombe replication origins is reported. These origins localize preferentially in the vicinity of promoters but transcription is not required for origin function. A detailed analysis of ars1 by RIP assay shows that replication initiates at a discrete site, as it is the case for S. cerevisiae ARS1.
81. Lucas I., Chevrier-Miller M., Sogo J.M., Hyrien O. Mechanisms ensuring rapid and complete DNA replication despite random initiation in Xenopus early embryos. J Mol Biol. 296:2000;769-786.