{"id":443,"date":"2014-03-16T23:07:09","date_gmt":"2014-03-16T14:07:09","guid":{"rendered":"http:\/\/localhost:8888\/wordpress\/essey-2"},"modified":"2014-03-24T15:43:29","modified_gmt":"2014-03-24T06:43:29","slug":"essey","status":"publish","type":"page","link":"https:\/\/www.igakuken.or.jp\/genome\/?page_id=443","title":{"rendered":"Essey"},"content":{"rendered":"<div id=\"toc-np-container\"><p id=\"toc-np-title\">Contents<\/p><ul class=\"no-bullets\"><li class=\"header-level-1\"><a href=\"https:\/\/www.igakuken.or.jp\/genome\/?page_id=443#A-personal-reflection-on-the-Replicon-Theory-50\">\u201cA personal reflection on the Replicon Theory\" \u30ec\u30d7\u30ea\u30b3\u30f3\u30e2\u30c7\u30eb\u3068\u3068\u3082\u306b50\u5e74\u9593<\/a><\/li><li class=\"header-level-1\"><a href=\"?p=443&page=2#Replication-timing-regulation-of-eukaryotic-repliconsRif1-as-a-global-regulator-of-replication-timing\">Replication timing regulation of eukaryotic replicons:Rif1 as a global regulator of replication timing<\/a><\/li><\/ul><\/div><h2>\u201cA personal reflection on the Replicon Theory&#8221;<span class=\"GINGER_SOFTWARE_mark\" id=\"8cbcefc3-511d-4f5b-80ca-a7d1c4bafda1\"> <br \/><\/span>\u30ec\u30d7\u30ea\u30b3\u30f3\u30e2\u30c7\u30eb\u3068\u3068\u3082\u306b50\u5e74\u9593<\/h2>\n<p>J. Mol. Biol.\u00a0<br \/>(to be published)<br \/>Perspectives<\/p>\n<p>Journal of Molecular Biology\u00a0<br \/>A special issue on the Replicon<br \/>Symposium celebrating the 50th anniversary of the Replicon theory<\/p>\n<p>Hisao Masai<br \/>Department of Genome Medicine, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamkitazawa, Setagaya-ku, Tokyo 156-8506<br \/>Japan<\/p>\n<p>*Correspondence: masai-hs@igakuken.or.jp<\/p>\n<p>Summary<\/p>\n<p>Fifty years after the Replicon theory was originally presented, detailed mechanistic insight into prokaryotic replicons has been obtained and rapid progress is being made to elucidate the more complex regulatory mechanisms of replicon regulation in eukaryotic cells. Here I present my personal perspectives on how studies of model replicons have contributed to our understanding of the basic mechanisms of DNA replication as well as the evolution of replication regulation in human cells. I will also discuss how replication regulation contributes to the stable maintenance of the genome and how disruption of replication regulation leads to human diseases.<\/p>\n<p>(Key words: Replicon Theory, R1 plasmid, Replication origin, Initiator, Replication timing)<\/p>\n<p>The beauty of the Replicon Theory is its amazing simplicity. Only two loci, SGI (structural gene for initiator) and replicator, were hypothesized as critical elements for DNA replication and it was proposed that the initiator positively regulates initiation at the replicator (1). Another important aspect of the Replicon Theory was the proposal that membranes play crucial roles in regulation of initiation of DNA replication. The latter issue, which has not yet been completely resolved, will not be dealt with in this article but will be briefly touched on later. Discovery of oriC and dnaA in the following years (2, 3) upheld the model as an accurate description of replication in bacteria, although biochemical evidence had to wait for another decade or more (4). Although it was evident from earlier studies that eukaryotic chromosomes contain multiple replicators, the nature of the initiator remained elusive until Orc was discovered in yeast (5). Now, the basic concept of the Replicon Theory has proven to apply for both prokaryotic and eukaryotic replicons (6).<\/p>\n<p>As in Arthur Kornberg\u2019s famous 10 commandments (VI. DEPEND ON VIRUSES TO OPEN WINDOWS) (7), viruses played crucial roles in elucidating mechanisms of DNA replication. Resolution and reconstitution studies saw first triumph when replication of small single-stranded virus DNAs was reconstituted with purified enzymes (8). This approach was quickly adopted for studies of animal virus replication, which provided important insight into the DNA chain elongation stages of eukaryotic DNA replication (9).\u00a0<br \/>The enzymatic studies on initiation at the chromosome replicator had to wait for development of a soluble enzyme system, which was made possible only after careful fractionation of crude extracts and inclusion of a polymer (polyethylene glycol or polyvinyl alcohol), which presumably caused a molecular crowding effect (10). Once a system was available, the rest followed rapidly. In only a few years, initiation at oriC was reconstituted with purified proteins (11), and the nature of dnaA (initiator)-oriC (replicator) interaction was resolved (4,12).<\/p>\n<p>R1 plasmid as a model for regulation of DNA replication<br \/>The presence of the bacterial chromosome replicator, oriC, was suggested in the 1960\u2019s by marker transfer experiments in B. subtilis and identified genetically in Escherichia coli in the early 70\u2019s (3,13). However, the molecular details were unknown until it was cloned as an autonomously replicating sequence supporting the episomal maintenance of the linked antibiotic resistance gene (14). oriC was also isolated as a novel F\u2019 DNA segment capable of replicating on Hfr or as a DNA segment capable of bypassing lambda immunity (15-17).\u00a0<br \/>Plasmids are generally circular double-stranded DNA stably maintained in the cytoplasm of bacterial cells independent of the host chromosome. Plasmids can confer drug resistance or fertility to host cells, but are normally non-essential for host maintenance. It has become obvious that each plasmid is an independent replicon composed of its own initiator and replicator. Since replication of most plasmid DNA depends almost entirely on the host replication machinery, plasmids have served as excellent model systems for host chromosome replication (18).<br \/>The R1 plasmid, belonging to the IncFII incompatibility group, encodes multiple drug resistance genes and is strictly maintained at one to two copies per host cell (19). The minimum segment required for replication contains a gene called RepA, which was likely to be an initiator for R1 plasmid replication (20). We first used an in vitro replication system that depends on de novo protein synthesis (21). The replicator, oriR, was mapped using the in vitro trans-complementation assay (22). The purified RepA protein can bind specifically to the sequences within oriR, and induces partial melting (or structural change) of duplex DNA at the AT-rich segment within oriR (23). Similar studies were conducted at the replicators of various plasmid replicons as well as at oriC, leading to the unified view on the prokaryotic mode of initiator-replicator interaction and function (24; Fig. 1).<br \/>During the course of our study, it became evident to us that RepA protein needs to be synthesized each time before initiation. Furthermore, it can activate only the origin present on the same template from which it was synthesized (22). It is inactivated each time after it triggers initiation at the replicator. Further analyses showed the requirement of the CIS segment downstream of the repA gene carrying a rho-dependent transcriptional terminator for the cis-action (25). The RepA trapping model was proposed in which the nascent RepA protein, synthesized on the paused mRNA, is first trapped at the entry sites present at the C-terminal coding segment of repA, followed by its one-dimensional diffusion to locate oriR (25, 26). After RepA fires initiation at oriR, it is inactivated or made unavailable for reuse by some unknown mechanism. The cis-action of the RepA protein is the central system to ensure once and only once replication of the R1 plasmid, reminiscent of the origin licensing system in eukaryotes (27) (Table 1). Multiple mechanisms have been shown to operate to inhibit re-replication at oriC (28) as well as that of eukaryotic chromosomes (29), pointing to the critical importance of the system to ensure \u201conly once\u201d replication in all organisms.<br \/>Although my comments were focused on plasmid R1, I should mention that studies of replication on other plasmids (ColE1, R6K, \uf06cdv, F, pSC101, RK2 and so forth) played very important and pioneering roles in dissecting the structure of replicators, initiator-replicator interactions, events during initiation and molecular mechanisms of regulation (30-35).\u00a0<\/p>\n<p>Replicator-initiator interactions in prokaryotes and eukaryotes<br \/>As discussed in the previous section, prokaryotic replicator-initiator interactions are highly replicon-specific, meaning each initiator will only interact with its appropriate replicator. In addition, the replicator-initiator interaction is sequence-specific. Generally, one base substitution abrogates the interaction, but once the initiator binds to the replicator and an initiation complex is assembled, initiation efficiency is almost 100%. In contrast, recognition of eukaryotic replicators by the eukaryotic initiator, Orc, appears to be much less specific. Although Orc in lower eukaryotes (e.g. budding yeast) was reported to bind to the cognate replicator in a sequence-specific manner (5), Orc from human binds to DNA in a sequence-independent manner in vitro (36). Whether Orc binds to chromosomes with some preference is still under debate (37). Genome-wide mapping of Orc and Mcm would provide some insight into this subject (37). Further contrasting prokaryotic replicons, efficiency of firing at each replicator is fairly low in eukaryotes, perhaps even occurring in a stochastic manner to a certain extent (39-41; Fig. 1).<br \/>DnaA protein can induce localized melting (or structural changes detectable with single-stranded DNA-specific probes) within the oriC (42), and other prokaryotic initiators can exert similar effects within the cognate replicators. In archaea, whose replication machinery shares more similarity with that of eukaryotes than the prokaryotic counterpart, Orc binds specifically to the repeat sequences present in the origins and induce distortion of duplex DNA which leads to localized unwinding at DUE (DNA Unwinding Element; 43-45). However, similar effects have not been detected with Orc or pre-RC bound at eukaryotic origins, although RPA binding can be detected which indirectly shows the generation of single-stranded DNA (46). Mcm has been shown to bind to origin as a double-hexamer encircling the duplex DNA (47,48), which is converted to two CMG helicase complexes (49) that encircle the single-stranded leading-strand template DNAs at each replication fork (50). A recently developed in vitro replication system dependent on a pre-RC generated at a specific origin sequence (51) holds great promise to clarify the structural change of template DNA during initiation steps.<\/p>\n<p>Regulation of origin selection and timing of the firing<br \/>In eukaryotes, DNA replication starts at multiple sites along the chromosomes. The numbers of the replicators (defined as the locations of pre-RC assembly) exceed those of actual initiation events occurring within S phase. Many replicators are dormant during normal S phase, and may be used only in an emergency when normally used replicators are unavailable (52,53). Even the active replicators are used only in a fraction of cells, a fraction of time, reflecting the inefficiency of firing (40,54). Replicator choice and timing of their firing are coordinated with S phase progression and appear to be predetermined in the population of cells (Fig. 1). How do cells determine the program of DNA replication?\u00a0<br \/>Replication stress activates checkpoint that suppresses the firing of late origins and\/ or slows down ongoing replication fork through the actions of checkpoint sensor and effector kinases. Studies in yeasts showed that suppression of the replication checkpoint (by mutation in Mec1 or Rad53 kinase) leads to the precocious firing of otherwise late-firing or dormant replicators (55,56). This could be due to loss of inhibitory signals that suppress the firing of these inactive origins. Some of the targets of this checkpoint may be Sld3 and Dbf4, which are crucial for conversion of the pre-RC into an active helicase complex at the fork (57,58). It was also proposed that the more efficient recruitment of pre-RC components may facilitate earlier firing of replicators (52). It was reported in yeasts that replicators compete for limiting replication factors including Cdc7-Dbf4 kinase and Sld3 and overexpression of these factors promotes firing of late\/dormant replicators in early S phase (59,60).\u00a0<br \/>Other classes of proteins that affect the replication program are modifiers of chromatin including histone deacetylase and methylase. Among them, the Rpd3 histone deacetylase affects the timing of origin firing genome-wide, since rpd3\u2206 mutation advances the firing of many late\/dormant replicators (61). This suggests that chromatin structure affects replication initiation. HBO1 histone acetylase facilitates pre-RC formation through interaction with Cdt1 (62), thus indirectly affecting firing efficiency. Recently, Forkhead transcription factors, Fkh1 and Fkh2, were shown to confer early, efficient firing of sets of origins by inducing clustering of the origins through its interaction with Orc in budding yeast. The origin clustering would increase the effective concentrations of initiation factors around origins, facilitating initiation (63). This appears to be a novel mechanism for marking early-firing origins.<br \/>Studies in yeast have shown that some sequences associated with late-firing origins can enforce late replication on otherwise early-firing origins (64). Further dissection showed that telomere-like repeats are responsible for this suppression. Indeed, deletion of Taz1, a telomere binding factor, caused the subtelomere regions as well as some arm segments to replicate early, presumably by binding to the telomere-like sequences (65).\u00a0<br \/>Cdc7 kinase plays a crucial role for initiation of DNA replication at each replicator by phosphorylating the Mcm subunit of the pre-RC (66,67). Although essential for normal growth, Cdc7 function could be bypassed in budding yeast by mutations in Mcm; they are mcm5 P83L mutation (bob1) (68,69) and the internal deletion at the N-terminal segment of mcm4 (70). These presumably change the conformation of Mcm complex, relieving the requirement of Cdc7 for initiation. Although similar mcm mutants that could bypass Cdc7 in other species have not been reported, we found checkpoint mutations including cds1 and mrc1 that could restore the growth of hsk1\u2206, the fission yeast homologue of Cdc7 (71,72). Screening of bypass mutants for hsk1\u2206 lead to isolation of rif1\u2206, which very efficiently rescued growth of hsk1\u2206 cells (73). Rif1, originally identified as a telomere binding protein in budding yeast, binds to telomere\/subtelomere regions in fission yeast and suppresses the early replication of these segments. We found that the replication timing of arm segments is also dramatically affected in rif1\u2206 cells. Importantly, not only late\/dormant origins were prematurely activated, but also early-firing origins were suppressed in rif1\u2206, causing the firing of most origins to shift toward mid-S (73).<br \/>In higher eukaryotes, it has been known that clusters of replicators are fired at similar timing during S phase (74,75; Fig. 1). Recent genome-wide studies revealed the presence of \u201creplication-timing domains\u201d which are replicated at specific timing of S phase (76,77). Earlier studies have shown that replication timing of metazoan chromosomes is determined at a timepoint in early G1 phase termed TDP (Timing Decision Point) (78), which was shown to be associated with chromatin repositioning. We and others found that mammalian Rif1 protein plays a major role in setting up the replication-timing domains (79,80). It plays an essential role in generating the mid-S replication domains by binding to nuclease-resistant nuclear structures at late M\/early G1 and presumably facilitating the formation of chromatin loop structures (Rif1 loop; see graphic abstract; 79). The origins present in these mid-S replication domains are somehow sequestered from firing until mid-S phase (Fig. 1 &amp; graphic abstract). Rif1 forms nuclear foci, enriched at the nuclear membrane and the nucleolar peripheries, coinciding with the locations of mid-S replication foci (81). Rif1 may facilitate association of chromatin with the nuclear membrane to enforce mid-to-late replication. In fact, budding yeast Rif1 protein localizes to the nuclear periphery dependent on a palmitoyltransferease (82), suggesting that Rif1 may be anchored to nuclear membrane through lipid modification. Thus, critical roles of membranes in regulation of DNA replication could be conserved from prokaryotes to eukaryotes.<br \/>It is interesting to note that two critical events for DNA replication in S phase, namely the origin licensing (helicase loading) and mid-S replication domain licensing (generation of special replication domains for mid-S) independently occur at late M\/early G1 (see graphic abstract).<\/p>\n<p>\u201cStrict\u201d and \u201csoft\u201d regulations of eukaryotic DNA replication<br \/>As discussed above, the inhibition of re-replication is strictly enforced in every species and aberrancy in this regulation can lead to increased genome instability and ultimately to various diseases, most notably cancer\/tumor formation (83-85). In contrast to this strict regulation, once cells enter S phase, regulation at the level of active origin selection or firing time appears to be rather flexible, representing the \u201csoft\u201d regulation of eukaryotic DNA replication. Indeed, origin firing patterns can be simulated by a stochastic firing model if one accepts two assumptions; the difference in firing probability between replicators and the increase in firing probability during S phase (86). The firing probability of each replicator may be affected by many genetic, epigenetic and physiological parameters.<br \/>Although replication-timing regulation is largely lost in rif1\u2206 fission yeast cells, S phase progression is almost normal and the mutant is resistant to various genotoxic stresses including HU and MMS (73). Although Chk1 is slightly activated (79,80), Rif1 depletion in HeLa cells similarly does not cause much effect on S phase progression in spite of major structural changes to replication-timing domains. These observations suggest that cells are quite tolerant to perturbation of replication timing. They are programmed to complete the S phase once it initiates the process of DNA replication, since obviously the abortion of DNA replication in the middle of the process would be devastating to cells\u2019 fate. hsk1\u2206 cells are not viable at 30\u00b0C or below, but is viable at 37\u00b0C, at which some dormant origins are fired. These results indicate that the origin firing program is plastic and could change under various physiological conditions (72).\u00a0<\/p>\n<p>Replication fork arrest and maintenance of genome stability<br \/>Replication fork progression can be disturbed by many internal or external causes including reduced level of nucleotide supply, proteins bound to the template, unusual DNA structures, agents influencing the replication machinery, and DNA damage etc. Stalled replication forks need to be detected and protected to avoid disintegration of the fork structure that could lead to DNA lesions and eventually to unwanted rearrangement or mutagenesis. In prokaryotes, there are only two replication forks on a single replicating chromosome, and stalling of one fork would leave a large unreplicated segment. Thus, in the event of replication fork stalling, restoration of the active fork is essential.<br \/>Resolution and reconstitution studies on \uf066X174 phage DNA in the 1970\u2019s lead to the discovery of a set of genetically unidentified proteins (87). Proteins i and n\u2019 were among them, and their identities had not been discovered until the genes for these proteins were cloned. Protein i is encoded by dnaT (88) and n\u2019 was a novel gene designated priA (89,90). The physiological role of \uf066X174-type primosome, a protein complex capable of duplex unwinding and primer RNA synthesis for lagging strand) was unclear but identification of protein i as DnaT (essential for inducible stable DNA replication, an oriC- and dnaA-independent mode of DNA replication of the E. coli chromosome induced by DNA damages) suggested a possibility that it plays a role in the alternative mode of chromosome replication. Indeed, PriA is also essential for the oriC-independent mode of chromosome replication (91). However, severe growth defects of priA\u2206 cells suggested essential roles of PriA during normal course of replication (92-94). Purified PriA protein bound to D-loop like structures or arrested fork-like structures in vitro (95). priA\u2206 cells are extremely sensitive to fork stalling agents, and thus it was proposed that PriA recognizes the stalled replication fork and promotes assembly of \u201cprimosome\u201d for fork restart (94,96). In fact, the fork restart process has been reconstituted with purified proteins (97).<br \/>Detailed analyses of how PriA interacts with the stalled fork revealed the presence of a pocket structure (TT-pocket; Three-prime Terminus recognition pocket) that accommodates the 3\u2019-terminus of DNA (98,99). PriA specifically recognizes the 3\u2019-terminus of the nascent leading strand at the stalled replication fork, stabilizes it and triggers reassembly of the active replication fork (100).\u00a0<br \/>On eukaryotic chromosomes, chances of fork stall are larger due to the presence of multiple replication forks at any given time during S phase. Therefore, the stalled fork needs to be swiftly dealt with to prevent fork collapse or aberrant fork structures (101), which would be most detrimental to the cell survival and maintenance of genome stability. Thus, eukaryotic replication machinery is equipped with many auxiliary factors that facilitate the stabilization and protection of the fork in cases where fork progression is blocked (102). These factors not only protect the fork but also send out signals to promote any necessary cell cycle arrest or DNA damage repair. The fork could be restarted at the stalled site, but restart may not be as crucial as in prokaryotes since nearby dormant origins can be activated to finish any unreplicated segments (103).\u00a0<br \/>In carcinogenesis, initial oncogenic stress may cause reduction in the level of cellular nucleotide precursors, leading to the blockage of replication fork progression (104-106). This can be detected by the appearance of DNA damage foci. Cells in this precancerous state may be converted to a malignant state if the DNA damage response system fails and large-scale genomic instability is induced (105). These reports underpin the importance of the cellular response to stalled replication forks. Indeed, mutations in various checkpoint\/ replication\/ repair factors have been reported to cause chromosome instability syndrome most of which are cancer-predisposed (107,108).\u00a0<\/p>\n<p>Concluding remarks<br \/>It is amazing that one simple theory provided guidance for the research that followed it for 50 years, which have now proven that the basic concept of the theory is correct from E. coli to humans. The structures of bacterial replicators have long been dissected (109), and those of lower eukaryotes have been analyzed in depth (110). Until now, however, the structures of mammalian replicators have been rather elusive (111), due to the lack of a consensus sequence, and to the lack of a convenient system to assay origin function. Recent technological advances for mapping replication origins or initiator binding sites genome-wide has made it possible to catalogue all the potential replicators and deduce some common structures (38,112,113 see the article by Mechali et al. in this issue). It was indeed suggested that replicators may be generated at any nucleosome-free intergenic segment to which Orc has high affinity (114). Future studies will uncover the nature of the determinants for the replicators in higher eukaryotes.<br \/>Initiators have been identified in both prokaryotes and eukaryotes. The site-specific binding of DnaA and Orc determines where replication is initiated and their functions are regulated by bound nucleotides (5, 115). High specificity of prokaryotic replicator-initiator interactions is associated with highly efficient initiation, while the specificity generally decreases with the increase of genome complexity and this probably contributes to more plastic and adaptive regulation of initiation events in higher eukaryotes. The attempt to determine the genome-wide distribution of Orc binding sites could provide important clues as to the determinants for recognition by Orc in mammalian cells (38,116).<br \/>Inhibition of re-replication appears to be of central importance for regulation of DNA replication in both prokaryotes and eukaryotes. Multiple mechanisms operate to ensure that re-replication is avoided (29). In contrast, origin selection or timing determination in eukaryotes appears to be under more plastic regulation (6). The firing may occur stochastically, with firing probability of each origin being affected by genetic\/ epigenetic environment or physiological conditions. This sort of plasticity may permit complex cellular systems to respond to a varied physiological conditions in a robust and adaptive manner and to complete S phase once committed.<br \/>Genomic DNA is very vulnerable during the course of DNA replication, since chromatin proteins are removed and duplex DNA becomes single-strand. Stalled replication forks can turn into catastrophic DNA lesions if not corrected immediately. Initial oncogenic stress was reported to reduce the level of nucleotide supply, causing replication stress reactions (104,105).\u00a0<br \/>Stalled replication forks are quickly recognized in both prokaryotes and eukaryotes. Fork restart by PriA is crucial in prokaryotes (94), whereas stabilization and protection of stalled forks by replication fork auxiliary factors may be important in eukaryotes to prevent fork collapse or fork inactivation.\u00a0<br \/>The role of membranes in replication and partition remains elusive even after 50 years. However, recent studies suggest the importance of nuclear membranes for regulation of chromosome dynamics including replication, repair and transcription (117, 118). After all these years, we are still being guided by the great Replicon Theory.<\/p>\n<p>Acknowledgments<br \/>I would like to deeply thank Dr. Ken-ichi Arai for leading me to the field of DNA replication 32 years ago and his continuous guidance and support through all the past years. I would also like to thank all my collaborators in and out of my laboratory who contributed to the story I described here. Finally, I would like to dedicate this article to my late mother, Yaeko Masai (passed away on October 23, 2012), who did so much for me and for whom I did so little.<\/p>\n<p>References<\/p>\n<p>1. Jacob, F., Brenner, C. &amp; Cuzin, D. (1963) On the regulation of DNA replication in bacteria. Cold Spring Harbor Quant. Biol. 28, 329-348.<\/p>\n<p>2. Kohiyama, M., Cousin, D., Ryter, A. &amp; Jacob, F. 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However, we would like to emphasize the conceptual similarity between the two regulatory systems.)<\/p>\n<p>Legend to Figure<\/p>\n<p>Figure 1 Initiation at the replicators of various organisms<br \/>(Upper left) In prokaryotes, replicators (origin) are composed of the initiator binding sequence and the adjacent AT-rich sequences that are melted upon initiator binding. In most cases, there is only one replicator on each genome, and efficiency of initiation is nearly 100%. (Lower center) In lower eukaryotes (e.g. yeast), origins fire at different timing. Firing at each origin can be stochastic. The efficiency of firing at each origin is low and which origin is fired may vary from one cell to another. (Upper right) In higher eukaryotes with longer chromosomes, the neighboring origins (shown by the same colors) are activated at a similar timing, forming replication timing domains. Mid-S replication domains (containing blue origins) are sequestered from activation in early S, and cannot be fired until mid-S. This temporal inhibition is mediated by the Rif1 protein. Late-S replication domain (containing yellow origins; mainly composed of heterochromatin region) may be similarly sequestered until late-S, but what mediates the suppression is not known. The efficiency of firing of each origin is low and which origins in the same replication domain are activated may be under stochastic regulation.<\/p>\n<p>Figure 1<\/p>\n<p><a href=\"http:\/\/localhost:8888\/wordpress\/wp-content\/uploads\/2014\/03\/JCFigure1.jpg\"><img decoding=\"async\" loading=\"lazy\" class=\"alignnone  wp-image-453\" alt=\"JCFigure1\" src=\"http:\/\/localhost:8888\/wordpress\/wp-content\/uploads\/2014\/03\/JCFigure1-300x225.jpg\" width=\"400\" height=\"225\" \/><\/a><\/p>\n<p><a href=\"http:\/\/localhost:8888\/wordpress\/wp-content\/uploads\/2014\/03\/JCTable1.jpg\"><img decoding=\"async\" loading=\"lazy\" class=\"alignnone  wp-image-454\" alt=\"JCTable1\" src=\"http:\/\/localhost:8888\/wordpress\/wp-content\/uploads\/2014\/03\/JCTable1-300x225.jpg\" width=\"400\" height=\"225\" \/><!--nextpage--><\/a><\/p>\n<h2><span id=\"Replication-timing-regulation-of-eukaryotic-repliconsRif1-as-a-global-regulator-of-replication-timing\"><span style=\"font-family: '\uff2d\uff33 \uff30\u30b4\u30b7\u30c3\u30af';\">Replication timing regulation of eukaryotic replicons:Rif1 as a global regulator of replication timing<\/span><\/span><\/h2>\n<p><span style=\"font-family: '\uff2d\uff33 \uff30\u30b4\u30b7\u30c3\u30af'; font-size: small;\"><span style=\"font-family: '\uff2d\uff33 \uff30\u30b4\u30b7\u30c3\u30af';\">DNA\u8907\u88fd\u30bf\u30a4\u30df\u30f3\u30b0\u5236\u5fa1\u306e\u5206\u5b50\u6a5f\u69cb<\/span><br \/><\/span><br \/><span style=\"font-family: \u30e1\u30a4\u30ea\u30aa;\">Abstract<\/p>\n<p>Origins of DNA replication on eukaryotic genomes have been observed to fire during S phase in a coordinated manner. In higher eukaryotes, they are clustered into sets of neighboring replication origins that fire at specific times within the S phase. Studies in yeast indicate that origin firing is affected by a number of factors, including checkpoint regulators and chromatin modifiers. However, it is unclear what the mechanisms orchestrating this coordinated process are. Recent studies have identified factors that regulate the timing of origin activation. Among them is Rif1, which plays crucial roles in the regulation of the origin firing program in fission yeast as well as in higher eukaryotes. In mammalian cells, Rif1 appears to regulate the structures of replication timing domains, each of which may replicate at defined timings during S phase, through its ability to organize the chromatin loop structures. Replication timing regulation would be associated with local chromatin structures as well as with more global chromatin architecture, and thus may be linked to regulation of other chromosome transactions including recombination, repair or transcription. This review summarizes recent progress in the effort to elucidate the mechanism of replication timing regulation of eukaryotic replicons.<\/p>\n<p>\u2003<br \/>Origins of DNA replication<br \/>Marking and activation-DNA replication is initiated at defined loci known as replication origins. In prokaryotic replicons, replication is initiated from a single locus in most cases, and the sequence specificity of origin activation is very high; generally one base substitution within origin leads to loss of initiation [1]. By contrast, replication initiates at multiple loci on eukaryotic genomes [2]. Although initiation occurs within specific loci on each genome at specific times during S phase, the sequence specificity can be significantly relaxed compared to the bacterial systems. It appears that cells prepare many potential origins for possible uses during S phase, but only a subset are utilized during the normal course of S phase. Other origins may be used at later stages of S phase or may not be used at all (dormant origins).\u00a0<br \/>It is well established now that preparation for DNA replication starts as early as late M or early G1 with assembly of pre-RCs (pre-replicative complexes) at the selected locations on chromosomes. This step, also called origin licensing, proceeds through the step-wise assembly of Orc, Cdc6 and Cdt1-Mcm, resulting in the loading of Mcm (helicase loading) onto the chromatin. The selected pre-RCs are activated by the actions of Cdc7 kinase and Cdk when cells enter S phase [2,3]. Once in S phase, origin licensing is strictly inhibited by layers of mechanisms that prevent rereplication [4]. These mechanisms are largely conserved from yeasts to human. Regulation of origin firing during S phase-Once S phase is initiated, origins are fired in a coordinated and regulated manner, until the entire genome is replicated. There are origins every 50-150 kb; about 300 in budding yeast, about 1100 in fission yeast, and more than 20,000 in human (Figure 1). These origins are fired in specific orders. Yeasts (budding and fission yeasts) have served as excellent model organisms for the study of regulation of origin firing due to its small genome size and ease of genetic manipulation. Thus, the precise locations of all the origins and the order by which they are fired have been known (Figure 1). Mechanisms that may regulate the timing of origin firing in yeasts have been the focus of many studies and are discussed below. In metazoans, firing of origins appears to be regulated on a domain basis [5,6], that is, clusters of nearby origins present in the same domain may be spatially and temporally coregulated [7,8]. Recent genomics studies demonstrated the presence of cell-type specific \u201creplication domains\u201d that define the segments of the chromosomes containing the coregulated origins (ranging in size from several hundred kilobases to megabase) [9]. It has been elusive how these replication domains are generated on chromosomes and how they are regulated in different cell types [9,10]. Rif1, originally identified in budding yeast as a Rap1-interacting factor involved in telomere length regulation [11], has recently come into the spotlight because of the unexpected discovery of its participation in origin regulation.\u00a0<br \/>In this article, we will first summarize various factors and conditions that regulate the origin firing\/ replication timing program in various eukaryotes (see Table 1 for a list; see Supplemental Table for factor nomenclature in various species.) We will then discuss the newly identified Rif1, which may be a global regulator of replication timing domains in metazoans.<\/p>\n<p>Regulation of the origin firing\/ replication timing program<br \/>Origin firing timing and replication timing-Origin firing timing and replication timing are not identical. One can determine the firing timing of a given origin in a given cell, which can be at a certain time during S phase. Replication timing at a certain genome position could be similar to the firing time of the nearest origin, but could be quite different if the nearest origins are far away. Firing efficiency of an origin can be very low, and a given origin may be fired in only a small subset of the cells during a given S phase. Thus, the average timing of DNA replication (usually replicated passively from origins further away) can be quite different from the time of the origin firing. However, firing occurs more or less simultaneously in the clusters of the neighboring origins. Thus, even though firing efficiency of each origin is low, the segments harboring early-firing origins are most likely to be replicated early and those harboring late-firing origins late. Therefore, in this article, origin firing program and replication timing program are generally used interchangeably unless specified otherwise. However, special note should be made on the timing transition region (TTR) that separates two distinct timing zones. TTR, identified more notably on the chromosomes of higher eukaryotes, is generally composed of a long stretch of chromosome with suppressed origins and is replicated unidirectionally [12,13]. The replication timing of TTR can obviously be different from the firing timing of the even nearest origin.<br \/>Possible mechanisms-For simplicity, let us assume there are two classes of replication origins; early- and late-firing. There are two ways to distinguish these two classes of origins. The first is to mark the early-firing origins. The chromatin structures in which the origins are embedded could dictate early-firing. Alternatively, covalent modification of the pre-RC components or other factor(s) that selectively associate with specific origins prior to the initiation could mark early-firing origins (Figure 2A). This \u201cmarking\u201d step would be required or at least highly facilitating for firing at early-S. The late origins may need to be marked later in S phase for initiation. Alternatively, firing of early origins may somehow cause changes of chromatin structures of the late origin segments, leading to activation of late origins. Alternatively, the initiation factors, which are limiting, are available for late origins only after the early segments are replicated. The second is to assume that all the origins are in the state that are ready to fire at the onset of the S phase (the default state). Late origins are somehow actively prevented from firing by being sequestered from interacting with replication initiation factors (Figure 2B). At later S phase, this constraint is released and late origins are fired. Combinations of these two are also certainly possible. As explained below, data supporting both mechanisms have recently been reported.<br \/>Temporal and spatial consideration-Before discussing the factors involved in the regulation, we would like to discuss the cell cycle and spatial regulation of replication timing. Using the combination of nuclei isolated from various cell cycle stages of CHO cells and extracts from Xenopus eggs that are capable of replicating the added nuclei, it has been shown that the replication timing program is established in nuclei isolated from cells that traversed a discrete point during the early G1 phase [14,15]. At this point, termed the TDP (Timing Decision Point), major chromatin repositioning, where the chromatin is relocated to its respective sub-nuclear positions, takes place in nuclei.\u00a0<br \/>In mammals, it has been well known that chromatin in the nuclear interior is replicated in early S, whereas the chromatin at nuclear periphery is preferentially replicated at late S. The late-replicating inactive X chromosome allele is associated with nuclear periphery, while the early-replicating active chromosome is located in the interior of the nuclei [16]. Thus, spatial arrangement of chromosomes may play an important role in origin firing program. In budding yeast, forced cell cycle-specific dissociation of telomeres from the origins demonstrated that the decision for late activation is established between mitosis and START (corresponding to the restriction point in mammalian cells) in the subsequent G1 phase. It is also interesting to note that, once established, late origin activation can be enforced even if the telomere is released from the target origin [17,18]. Furthermore, late origins associate with the nuclear envelope during G1 phase, whereas early origins are randomly localized within the nucleus throughout the cell cycle [18]. However, the artificial tethering of an early replicating origin at nuclear membrane in budding yeast did not cause late replication of this origin [19], suggesting that the association with nuclear periphery alone is not sufficient for enforcing late replication on an early-firing origin.<\/p>\n<p>Factors involved in regulation of origin firing program<br \/>Checkpoint signaling-Treatment of cells with hydroxyurea (HU) or methyl-methane sulfate (MMS) delays or blocks the initiation from the late-firing origins [20,21]. However, they are precociously activated in checkpoint mutants, rad53 or mec1 [22-27], indicating that the origin firing program is regulated by the checkpoint pathway. Tof1-Csm3-Mrc1 are conserved factors that play important roles in mediating the stalled fork signal to downstream, checkpoint machinery [28]. In mrc1\u2206, late or dormant origins are activated in the presence of HU or in early S phase both in budding and fission yeasts. Further analyses of various checkpoint mutants for their ability to suppress late origin firing showed that the RFCctf18 clamp loader (a complex consisting of RFC, Ctf18, Ctf8 and Dcc1) plays an important role in replication stress-induced repression of late origin firing [29]. This complex is essential for mediating replication-stress induced checkpoint signaling that results in Rad53 kinase activation, and thus plays important roles in late origin suppression. Suppression of dormant or late-firing origins by the checkpoint pathway was also reported in human cells [30].<br \/>Transcription and transcription factors-Although not very obvious in yeast [31], the correlation between active transcription and early replication has been shown in other organisms including Drosophila and mammals [32-35]. DNA combing analyses of replicated molecules at the human IgH locus showed that replication origin activation correlated with changes in the chromatin structure and transcriptional activity at different stages of B cell differentiation [36]. It appears that open chromatin or a potential for transcription activity, rather than active transcription per se, is related to replication in early S phase.\u00a0<br \/>Recently, Forkhead transcription factors, Fkh1 and Fkh2, were shown to regulate the origin firing time of many replication origins in budding yeast by binding near a subset of early-firing origins and facilitating the association of Cdc45 with these origins in G1 phase, possibly by promoting origin clustering. Fkh1\/2 binding sites are enriched near Fkh-activated origins and depleted near Fkh-repressed origins. Fkh1\/2 interacts with ORC and potentially tether together Fkh-activated origins in trans, which may facilitate the early firing of these origins by increasing the effective concentration of replication factors [37]. These functions of Fkh1\/2 are independent on their transcription activity. Although it is not known whether similar mechanisms operate in other species, transcription factors have been implicated in origin activation\/ selection in mammals [38,39]. In replication initiation at oriP, the origin of Epstein Barr Virus, EBNA1 transcription factor was shown to facilitate the recruitment of ORC [40].<br \/>Chromatin structures-Histone modifiers play crucial roles in regulation of origin firing program. For example, Sir3, which interacts with the histone deacetylase Sir2, was shown to delay or suppress firing of subtelomeric origins in budding yeast [41]. Sir2 suppresses pre-RC formation through a specific sequence present near a subset of origins by generating unfavorable chromatin structures for pre-RC assembly [42]. In human cells, HBO1, a histone acetylase, that binds to Cdt1 [43], was shown to promote pre-RC assembly through acetylation of histone H4, which is inhibited by Geminin known to play a crucial role in prevention of rereplication [44]. It was also reported that the artificial tethering of HAT1, the Drosophila homologue of HBO1, to an origin presumably leads to efficient pre-RC formation, which could in turn cause early-firing at this origin in S phase [45] (see below). The histone H3 deacetylase Rpd3, a component of a gene-specific transcriptional repressor, is arguably the most important histone modifier with regard to origin firing. It exerts a profound effect on genome-wide origin firing profiles in a manner independent of checkpoint signaling [46-48]. Correlations between regional transcriptional activation and origin deregulation suggested that hyper-acetylation near origins caused by the lack of Rpd3 facilitates both transcription and the origin activation process potentially through induction of open chromatin structures. It should be noted that chromatin structures can affect efficiency of both formation and activation of pre-RC. Although the efficient pre-RC formation can lead to early-firing events, its mechanism is elusive. Therefore, we will mostly focus on replication timing regulation that acts at the stage of the pre-RC activation (firing step).<br \/>Replication timing of chromosomes with special chromatin structures may be regulated by distinct mechanisms. The fission yeast centromere segments are replicated early in spite of their heterochromatin character (Figure 1). This is made possible by Swi6\/HP1 which recruits the kinase Hsk1 (the Cdc7 homologue) through interacting with the Dfp1\/Him1 subunit (Dbf4 homologue) of the Hsk1 kinase complex [49]. Telomere\/ subtelomere segments are replicated very late in S phase in spite of the presence of clusters of pre-RCs [50] (Figure 1). Transfer of an early-firing origin on the arm region to the vicinity of the subtelomere region renders it late-firing. Special chromatin structures in the subtelomere regions may be responsible for the suppression of initiation on these regions, since disruption of telomere binding factors, known to be required for generation of telomere-specific chromatin structures, deregulates replication and causes theses regions to replicate early [51-53]. It was also reported that telomere shortening by the yKu mutation stimulates the firing in the subtelomere segment up to ~40 kb from the end [54]. These results indicate chromatin structures are major regulatory elements for origin firing.\u00a0<br \/>On the other hand, effect of replication timing on chromatin structures have been argued [35]. The process of DNA replication can induce changes of chromatin status by altering the histone modification patterns or spatial arrangement of chromatin in nuclei. The detailed genome-wide studies on replication timing and chromatin features in human cells indicated the presence of the correlation of chromatin modification features and replication timing in TTR [55], a long stretches of chromosome that is unidirectionally replicated. This correlation is very similar to what is found in the other constant replication timing segments, arguing in favor for the idea that replication timing affects the chromatin structures.<br \/>Other factors-Mrc1 is a checkpoint adaptor protein and late origins are fired in the presence of HU in mrc1\u2206 due to loss of checkpoint. It was reported that weak early-firing origins are stimulated in the fission yeast mrc1 mutant proficient in checkpoint responses, suggesting that Mrc1 regulates early origin firing also in a checkpoint-independent manner as well, although its precise mechanism is unknown. Mrc1 selectively binds to early-firing origins at the pre-firing stage at the onset of S-phase, leading to a proposal that Mrc1 may mark the early-firing origins [56].<br \/>A simpler model was also proposed. In fission yeast, early recruitment of Orc to chromatin during the preceding M phase facilitates pre-RC formation in G1 as well as early firing of these origins in S phase presumably through increased recruitment of initiation factors [57]. Extension of M phase resulted in more uniform levels of initiation at \u201cearly\u201d and \u201clate\u201d origins, leading the authors to conclude that differential recruitment of ORC during M phase determines the timing and efficiency of origin firing. However, a possibility remains that the altered chromatin structures caused during the extended M phase are responsible for the change of origin activation.\u00a0<br \/>In both budding and fission yeasts, late-firing or dormant origins can be precociously activated by overexpression of some of the replication factors including Cdc7-Dbf4, Sld3 and Cdc45, which are limiting in numbers and appear to be preferentially utilized by \u201cearly\u201d origins [58,59]. It is not known whether this is the case in higher eukaryotes.<br \/>In summary, whereas a number of factors may regulate the origin firing program, local chromatin structures are likely to be one of the most crucial elements in its determination. In yeasts, preferential recruitment to the early-firing origins of the limiting factors could determine the origin firing pattern. This could be facilitated not only by preferred chromatin structures but also by origin tethering mediated by a factor like Fkh or by origin marking with some early origin-specific binding factor such as Mrc1. Late origins may be available for firing at early-S phase but are fired only after early origins are fired and limiting factors become available. On the other hand, another model, which is not exclusive with above, hypothesizes that the late origins are actively suppressed by some unknown mechanisms, permitting the firing of only the origins present in the early-replicating segments. In the following sections, we will discuss a novel factor that might participate in this latter mechanism.<\/p>\n<p>Rif1 as a novel regulator of genome-wide replication timing<br \/>Fission yeast cells-In order to understand the molecular basis for the replication origin firing program, it would be important to identify the crucial factors that may determine the genome-wide pattern of early- and late-firing of origins. In budding yeast, the Cdc7 kinase is a critical regulator of origin firing and may determine which potential origins are activated. In the absence of Cdc7, firing efficiency is too low to support the completion of S phase under normal growth conditions. Similarly, fission yeast hsk1\u2206 (encoding the fission yeast homologue of Cdc7) exhibit a delayed S phase and eventually die [60,61]. However, a variety of genetic or physiological conditions (mrc1\u2206, cds1\u2206, and high temperature etc.) allow hsk1\u2206 cells to survive and form colonies ([62]; see Box 1). Under these conditions, the origin-firing program is modified and firing occurs in the absence of Hsk1 kinase. Systematic screening for mutations that permit growth of hsk1\u2206 cells lead to the identification of Rif1 (Rap1 interacting factor). rif1\u2206 efficiently restores growth of hsk1\u2206, and this bypass appears to be distinct from that of checkpoint mutants (Box 1)[63].\u00a0<br \/>Rif1 was originally discovered as a telomere binding factor in budding yeast, and is involved in telomere length regulation in both budding and fission yeasts (telomere elongation in the absence of Rif1). It binds to subtelomere regions and suppresses their replication. In rif1\u2206 of fission yeast, not only the subtelomere regions but also many late or dormant origins on the arms of the chromosome are vigorously fired in the presence of HU (Figure 1). Some origins that are not fired in checkpoint mutants are fired in rif1\u2206. Indeed, twice as many origins are activated in rif1\u2206 than in checkpoint mutants, including 90% of those activated in the latter. More interestingly, more than 100 early-firing origins are downregulated or their firing timing is delayed in rif1\u2206 cells, suggesting that Rif1 negatively regulates late-firing origin and positively regulates early-firing origins. Thus, it appears that the combination of these opposing effects results in replication timing that occurs most frequently in mid-S phase in rif1\u2206. A possibility remains, however, that extensive deregulation of late-firing\/ dormant origins in rif1\u2206 causes titration of limiting initiation factors, resulting in reduced firing of origins normally fired early. Together, these results indicate that Rif1 is required for establishing genome-wide origin firing program in fission yeast [63].\u00a0<br \/>Rif1 binding sites identified by ChIP-chip analyses do not generally overlap with the known origins in yeast. However, Rif1 tends to bind at sites closer to the upregulated origins than to the downregulated origins. Analyses of sequences of the binding segments near the upregulated origins revealed the presence of a conserved sequence, although its functional significance is not clear. Rif1 deletion does not affect the pre-RC formation but the loading of Cdc45 onto the pre-RC is either stimulated or decreased at the upregulated or downregulated origins, respectively [63].\u00a0<br \/>The telomere binding protein, Taz1 (a homologue of human TRF1\/2), is also involved in the origin activation program in fission yeast [53]. Deregulation of origin firing was observed at telomeres as well as some of the non-telomeric late origins in the arm of the chromosomes in taz1\u2206. Sequences similar to Taz1 binding sites were identified near the suppressed origins and these sequences were shown to be required for suppression. Taz1 may enforce late origin firing through binding to these telomere-like repeats present near the affected origins [53]. Unlike rif1\u2206, suppression of early-firing origins was not observed in taz1\u2206. All the Taz1-regulated origins are also regulated by Rif1, suggesting that suppression of late origins by Taz1 involves the function of Rif1.<br \/>Mammalian cells- In mammalian cells, clusters of origins are activated at distinct times [64], and they constitute chromosome, so called \u201creplication (timing) domains\u201d that are replicated at specific timings within the S phase. Replication domains were initially observed as adjacent chromosome segments that incorporated [3H]-thymidine asynchronously during the S phase. Recent genome-wide studies revealed the presence of distinct replication timing domains that presumably contain sets of temporally coregulated origins and their distribution pattern on the entire chromosomes has been clarified in many cell types [65](Figure 1).\u00a0<br \/>It has long been known that replication occurs at specific locations within nuclei [66]. These foci, detectable by incorporated nucleotide analogues, adopt characteristic patterns at distinct stages of S phase. Most notably, foci are detected at nuclear and nucleoli periphery at the mid-S phase stage, whereas foci are scattered uniformly over nuclei during early-S phase [64]. These foci may correspond to each replication domain replicating at a distinct time within S phase. Recent studies demonstrate that replication timing profiles change during development [9]. During the differentiation of ES cells into neural stem cells, changes in replication timing occur in ~ 20% of the genome, often resulting in a shift of the boundary or consolidation of replication domains into a large coordinatedly regulated regions [9]. Similarly, the genome-wide patterns of replication domains change significantly in different cell types [10].\u00a0<br \/>Mammalian Rif1 does not play a role in telomere maintenance but it is required for establishment of replication timing domains in mammalian cells [67,68]. The most striking effect of Rif1 depletion in mammalian cells is the loss of the mid-S phase specific replication foci pattern. In Rif1-depleted cells, an early-S phase like foci pattern prevails throughout S phase, and late-S phase pattern, characterized by replication foci at the heterochromatin segments, appears at the end of S phase. Replication timing profiles also undergo dramatic changes. Genome-wide analyses in Rif1 knockout mouse MEF cells indicated that both early to late and late to early changes in replication timing occurred in over 40% of the replication segments, resulting in fragmentation of replication timing domains [68]. Knockdown of Rif1 in human cancer cells led to marked alteration of replication domain profiles in the 42Mb segment of the human chromosome 5. These changes are complex, including late to early and early to mid conversion and consolidation of domains (fusion of smaller domains)[67]. Generally, the distribution of replication timing occurred on average in mid S-phase in the absence of Rif1, suggesting that the replication timing regulation is generally lost, consistent with the analyses of rif1\u2206 fission yeast cells. During early-S phase, Cdc7-mediated phosphorylation and chromatin loading of replication factors are enhanced in Rif1-depleted cells, most likely pointing to the fact that more pre-RCs gain access to replication initiation factors and are fired, albeit during a limited time window. Rif1 is localized in nuclease-insoluble structures within nuclei and maintains chromatin loops and facilitates nucleosome formation. Rif1 localization overlaps with mid-S replication foci, suggesting that Rif1 generates nuclear structures specifically required for establishing mid-S replication domains (see Figure 3). Chromatin loop sizes increase in Rif1-depleted cells, indicating that Rif1 is required for correct chromatin loop formation. Chromatin loop sizes may be related to the sizes of the replication timing domains, since chromatin loops can generate chromatin domains that contain synchronously firing origins and are insulated from the neighboring segment. Rif1 is highly expressed in undifferentiated ES cells, which may be related to the smaller replication domain sizes observed in these cells. In differentiated cells, Rif1 levels are much lower, while replication domain sizes are increased [9].\u00a0<br \/>During the mitotic phase, Rif1 dissociates from chromatin and re-associates with it at late M\/ early G1 in a manner that is resistant to nuclease treatment. Thus, Rif1 must generate mid-S replication domain structures by early G1. The TDP was reported to occur during early G1 concomitant with chromatin repositioning [14,15]. It is an intriguing possibility that Rif1 may play a role at the TDP in establishing replication timing domains.<\/p>\n<p>A model for regulation of replication timing domains in mammalian cells\u00a0<br \/>We propose that the association of potential origins with replication factories triggers initiation and that this process is dynamic and stochastic to some extent (Figure 3). In early-S phase replicating domains, replication origins are not constrained in G1 phase, and upon entering S phase, multiple origins may be tethered at a replication factory and simultaneously fired for initiation. The choice of which origins are brought to the factories may be made stochastically, although origins in \u201copen chromatin\u201d structures may have higher probability of being recruited and firing. In mid-S phase replicating domains, Rif1, which is bound to nuclear insoluble structures (e.g. nuclear lamina), tethers the chromatin and forms a loop (\u201cRif1-loop\u201d) containing many potential origins. Association of these origins with replication factories (similarly present at the nuclear insoluble structures) may be inhibited by Rif1. At mid-S phase, this inhibition is released and the mid-S phase origins are now able to interact with the replication factories to initiate replication. The choice of origins to be activated could be stochastic but might be influenced by various local epigenetic factors, as in the case for early-firing origins. In the absence of Rif1, the Rif1-loops are not generated and the mid-S phase replication domains are released from constraint, and thus the origins, normally prevented from being activated, are brought to replication factories and fired at early S phase. Under these conditions, more origins would compete for limiting replication factors and some of the origins normally firing early may be competed out by more \u201cactive\u201d origins, resulting in delayed or reduced firing, although more positive roles of Rif1 in generating early-S replication domains cannot be ruled out. Several unsolved issues include (i) what tethers replisomes to the replication factories in early S phase? (ii) how does the Rif1-loop prevent the origins from firing in early S phase? (iii) How is this inhibition released to permit the firing of origins in mid-S phase? (iv) How does Rif1 (close to 2500 amino acids in human Rif1 without any notable motifs except for Heat repeats at the N-terminus) promote chromatin loop formation? (v) Does localization of Rif1 at nuclear periphery play any roles in preferred localization of late replicating chromatin close to nuclear membrane? and (v) are there similar mechanisms by which the firing of late-firing origins (associated with heterocrhomatin) are inhibited until late S phase?<\/p>\n<p>Biological significance of replication timing<br \/>The regulation of the replication timing program is likely to be evolutionarily conserved because (i) factors regulating origin firing\/ replication timing are conserved, and (ii) in animal cells, replication domain structures are conserved between different species in spite of significant divergence of primary sequences and rearrangement of genomic segments [65,69]. However, the apparent normal growth of rif1\u2206 fission yeast despite severely misregulated origin firing patterns suggests that cells can tolerate aberrant origin activation. Similarly, Rif1-depleted cancer cells do not show apparent defects in S phase progression despite the genome-wide alteration of replication timing program. These observations are in line with the speculation that the order and spatial location of origin firing in nuclei are quite flexible and adapt to changes in the environment.\u00a0<br \/>However, the biological significance of replication timing regulation in eukaryotes should not be dismissed altogether. Regulated timing may function to avoid too many replication forks at one time that would cause a shortage of nucleotide pools, resulting in stalled replication forks and eventually in DNA damage. Precise replication timing may facilitate the coordination with transcription timing, which may be required for proper progression of the cell cycle. Early replication may facilitate more protein synthesis of the genes present on the early-replicating segments due to doubled copy numbers and this increased expression may be needed for progression through the cell cycle. As discussed above, replication timing could affect the chromatin structures, which may in turn affect various chromatin activities including transcription, recombination and repair. Special mid-S phase replication timing domain may play roles for cell-type specific chromatin packaging or efficient condensation of chromosomes. Indeed, the Rif1 knockout MEF cells grow poorly with extended S phase [68]. Finally, it was reported that the mutation frequency varies during S phase. (see Box 2) It is higher in late S phase than in early S phase, although the significance of differential mutation rates regulated by replication timing is not clear.<\/p>\n<p>Concluding remarks<br \/>Precise and complete replication of the entire genome is crucial for cell growth and survival. Many different measures are taken to ensure that replication occurs once and only once. After entering S phase, long chromosomes need to be replicated in an ordered manner. Regulation of the timing of replication and the nuclear localization of chromosome are both important for efficient and coordinated replication of the entire chromosome.<br \/>In contrast to the prokaryotic genomes which typically carry only a single, very efficient origin, eukaryotic chromosomes carry many potential origins that are in many cases much less efficient. In addition, any given origin is utilized only in a fraction of the cells and cell cycles [7,8]. These features may provide eukaryotic cells with plasticity and adaptability to respond to various environmental or epigenetic changes [9,62,70]. Although the firing at each origin may be stochastic, the order of origin firing and timing of replication in a population of cells are predetermined and this feature is evolutionally conserved. Chromatin structures may play a major role in determination of the activity of each origin. Generally, \u201copen chromatin\u201d configurations characterized by \u201ctranscription-permissive\u201d histone marks promote early replication.\u00a0<br \/>We propose the presence of two layers of regulation for origin firing timing. The first is on the level of replication domains of several hundred kb to megabase. The domains that are insulated by Rif1 may be suppressive for initiation at early S phase and defines mid-S replication domains. There may be early-firing origin domains defined by factors such as Fkh1\/2. Another would be on the level of individual replicons or smaller clusters of replicons, the firing of which may be affected by local chromatin structures. This would determine which origin would be used for firing in each individual cell in each cell cycle, and the selection process could bear stochastic nature (Figure 3).<br \/>The mid-S replication \u201cfactories\u201d are very unique in their nuclear distribution and it is noteworthy that the factories appear to be generated as early as at late M\/ early G1. The pre-RC, which is generated also at a similar timing during the cell cycle, is not affected by the absence of Rif1 both in fission yeast and in human cells, and thus, origin licensing and so-called \u201cmid-S licensing\u201d occur independently at a similar timing during the cell cycle, and may constitute the two major steps for regulated and coordinated replication of the genome during S phase.\u00a0<br \/>Rif1 has been shown to interact with transcription factors, and was also implicated in DNA damage checkpoint pathways [71-78. The chromatin architecture generated by Rif1 may regulate not only DNA replication, but also other chromosome transactions including transcription. Indeed, the transcriptional profile changes significantly in Rif1-depleted cells in fission yeast and human cancer cells (although significant changes were not observed in MEF cells). Thus, it would be interesting to examine the roles of Rif1 in transcription, recombination, repair and other related processes. The roles of mammalian Rif1 in DNA damage responses have been investigated in detail and is well-established. It was recently reported that Rif1 is required for non-homologous end joining and inhibits DSB end resection [73-76].\u00a0<br \/>Rif1 is essential for embryonic development (Note: in one genetic background, male Rif1 knockout mice were viable for reasons still unclear [75]), and this may be due to aberrant transcription profiles in knockout mice. The importance of Rif1 in early development is further underscored by its high expression level in undifferentiated embryonic stem cells [77; its level quickly decreases upon induction of differentiation [78]. This high level of Rif1 protein is required to maintain the undifferentiated state [77,78]. Thus, the chromatin architecture defined by Rif1 may be established early in development and continues to influence various biological events throughout the life of the organism. Further elucidation of its roles would help clarify the biological significance of replication timing regulation and explain the connection between the Rif1-generated chromatin loops and origin firing.<\/p>\n<p>Box 1<br \/>Cdc7 kinase and its bypass mutations<br \/>Cdc7, originally identified in budding yeast in the Hartwell collection, encodes a serine-threonine kinase that seems to be essential for initiation of S phase, and was later shown to form a complex with Dbf4, identified in an independent screening as another mutant defective in S phase initiation. The complex formation activates Cdc7 kinase. Cdc7 phosphorylates Mcm in the pre-RC on the chromatin and this phosphorylation stimulates interaction of Cdc45with the pre-RC [79,80], facilitating the generation of an active helicase complex (CMG [Cdc45-Mcm-GINS] helicase) [81]. The haploid budding yeast cdc7(ts) mutant cells arrest with 1C DNA content (representing the cell cycle stage before DNA replication) at the non-permissive temperature and can be released into the cell cycle upon return to the permissive temperature. In fission yeast, hsk1(ts)\uff08encoding the Cdc7 homologue\uff09, at the non-permissive temperature, temporarily arrests with 1C DNA, later starts to synthesize DNA, arrest in S phase and eventually undergoes cell death [61]. In embryonic stem cells, Cdc7 knockout results in S phase arrest, G2-M checkpoint induction, and eventually in p53-dependent cell death [82]. Thus, Cdc7 is generally essential for DNA replication and for growth under normal conditions.<br \/>The first bypass mutation of cdc7\u2206, bob1, was isolated in budding yeast and was identified as mcm5P83L. This mutation appears to bypass the requirement for Cdc7 through modifying the structure of the Mcm complex so it can initiate replication in the absence of Cdc7-mediated phosphorylation [83]. More recently, deletion within the N-terminal segments of Mcm4 were found to bypass cdc7 [84]. In fission yeast, checkpoint mutants, mrc1\u2206 and cds1\u2206, were found to bypass hsk1\u2206. In mrc1\u2206 and cds1\u2206, late or dormant origins were activated early presumably due to loss of inhibitory checkpoint signals preventing replication [56,62]. Thus, increased initiation potential in checkpoint mutants appears to reduce the requirement of Cdc7 for initiation. Notably, growth at a higher temperature (37\u00b0C) also restores the growth of hsk1\u2206 cells, which do not grow at 30\u00b0C or lower. Firing of some dormant origins was observed in the wild-type cells at 37\u00b0C, although the precise mechanisms of this deregulation of origin firing at a higher temperature are not known [62]. Random screening of bypass mutants of hsk1\u2206 yielded rif1\u2206, which restores the growth of hsk1\u2206 most efficiently [63]. The efficiency of bypass appears to be correlated with the extent of late\/ dormant origin firing in the presence of HU. Indeed, about two times more origins are activated in rif1\u2206 than in checkpoint mutants.<\/p>\n<p>Box 2<br \/>DNA replication timing and diseases<br \/>Altered replication timing was reported in various diseases such as ICF immunodeficiency syndrome, Gilles de la Tourette syndrome and DiGeorge and Velocardio facial syndromes [85-87]. In some cases, transcriptional silencing and late replication were strongly correlated.<br \/>Genome-wide analyses of DNA replication timing in cancer cells from patients indicated the alteration of DNA replication timing pattern specific to cancer [88]. The changes occur along the developmentally regulated boundary of replication timing domains, supporting the concept that replication domains represent units of chromosome structure and function. In cancer cells, misregulation occurs at the level of these units [88].<br \/>Replication timing profile data are useful to detect karyotypic abnormalities and copy-number variation. Aneuploidies or translocation breakpoints could be detected as lower signal or unnaturally sharp transitions in replication. Cancer SCNAs (Somatic copy-number alterations) arise preferentially in genomic regions that have both the same replication timing and share long-range interactions in the nucleus [89].<br \/>All these data indicate that the genome rearrangements responsible for genome duplications, deletions or translocations tend to occur at the boundaries of replication domains or chromosome interaction units. These results suggest the potential of replication timing profiles as a diagnostic biomarker.\u00a0<br \/>It has been suggested that there is an association between mutation rate and DNA replication timing. Initial observation suggested that cancer-related genes are frequently present at the TTR (boundary of domains with different replication timing) [90]. Similarly, SNPs are more enriched in the TTR and late replicating regions [90]. Analyses using ENCODE comprising 1% of the human genome revealed that mutation rate, as reflected in evolutionary divergence [91] and human nucleotide diversity (SNPs), is markedly increased in later-replicating segments of the human genome [92]. Analyses of somatic mutations in &gt; 400 cancer genomes also showed that the frequency of somatic SNPs increases with replication time during the S phase. The late-replicating regions are enriched for recurrently mutated genes but are depleted of cancer driver genes. A higher mutation rate in the late replicating chromosome segments was also reported in yeast [93].<br \/>The mechanisms for increased mutation rate in the late replicating regions are not clear. The depletion of nucleotide pools in late S phase may cause increased incidences of fork arrest. Alternatively, replication through heterochromatic regions may pause at obstacles for fork progression. The stalled replication fork could generate unstable single-stranded DNA segments that could be the cause for mutagenesis.<\/p>\n<p>Acknowledgments<\/p>\n<p>We would like to thank Dave Gilbert for critical reading of the manuscript and discussion, Yutaka Kanoh for the original drawing used in Figure 1, and Seiji Matsumoto, Yutaka Kanoh, Michie Shimmoto and all other members of our laboratory for collaboration and helpful discussion. We also thank Claire Renard-Guillet and Katsuhiko Shirahige for informatics analyses of microarray data in fission yeast. Finally, we would like to express our deepest gratitude to the editor and reviewers for comments and extensive corrections of the manuscript. The research in our laboratory was supported by grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.<\/p>\n<p>\u2003<br \/>References<\/p>\n<p>1. Katayama, T., et al. (2010) Regulation of the replication cycle: conserved and diverse regulatory systems for DnaA and oriC. Nat. Rev. Microbiol. 8, 163-170<\/p>\n<p>2. Masai, H., et al. (2010) Eukaryotic chromosome DNA replication: where, when, and how? Annu. Rev. Biochem. 79, 89-130<\/p>\n<p>3. Labib, K. (2010) How do Cdc7 and cyclin-dependent kinases trigger the initiation of chromosome replication in eukaryotic cells? Genes Deve. 24, 1208-1219<\/p>\n<p>4. Arias, E.E., and Walter, J.C. (2007) Strength in numbers: preventing rereplication via multiple mechanisms in eukaryotic cells. Genes Deve. 21, 497-518<\/p>\n<p>5. 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Proc. Natl. Acad. Sci. U. S. A. 106, 3184-3189<\/p>\n<p>103. Jorgensen, H.F., et al. (2007) The impact of chromatin modifiers on the timing of locus replication in mouse embryonic stem cells. Genome Biol. 8, R169<\/p>\n<p>104. Yokochi, T., et al. (2009) G9a selectively represses a class of late-replicating genes at the nuclear periphery. Proc. Natl. Acad. Sci. U. S. A. 106, 19363-19368<\/p>\n<p>Legends to Figures<\/p>\n<p>Figure 1 Replication programs of budding yeast, fission yeast, and human chromosomes<br \/>A. The locations of replication origins on chromosome VI (0.27 Mb) of budding yeast. Red, early-firing origins; green, mid-firing origins; blue, late-firing origins [93]. B. The locations of replication origins on the chromosome II (4.5 Mb) of fission yeast. Red vertical lines, early-firing origins (origins firing in the presence of HU); blue vertical lines, late-firing or dormant origins [50]. C. Replication timing profile of the human chromosome 20 (62 Mb) in K562 cells is shown. Early-, mid- and late-replicating domains are deduced from data in [10] and are shown by red, green and blue horizontal bars, respectively.<\/p>\n<p>Figure 2 Possible mechanisms for determination of origin firing timing,<br \/>A. Early-firing origins are molecularly marked. Marking can be chromatin structures favorable for firing, covalent modification of pre-RC components, or association of other factor(s) that facilitate recruitment of initiation proteins. B. A hypothetical Factor X inhibits firing of late origins by preventing the recruitment of initiation factors. As a result, only the early-firing origins can get access to initiation factors during early S phase.<\/p>\n<p>Figure 3 A model for regulation of replication timing domains in higher eukaryotes<br \/>Replication occurs at factories where two replisomes are held together and replicating DNA strands are passed through as bidirectional DNA synthesis proceeds, generating the loop consisting of the replicated daughter molecules (replication loops; shown in gray) [94]. In the early-replicating domains (upper), chromosomes, whose conformations are not constrained during G1, can associate with replication factories where the clusters of early origins are simultaneously replicated. In the mid-replicating domains (lower), Rif1 generates specific chromatin loop structures (which we call \u201cRif1-loops\u201d to distinguish from replication loops) in G1, and origins present in the Rif1-loops are sequestered and kept inactive until mid-S phase. Rif1 associates with nuclear insoluble structures, which could hold together multiple Rif1-loops. At mid-S phase, the origins in the Rif1-loop are activated through association with the axis of the Rif1-loop, generating an active replication factory. Multiple origins could be simultaneously activated within the Rif1-loop, generating multiple smaller replicating loops. Again, the selection of origins to be activated could be dynamic and stochastic, and thus, the sizes and numbers of replication loops generated from one Rif1-loop may vary from one cell to another and from one cell cycle to next. How the origins in the Rif1-loops are kept from activation in early S phase and how they get activated after mid-S phase are unknown. We also do not know if any factors are responsible for replication loop formation during early S phase and if any factors sequester the late-replicating domains, which are not described here but show distinct spatial distribution. In the absence of Rif1, the early S phase domains are intact but the Rif1-loops are disrupted, releasing mid-S phase origins from sequestration. Thus, the majority of the chromosomes (except for the late replicating heterochromatin segments) are replicated in the early-S phase pattern throughout the S phase except for very late S phase. The replication loops in the main part of the figure are shown by single lines, even though they are made up with two daughter molecules, and both replicated and unreplicated DNAs are shown in black.<\/span><\/p>\n<p><a href=\"http:\/\/localhost:8888\/wordpress\/wp-content\/uploads\/2014\/03\/table1.jpg\"><img decoding=\"async\" loading=\"lazy\" class=\"alignnone size-large wp-image-459\" alt=\"table1\" src=\"http:\/\/localhost:8888\/wordpress\/wp-content\/uploads\/2014\/03\/table1-994x1024.jpg\" width=\"640\" height=\"659\" srcset=\"https:\/\/www.igakuken.or.jp\/genome\/wp-content\/uploads\/2014\/03\/table1-994x1024.jpg 994w, https:\/\/www.igakuken.or.jp\/genome\/wp-content\/uploads\/2014\/03\/table1-291x300.jpg 291w\" sizes=\"(max-width: 640px) 100vw, 640px\" \/><\/a><\/p>\n<p><a href=\"http:\/\/localhost:8888\/wordpress\/wp-content\/uploads\/2014\/03\/TIGFigure1.jpg\"><img decoding=\"async\" loading=\"lazy\" class=\"alignnone size-large wp-image-460\" alt=\"TIGFigure1\" src=\"http:\/\/localhost:8888\/wordpress\/wp-content\/uploads\/2014\/03\/TIGFigure1-1024x814.jpg\" width=\"640\" height=\"508\" srcset=\"https:\/\/www.igakuken.or.jp\/genome\/wp-content\/uploads\/2014\/03\/TIGFigure1-1024x814.jpg 1024w, https:\/\/www.igakuken.or.jp\/genome\/wp-content\/uploads\/2014\/03\/TIGFigure1-300x238.jpg 300w\" sizes=\"(max-width: 640px) 100vw, 640px\" \/><\/a><\/p>\n<p><a href=\"http:\/\/localhost:8888\/wordpress\/wp-content\/uploads\/2014\/03\/TIGFIgure2.jpg\"><img decoding=\"async\" loading=\"lazy\" class=\"alignnone size-full wp-image-461\" alt=\"TIGFIgure2\" src=\"http:\/\/localhost:8888\/wordpress\/wp-content\/uploads\/2014\/03\/TIGFIgure2.jpg\" width=\"960\" height=\"720\" srcset=\"https:\/\/www.igakuken.or.jp\/genome\/wp-content\/uploads\/2014\/03\/TIGFIgure2.jpg 960w, https:\/\/www.igakuken.or.jp\/genome\/wp-content\/uploads\/2014\/03\/TIGFIgure2-300x225.jpg 300w\" sizes=\"(max-width: 960px) 100vw, 960px\" \/><\/a><\/p>\n<p><a href=\"http:\/\/localhost:8888\/wordpress\/wp-content\/uploads\/2014\/03\/TIGFigure3.jpg\"><img decoding=\"async\" loading=\"lazy\" class=\"alignnone size-large wp-image-462\" alt=\"TIGFigure3\" src=\"http:\/\/localhost:8888\/wordpress\/wp-content\/uploads\/2014\/03\/TIGFigure3-918x1024.jpg\" width=\"640\" height=\"713\" srcset=\"https:\/\/www.igakuken.or.jp\/genome\/wp-content\/uploads\/2014\/03\/TIGFigure3-918x1024.jpg 918w, https:\/\/www.igakuken.or.jp\/genome\/wp-content\/uploads\/2014\/03\/TIGFigure3-268x300.jpg 268w, https:\/\/www.igakuken.or.jp\/genome\/wp-content\/uploads\/2014\/03\/TIGFigure3.jpg 1527w\" sizes=\"(max-width: 640px) 100vw, 640px\" \/><\/a><\/p>\n<p>&nbsp;<\/p>\n<p><a href=\"http:\/\/localhost:8888\/wordpress\/wp-content\/uploads\/2014\/03\/tableS3.jpg\"><img decoding=\"async\" loading=\"lazy\" class=\"alignnone size-large wp-image-463\" alt=\"tableS3\" src=\"http:\/\/localhost:8888\/wordpress\/wp-content\/uploads\/2014\/03\/tableS3-928x1024.jpg\" width=\"640\" height=\"706\" srcset=\"https:\/\/www.igakuken.or.jp\/genome\/wp-content\/uploads\/2014\/03\/tableS3-928x1024.jpg 928w, https:\/\/www.igakuken.or.jp\/genome\/wp-content\/uploads\/2014\/03\/tableS3-272x300.jpg 272w\" sizes=\"(max-width: 640px) 100vw, 640px\" \/><\/a><\/p>\n","protected":false},"excerpt":{"rendered":"Contents\u201cA personal reflection on the Replicon Theory&#8221; \u30ec\u30d7\u30ea\u30b3\u30f3\u30e2\u30c7\u30eb\u3068\u3068<a href=\"https:\/\/www.igakuken.or.jp\/genome\/?page_id=443\">&nbsp;&nbsp;[&#8230;\u7d9a\u304d\u3092\u8aad\u3080]<\/a>","protected":false},"author":3,"featured_media":0,"parent":0,"menu_order":8,"comment_status":"closed","ping_status":"closed","template":"","meta":[],"_links":{"self":[{"href":"https:\/\/www.igakuken.or.jp\/genome\/index.php?rest_route=\/wp\/v2\/pages\/443"}],"collection":[{"href":"https:\/\/www.igakuken.or.jp\/genome\/index.php?rest_route=\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/www.igakuken.or.jp\/genome\/index.php?rest_route=\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/www.igakuken.or.jp\/genome\/index.php?rest_route=\/wp\/v2\/users\/3"}],"replies":[{"embeddable":true,"href":"https:\/\/www.igakuken.or.jp\/genome\/index.php?rest_route=%2Fwp%2Fv2%2Fcomments&post=443"}],"version-history":[{"count":10,"href":"https:\/\/www.igakuken.or.jp\/genome\/index.php?rest_route=\/wp\/v2\/pages\/443\/revisions"}],"predecessor-version":[{"id":566,"href":"https:\/\/www.igakuken.or.jp\/genome\/index.php?rest_route=\/wp\/v2\/pages\/443\/revisions\/566"}],"wp:attachment":[{"href":"https:\/\/www.igakuken.or.jp\/genome\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=443"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}