next-generation sequencing and dna replication in human cells: the future has arrived

683ISSN 1479-6694Future Oncol. (2014) 10(4), 683–693

Review

Eukaryotic DNA replication starts from a large number of genomic sites (origins of replication [ORIs]), ranging from few hundreds in yeast to tens of thousands in humans [1]. Following sequen-tial recruitment of the origin recognition complex (ORC1–6 proteins), Cdc6, Cdt1 and the mini-chromosome maintenance (MCM) complex (MCM2–7 proteins), which together constitute the pre-replication complex (pre-RC), ORIs are simultaneously licensed during the G1 phase of the cell cycle (at the origin decision point) [2]. However, during the S-phase, origin firing is activated by the cyclin-dependent kinases Cdk2 and Cdk1, in complex with cyclin E or cyclin A, and by Cdc7/Dbf4 [1,3], and it occurs following a specific temporally ordered program of initiation that is established during the G1 phase (at the replication timing decision point) [4].

In Saccharomyces cerevisiae, ORIs are characterized by a short sequence, 100–200 bp in length, termed the autonomously replicating sequence, which is composed of several specific elements such as the A element, an AT-rich 12-bp consensus-sequence for ORC binding that is essential for initia-tion [1,5]. By contrast, in metazoans, the pre-RC does not exhibit obvious sequence specificity, but shows a variable, and still unknown, combination of genetic and epigenetic features that vary from cell to cell. This further complexity might reflect the need of metazoans to adapt the distribution of ORIs to the unique expression pattern of individual cell types and/or levels of differentiation. Furthermore, yeast ORCs (ORC1–5) remain intact and stably bound to chromatin throughout the cell cycle, while the subunits undergo a cell cycle-dependent phosphorylation that prevents re-initiation prior to mitosis.

Mammalian ORCs also consist of a stable core complex (ORC2–5) that is bound to chromatin throughout the cell division. However, mammalian ORC activity is regulated, through a weak inter-action, by cell cycle-dependent post-translational modifications of ORC1, which is essential for the assembly of the pre-RC [1,6,7] and is bound to chromatin in the G1 phase, but not during mitosis [1,8].

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Review

Next-generation sequencing and DNA replication in human cells: the future has arrived

Next-geNeRatiON sequeNciNg & DNa Replica-tiON iN humaN cells

DelliNO & pelicci

Gaetano Ivan Dellino*,1,2 & Pier Giuseppe Pelicci1,2

1Department of Experimental Oncology, European Institute of Oncology, 20141 Milan, Italy 2Dipartimento di Scienze della Salute, University of Milan, 20122 Milan, Italy

*Author for correspondence: [email protected]

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AbstrAct: Accurate regulation of DNA replication ensures faithful transmission of eukaryotic genomes and maintenance of genomic stability and chromatin organization. However, by itself the replication process is a threat for both DNA and chromatin integrity. This becomes particularly relevant in cancer cells, where activated oncogenes induce replication-stress, including unscheduled initiation, fork stalling and collapse and, ultimately, genomic instability. Studies addressing the relationship between (epi)genome integrity and disease have been hampered by our poor knowledge of the mechanisms regulating where and when eukaryotic replication initiates. Recently developed genome-scale methods for the analysis of DNA replication in mammals will contribute to the identification of missing links between replication, chromatin regulation and genome stability in normal and cancer cells.

Keywords • DNA replication • origin recognition complex • replication stress • replication timing • transcription start sites

For reprint orders, please contact: [email protected]

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In Chinese hamster ovary cells, ORC1 levels do not change during the cell cycle, but mono-ubiquitylation during the S-phase and phospho-rylation during the G2-M phases can prevent a stable association of ORC1 with the chroma-tin, as suggested by ORC1 accumulation in the cytoplasm [1,9,10]. By contrast, in HeLa and other human cell lines, ORC1 undergoes ubiquitin-dependent proteolysis during the S-phase [1,11], consistent with the observation that ORC2, but not ORC1, can be crosslinked to known origins in S-phase cells [12,13]. It should be mentioned that all the studies investigating the kinetics of the interaction between the pre-RC and DNA at individual origins in mammalian cells have been hampered by the difficulty in immunoprecipitat-ing chromatin-bound pre-RC proteins, and by the limited number of origins that were, until very recently, available (see below).

Replication timing & transcriptionAnalysis of 40% of all the Drosophila genes showed a strong statistical correlation between DNA replication early in the S-phase and tran-scriptional activity [14]. In particular, it has been shown that early replicating genes can be either expressed or silent, while late replicating genes are generally, yet not always, silent. This correla-tion has been confirmed by microarray studies in plants and higher eukaryotes (human and mouse cells) [15–22], although it has not been observed in yeast (e.g., when considering the replication timing of ribosomal protein genes, accounting for 50% of the RNA polymerase II transcrip-tion) [23]. It is not known, however, whether replication timing is associated with the global transcriptional status of the replicating chromo-somal domains or, alternatively, with the genetic and/or epigenetic features of specific origins or gene promoters therein [24–26].

No doubt this issue will be accurately addressed in the near future, as it is becoming possible to analyze different cell types (both immortalized and primary cells at different stages of differentiation) using emerging new technologies for the genome-wide profiling of replication timing, ORIs, RNA and chromatin. The genome-wide analysis of replication tim-ing (Repli-Seq) is based on the sequencing of replicating (BrdU-labeled) DNA purified from cells sorted into six consecutive fractions span-ning the whole S-phase (S1–S6) of the cell cycle [27]. Thus, Repli-Seq allows the visualization of the replication progression and, indirectly, the

identification of ORI-containing regions (named inverted-V apexes) as genomic sites where the bidirectional replication initiates (symmetrical early-to-late transitions or inverted-Vs). The resolution of Repli-Seq (in the range of hun-dreds of kb) depends on both the duration of BrdU incorporation and the number of sorted fractions in which the S-phase is divided, and is much lower than a typical chromatin immu-noprecipitation (ChIP) or the isolation of small nascent leading strands (SNS; in the range of hundreds of bp). The combination of Repli-Seq and ORI-mapping technology, however, might allow unambiguous mapping of the temporal and spatial features of DNA replication.

Origin mapping by isolation of sNsSince the use of ChIP to identify pre-RC-bind-ing sites in metazoans has proved to be very dif-ficult, the most widely used approach for the identification of replication origins has been the isolation of SNS [28]. A more stringent version of this method uses λ-exonuclease (Lexo), an enzyme that selectively digests the 5´ ends of contaminating broken genomic DNA, but not the SNS, which are protected at their 5´ end by an RNA primer [29]. However, although this method is suitable for high-throughput approaches, previous genome-wide studies based on the hybridization of purified SNS to 1–2% of the human or mouse genomes showed modest reproducibility [24,30–32]; for example, using two different subclones of the HeLa can-cer cell line, two independent studies showed <14% overlap between the identified putative ORIs [31,32]. Several reasons have been hypoth-esized to explain this small overlap: differences in cell-culture conditions; variable degrees of purity in different SNS preparations; differences in experimental protocols; and, the possibility that not all the SNS-captured sequences will ever mature into finished replicons, as suggested by the identification of an apparently futile repli-cation cycle of double-stranded DNA sequences [33]. An alternative cause could be a consider-able cell-to-cell heterogeneity with respect to origin selection within the analyzed cell popu-lations: since most origins (in the order of tens of thousands in mammalian cells) are only used in a fraction of cells, each data set might merely capture a fraction of origins, due to lack of saturation. To test this hypothesis, Besnard and colleagues have subjected the purified SNS to deep sequencing, reaching saturation

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of origin peak detection at approximately two-thirds of the 150 × 106 sequence-tags that were obtained [26]. Surprisingly, although the number of ORIs per cell cycle is generally estimated to be around 20,000–50,000, the authors identi-fied a much higher number of putative ORIs (200,000–250,000 SNS peaks) in the HeLa cells and the other cell types that were analyzed. Consistently, the mean interorigin distance measured in this study was ∼11 kb, which is con-siderably shorter than that generally observed by DNA-combing experiments on individual DNA fibers (∼75 and ∼140 kb in Drosophila and mouse cells, respectively; ∼40 kb in the human HeLa cell line) [24,34]. The most obvi-ous interpretation of this discrepancy between genome-scale studies and DNA-combing data is that sequencing to saturation allows resolution of cell heterogeneity with respect to ORI selec-tion. Nevertheless, it should be noted that the data set from Besnard et al. identifies only half of the sites mapped by a very different approach for ORI identification (i.e., isolation of repli-cation-bubble-containing restriction fragments selectively trapped in gelling agarose, followed by hybridization to microarrays containing 1% of the human genome [35]). Thus, the sensitiv-ity of the SNS-sequencing approach, even when sequencing is pushed to saturation, might not be sufficient to detect all the ORIs that are enriched by bubble trapping.

Sequence analyses of the identified SNS-peaks showed a signif icant enrichment of G-quadruplex structures (G4 DNA), as previ-ously found by analyses of ORIs identified by hybridization of isolated SNS to tiling micro-arrays of 118 Mb of the mouse genome [36]. The G4 DNA consists of a sequence motif (G≥3

NxG

≥3N

xG≥3

NxG≥3

) that can fold into a stable four-stranded structure in which interactions among strands are stabilized by G-quartets. However, their role in the selection of replication origins remains to be elucidated, since a huge number of G4 DNA sequences have been identified in the human genome (∼1 million) and implicated in the regulation of transcription, translation and progression of the replication fork [37].

Origin mapping by chip of pre-Rc proteinsA contribution towards our understanding of the origin selection process might come from the identification of the binding sites of proteins of the pre-RC in mammalian cells. Earlier efforts suggested that the ORC complex associates with

chromatin over wide areas, and that the enrich-ment measured at ORIs was too low compared with other DNA-binding proteins, such as tran-scription factors. The first demonstration that ORC and/or MCM2–7 proteins are recruited to human origins was obtained for the MCM4 and TOP1 ORIs [13,38,39]. Since then, seven addi-tional human origins were shown to bind pre-RC proteins by ChIP assays [12,40–45]. Furthermore, in the same studies, ChIP was used to assess the presence of ORC proteins (or other pre-RC components) at sites that had already been identified as origins (or at least as good candi-dates), with one exception [45]. However, even in model systems with smaller genomes (i.e., yeast and Drosophila), ORC enrichment at ORIs, as revealed by hybridization to DNA microarrays following conventional ChIP (ChIP-on-chip), is often not more than twofold over background [46,47]. Thus, notwithstanding the current high-throughput approaches, the complexity of mam-malian genomes and the intrinsic flexibility in origin selection precluded the study of specific interactions between ORC proteins and DNA.

Very recently, however, ORC2- and MCM3-binding sites in G1 chromatin (following cen-trifugal elutriation) have been mapped to the 50–60 latent and predominantly extra-chro-mosomal Epstein–Barr virus genomes, which are replicated by the replication machinery of the Burkitt lymphoma-derived Raji cell line [48]. Interestingly, almost half (44%; n = 28/64) of the pre-RC bound regions were found to overlap with Epstein–Barr transcrip-tion start sites (TSS), a frequency that rises to 66% (n = 42/64) if one considers those peaks mapping to the proximal promoter of the viral genes (TSS ± 500 bp). This is in line with the previously published data sets of SNS peaks: 7–44% overlap with mammalian and 64% with Drosophila promoters [24,30–32,47,49].

The first successful attempt to perform genome-wide mapping of human DNA-replication origins in asynchronous popula-tions of logarithmically growing HeLa cells, by targeting pre-RC proteins (namely ORC1 and MCM5), was carried out by means of a slight modification of the conventional ChIP assays [25]. Usually, immunoprecipitation is performed using total chromatin, previously crosslinked and sheared by sonication or endonuclease (i.e., MNase) digestion. Instead, in this study, prior to immunoprecipitation, sheared crosslinked chromatin was subjected to equilibrium

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density-gradient centrifugation. Strikingly, both pre-RC proteins and DNA from known ORIs were found in the low-density gradient fractions, where the association of DNA frag-ments with very high molecular-weight protein complexes shifts their buoyant density to values lower than that of bulk chromatin. These low-density fractions were then used for anti-ORC1 or anti-MCM5 ChIP assays, which showed a tenfold enrichment for origin DNA compared with ChIP assays performed with the same antibodies on total (unfractionated) chromatin. High-throughput sequencing of the immuno-precipitated DNA fragments allowed the identi-fication of approximately 13,600 ORC1 binding sites. Most of these ‘peaks’ were sharp (gener-ally ∼500 bp in width) and localized to spe-cific regions of the human genome, thus show-ing the typical features of most transcription factors. The identified ORC1 sites contained known ORIs, and, among the new ones, several were validated for ORC1 and MCM5 binding and local enrichment of SNS. Most notably, the majority mapped within ORI-containing regions identified by Repli-Seq.

The huge difference between the ORC1 and SNS data sets [25,26], in terms of number of peaks obtained (∼14,000 ORC1 peaks vs ∼230,000 SNS peaks), opens new intriguing questions about the relationship between ORC-binding sites and origin-firing sites along the DNA fiber. Even conceding that saturation sequenc-ing of ORC1 ChIP-Seq will raise the number of ORC1 peaks and/or accepting that not all the SNS-captured sequences derive from origins [50], it hardly seems possible that two data sets will come to comparable numerousness. These findings suggest the possibility that the topo-graphical relationship between ORC sites and SNS peaks along the chromosome fiber is such that the chromatin-bound ORC may have the potential to associate with MCM complexes recruited to distal sequences by looping of the intervening chromosomal DNA. In a variable percentage of cells from a homogeneous popula-tion, this would cause activation of less efficient (or possibly dormant) ORIs at a distance. This hypothesis is consistent with studies showing that ORC and Cdc6 are not necessary for ori-gin activity after ORC-mediated MCM loading has taken place [51]. In addition, both in yeast and metazoans, the MCM complex is present in excess of approximately 20-fold molar com-pared with the ORC [52,53], and several SNS

peaks are in close proximity of a single ORC1 site (Figure 1).

Despite the difference in numbers of ORIs, both genome-wide studies showed that the den-sity of replication origins decreases while the S-phase progresses [25,26]. Interestingly, the den-sity of ORC1 sites was consistent with the replica-tion kinetics of HeLa cells only in early S-phase [25]. However, since both mean interorigin spacing (∼40 kb) and the average rate of fork progres-sion (∼0.7 kb/min) do not change in HeLa cells during the S-phase [34], the decreasing number of identified ORC1 and SNS sites suggests that both SNS-Seq and ChIP-Seq failed to identify a significant fraction of the late-S ORIs. This leaves unanswered the question of whether success in identifying individual origins may largely depend on ORI efficiency, such that any approach that uses entire cell-populations might fail to detect the least-efficient and latest-firing origins (see below). Recent work, in fact, emphasizes the highly vari-able percentage of cells in which a given ORI is selected and then activated (so-called ORI effi-ciency: 5–20% in metazoans) [54,55].

ORc1 meets transcriptionGene annotation of the ORC1 sites showed enrichment at proximal gene promoters (∼35% of all ORC1 peaks), particularly at the TSS of annotated genes, consistent with previous ChIP-on-chip studies using isolated SNS [30,31]. However, comparison of the ORC1 data set with RNA transcripts mapped in the same cell line (RNA-Seq) [25] and human functional TSS iden-tified in different human cell-types (TSS-Seq) [56] revealed that 72% of all ORC1 sites mapped to transcriptionally active TSS of known or not-yet annotated genes. Strikingly, a large fraction of the ORC1 sites located within gene bodies or putative intergenic regions mapped to active TSS of small noncoding RNAs. The remaining 28% of ORC1 sites, although apparently not associ-ated with transcription in HeLa cells, mapped to functional TSS and/or showed epigenetic marks of open chromatin (DNase I hypersensitive sites, H3K4me3 or RNA polymerase II occupancy), thus suggesting that they might be expressed in HeLa cells at levels below RNA-Seq sensitiv-ity. Therefore, the association between ORC1 binding and transcription initiation of known or unknown genes (transcribed in either coding or small noncoding RNAs) is a universal feature of ORC1 sites, rather than a specific property of a small subset of ORIs (as previously suggested



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in [57]). This opens new scenarios; transcription initiation, in fact, might favor recruitment of the pre-RC complex or be mechanistically linked to the initiation of DNA replication.

Levels of expression at ORC1-bound TSS, however, are highly variable (from one to sev-eral thousands of RNA copies per cells), and might influence the probability of firing during the subsequent S-phase and, consequently, rep-lication timing [25]. The origins mapping to the TSS associated with moderate/high transcrip-tion levels fire early during the S-phase, while origins mapping to the TSS associated with very low transcription levels (<1 RNA copy/cell) fire throughout the entire S-phase [25].

Levels of expression at ORC1-bound TSS might also influence the efficiency of pre-RC recruitment during the G1 phase. Comparison of ORC1 ChIP-Seq and Repli-Seq data showed that the highest ORC1 peaks are mainly found in the very early S-phase, compared with the lowest ORC1 peaks, suggesting that the highest

peaks are the first to fire [25]. Notably, also the genome-wide mapping of SNS showed that amplitude of the SNS peaks was reduced in the late S-phase, as compared with early firing ori-gins [26]. Although one cannot formally exclude that the differences in the height of ChIP-Seq peaks are due to technical variability, the finding that the height of ORC1 peaks correlate with the associated transcription levels (i.e., the high-est ORC1 peaks map to the TSS of the most expressed genes), while they decrease during S-phase progression [25], suggests that ORC1 peak amplitude is a measure of origin efficiency, thus providing a tool to investigate cell hetero-geneity with respect to ORC1 recruitment. This hypothesis and the reliability of such strategies for genome-wide studies can be experimen-tally tested, at least for some genomic regions (and regardless of their replication timing), by single-molecule approaches such as single-molecule analysis of replicated DNA, which provides a powerful combination of relatively



Figure 1. Differences in the distribution of origin recognition complex 1 and small nascent leading strands peaks. Chromatin immunoprecipitation sequencing profile of ORC1 [25], distribution of SNS peaks [26] and position of RefSeq genes within four regions of the human genome. The upper right panel shows a magnification of the MCM4 ORI (also shown within the green oval of the left panel). Y-axis values of the ORC1 and SNS tracks indicate the number of overlapping tags from the anti-ORC1 ChIP-Seq and the score of SNS peaks, respectively. Statistically significant ORC peaks are indicated by the black boxes below the ORC1 track [25]. Scale bar: 2, 20 or 50 kb, as indicated. ORC: Origin recognition complex; SNS: Small nascent leading strands.

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high-resolution origin mapping (∼5–10 kb) and a large number of DNA molecules from the chromosomal region of interest [58,59].

In summary, preliminary genome-wide analy-ses suggest the existence of two classes of ORC1-binding sites: those mapping to the TSS of cod-ing genes expressed at moderate/high levels, which favor the assembly of the pre-RC complex in the majority of cells and replicate early during the S-phase (most-efficient ORIs); and, those map-ping to the TSS of noncoding genes expressed at low level, which fire throughout the entire S-phase in a minority of cells (least-efficient ORIs). The latter might be activated in early S-phase by the incoming forks, while in late S-phase they repre-sent the only available option for replication initia-tion within gene-free or transcriptionally silenced regions. Thus, in early S-phase, high- and low-effi-cient ORIs might co-exist within the same chro-mosomal domains. Preliminary analyses of their relative contribution to the replication of a typi-cal inverted-V apex (by comparing ORI position, interorigin distances and average rate of fork pro-gression) suggest that the most-efficient ORIs are

sufficient for the full replication of the considered chromosomal domain (Figure 2). The availability in the mammalian genome, for a given cell cycle, of many more origins than actually needed, raises the possibility that the unscheduled activation of low-efficient (or dormant) origins might represent one of the major causes of genomic instability.

cancer & replication originsAccurate regulation of origin licensing is crucial to avoid genomic instability, which is a hallmark of tumorigenesis [60–63]. Thus, origin selection and the regulation of origin firing under nor-mal (unperturbed) or stress conditions are both biologically and clinically relevant. A number of ORIs lower than what is necessary to guar-antee precise chromosome duplication will lead to incomplete duplication of genomic DNA, thus resulting in DNA strand breaks and gross chromosomal rearrangements in the surviving daughter cells. On the other hand, origins firing more than once in a given cell cycle will lead to gene amplification, polyploidy and other kinds of genome instability. Not surprisingly, inappropri-ate expression of pre-RC proteins has been found in a wide variety of premalignant lesions and cancers [62,64–67]. Overexpression of MCM2–7 and other licensing factors (both at protein and mRNA levels) has been observed in oral, laryn-geal, lung, mammary, ovarian, prostatic, renal, colorectal and hematological cancers [64,65]. This could simply be the consequence of the failure of cancer cells to properly exit the cell cycle, with a consequent higher fraction of cells remaining in-cycle, hence expressing licensing proteins. However, no correlation was found between expression of the proliferation marker Ki-67 and overexpressed Cdt1 and Cdc6 in different pretu-moral and tumoral lesions of the lung, colon and head-and-neck, suggesting that overexpression of pre-RC proteins is critical to tumorigenesis rather than a mere consequence of increased prolifera-tion [68]. On the other hand, cells expressing mutants of MCM2–7 show normal proliferative potential, high sensitivity to replication inhibitors and increased levels of spontaneous DNA dam-age. Notably, approximately 80% of female mice heterozygous for an MCM4 hypomorphic muta-tion (affecting protein stability) showed high fre-quency of chromosome instability and developed mammary adenocarcinomas [69]. Similarly, mice with reduced MCM2 expression develop T- and B-cell lymphomas at an early age [70], consistent with the idea that insufficient origin licensing



Figure 2. ORc1 peaks with the highest or the lowest amplitude show different distribution within origins of replication-containing regions. Visualization of HeLa Repli-Seq in the University of California, Santa Cruz browser showing two genomic regions of approximately 500 kb in length from human chromosomes 11 and 16. Colored vertical lines indicate ORC1 peaks that show the highest (green) or the lowest (red) amplitude within the algorithmically identified inverted-V apexes (or origins of replication-containing regions; red filled boxes). S1–S6 sequence-tag densities are also shown (see details in [25]). Scale bar indicates 500 kb.

S6

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hampers cell response (e.g., by activation of dor-mant origins) to sporadic replication defects, leading to incomplete replication.

Paucity of initiation events has been proposed to contribute to genomic instability of common fragile sites (CFS), which have been identified as hotspots for chromosomal rearrangements in cancer [71] and preferential targets for oncogene-induced DNA-damage in preneoplastic lesions [72]. In cells where it is highly expressed, a large region (700 kb in length) of the FHIT tumor-suppressor gene was shown to be ORI-free (or to contain very few and inefficient ORIs), while multiple ORIs were found to be evenly distrib-uted within the fragile region in cells where this gene is poorly expressed [73,74]. Thus, ORI pau-city (in combination with late replication tim-ing) might explain instability of a subset of CFS under replication stress conditions. On the other hand, the supposed stability of CFS in normal cells in physiological conditions, thought to be maintained by activation of dormant ORIs that allow the completion of DNA replication, has recently been challenged [75].

Alternatively, the licensing system might be deregulated as consequence of oncogene expres-sion, and be responsible for cells re-replicating genomic DNA, or even only fractions of it. CDT1 stabilization during S-phase has been linked to origin re-licensing and re-replication observed upon overexpression of a mutant form of cyclin D1 [76]. Similarly, ectopic expression of hyper-rep-lication , as in primary cells, induces senescence, with arrested senescent cells showing more than two copies of specific chromosomal loci and a strong induction of DNA damage response [77]. Finally, collisions of the transcription machinery with a replication fork, by causing RNA:DNA hybrid (R-loop) formation and consequent DNA breakage at very long genes (>800 kb), have been shown to contribute to CFS instability, which again depends on the expression of the underly-ing genes [78]. A similar mechanism may be at the basis of the genomic instability promoted by oncogenes in cancer cells (i.e., by enhancing the interference between replication and transcrip-tion). However, while a coherent model to explain how oncogenes induce replication stress and DNA damage is still missing, it has been shown that experimental overexpression of c-Myc or H-Ras increases levels of replication initiation [77,79]. Consequently, in a given S-phase window, more active replication forks than expected may deplete necessary replication factors, such as nucleotides.

Alternatively, and more intriguingly, oncogene-dependent changes in origin distribution and/or density may have an impact on the coordination of replication initiation and transcription. Thus, increased collisions between replication and tran-scription complexes may cause DNA damage and genomic instability [78,80–82]. Interestingly, over-expression of cyclin E (which directly activates Cdk2) increases origin firing, alters replication-fork progression and induces DNA damage and eventually senescence [83,84], while leading to chromosomal instability in cells that do not enter senescence [85]. Recent evidence suggests that cyclin E-induced replication-stress results from increased interference between replication and transcription, which slows down replication forks by increasing the number of collisions of replication forks with the transcription machinery itself or the RNA–DNA hybrids (R-loops) left behind by the transcription machinery [86].

In conclusion, genome-wide studies of repli-cation initiation sites significantly profited from massive sequencing. Since replication licensing is essential for cell proliferation, our understanding of the mechanisms underlying origin selection and replication timing in normal and cancer cells, and of the interference between DNA rep-lication and gene transcription, might represent a fundamental step towards understanding basic mechanisms of tumor growth, possibly leading to the identification of new anti-cancer molecular targets.

Future perspectiveIn the near future, genomic fingerprints of DNA replication in cancer cells will probably represent a powerful tool to predict biological properties of tumors and response to cancer drugs.

acknowledgementsThe authors thank L Luzi and L Riva for helpful discussions; and P Dalton for critical review of the manuscript.

Financial & competing interests disclosureThis study was supported by grants from the Associazione Italiana Ricerca sul Cancro (AIRC RF-2009–1470484) to PG Pelicci and the Fondazione Giancarla Vollaro to GI Dellino. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.



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ReferencesPapers of special note have been highlighted as:• of interest

1 Sacco E, Hasan MM, Alberghina L, Vanoni M. Comparative analysis of the molecular

mechanisms controlling the initiation of chromosomal DNA replication in yeast and in mammalian cells. Biotechnol. Adv. 30(1), 73–98 (2012).

2 Wu JR, Gilbert DM. A distinct G1 step required to specify the Chinese hamster

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3 Woo RA, Poon RY. Cyclin-dependent kinases and S phase control in mammalian cells. Cell Cycle 2(4), 316–324 (2003).

executive summAryOrigin recognition complex activity is regulated by cell-cycle-dependent post-translational modifications of ORc1

● Origin recognition complex (ORC)1 is bound to chromatin in the G1 phase, but not during mitosis.

● In Chinese hamster ovary cells, ORC1 levels do not change during the cell cycle, but a cytoplasmic mono-ubiquitinated form of ORC1 appears during the S-phase.

● By contrast, in HeLa and other human cell lines, ORC1 undergoes ubiquitin-dependent proteolysis during the S-phase.

high-resolution approaches for origins of replication identification: isolation & mapping of small nascent leading strands

● There are two leading strands that stem, bidirectionally, from origins of replication (ORIs), and their 5´ ends define the initiation site of replication.

● Before the advent of next-generation sequencing: genome-wide studies performed by hybridization of purified small nascent leading strands (SNS) to arrays containing 1–2% of the human or mouse genomes showed little overlap.

● After the advent of next-generation sequencing: genome-wide studies performed by deep sequencing of human SNS preparations revealed 200,000–250,000 peaks in all the analyzed cell types. However, this data set does not capture all human origins previously identified by bubble trapping methodology.

high-resolution approaches for ORi identification: isolation of pre-replication complex binding proteins

● ORIs are licensed during the G1 phase of the cell cycle, following sequential recruitment of the ORC1–6 proteins, Cdc6, Cdt1 and the mini-chromosome maintenance (MCM) complex (MCM2–7 proteins), which all together constitute the pre-replication complex (pre-RC).

● In the past, conventional chromatin immunoprecipitation methods for the genome-wide identification of pre-RC protein binding sites in mammalian cells have been unsuccessful.

● Upon equilibrium density-gradient centrifugation of sheared cross-linked chromatin, both pre-RC proteins and DNA from known ORIs are found in the low-density fractions, where the association of DNA fragments with very high-molecular-weight protein complexes shifts their buoyant density to values lower than that of bulk chromatin. This produces a significant ORC and MCM enrichment over background. High-throughput sequencing of the immunoprecipitated DNA fragments has allowed the identification of approximately 13,600 ORC1 binding sites.

Features of ORc1-binding sites

● The association between ORC1 binding and transcription initiation of known or unknown genes (transcribed into either coding or small noncoding RNAs) seems a universal feature of ORC1 sites, although levels of transcription differ significantly among them.

● ORC1 sites coincide with ORIs.

● The density of ORIs decreases while the S-phase progresses.

● ORI efficiency correlates with replication timing.

● The topographical relationship between ORC sites and SNS peaks along the chromosome fiber suggests that chromatin-bound ORC may associate with MCM complexes recruited to distal sequences by looping of the intervening chromosomal DNA.

cancer & ORis

● Inappropriate expression of pre-RC proteins was found in a wide variety of premalignant lesions and cancers.

● Oncogenes might favor genomic instability in cancer cells by enhancing the interference between replication and transcription.



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next-generation sequencing and dna replication in human cells: the future has arrived

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