Genome-wide localization of pre-RC sites and identification of replication origins in fission yeast
2007; Springer Nature; Volume: 26; Issue: 5 Linguagem: Inglês
10.1038/sj.emboj.7601585
ISSN1460-2075
AutoresMakoto Hayashi, Yuki Katou, Takehiko Itoh, Mitsutoshi Tazumi, Yoshiki Yamada, Tatsuro Takahashi, Takuro Nakagawa, Katsuhiko Shirahige, Hisao Masukata,
Tópico(s)Genomics and Chromatin Dynamics
ResumoArticle15 February 2007free access Genome-wide localization of pre-RC sites and identification of replication origins in fission yeast Makoto Hayashi Makoto Hayashi Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka, Japan Search for more papers by this author Yuki Katou Yuki Katou Riken Genomic Science Center, Human Genome Research Group, Genome Informatics Team, Tsurumi-ku, Yokohama, Kanagawa, Japan Search for more papers by this author Takehiko Itoh Takehiko Itoh Research Center for Advanced Science and Technology, Mitsubishi Research Institute Inc., Chiyoda-ku, Tokyo, Japan Search for more papers by this author Mitsutoshi Tazumi Mitsutoshi Tazumi Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka, Japan Search for more papers by this author Yoshiki Yamada Yoshiki Yamada Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka, JapanPresent address: The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA Search for more papers by this author Tatsuro Takahashi Tatsuro Takahashi Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka, JapanPresent address: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA Search for more papers by this author Takuro Nakagawa Takuro Nakagawa Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka, Japan Search for more papers by this author Katsuhiko Shirahige Katsuhiko Shirahige Riken Genomic Science Center, Human Genome Research Group, Genome Informatics Team, Tsurumi-ku, Yokohama, Kanagawa, Japan Center for Biological Resources and Informatics, Division of Gene Research, and Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Midori-ku, Yokohama, Japan Search for more papers by this author Hisao Masukata Corresponding Author Hisao Masukata Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka, Japan Search for more papers by this author Makoto Hayashi Makoto Hayashi Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka, Japan Search for more papers by this author Yuki Katou Yuki Katou Riken Genomic Science Center, Human Genome Research Group, Genome Informatics Team, Tsurumi-ku, Yokohama, Kanagawa, Japan Search for more papers by this author Takehiko Itoh Takehiko Itoh Research Center for Advanced Science and Technology, Mitsubishi Research Institute Inc., Chiyoda-ku, Tokyo, Japan Search for more papers by this author Mitsutoshi Tazumi Mitsutoshi Tazumi Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka, Japan Search for more papers by this author Yoshiki Yamada Yoshiki Yamada Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka, JapanPresent address: The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA Search for more papers by this author Tatsuro Takahashi Tatsuro Takahashi Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka, JapanPresent address: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA Search for more papers by this author Takuro Nakagawa Takuro Nakagawa Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka, Japan Search for more papers by this author Katsuhiko Shirahige Katsuhiko Shirahige Riken Genomic Science Center, Human Genome Research Group, Genome Informatics Team, Tsurumi-ku, Yokohama, Kanagawa, Japan Center for Biological Resources and Informatics, Division of Gene Research, and Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Midori-ku, Yokohama, Japan Search for more papers by this author Hisao Masukata Corresponding Author Hisao Masukata Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka, Japan Search for more papers by this author Author Information Makoto Hayashi1, Yuki Katou2, Takehiko Itoh3, Mitsutoshi Tazumi1, Yoshiki Yamada1, Tatsuro Takahashi1, Takuro Nakagawa1, Katsuhiko Shirahige2,4 and Hisao Masukata 1 1Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka, Japan 2Riken Genomic Science Center, Human Genome Research Group, Genome Informatics Team, Tsurumi-ku, Yokohama, Kanagawa, Japan 3Research Center for Advanced Science and Technology, Mitsubishi Research Institute Inc., Chiyoda-ku, Tokyo, Japan 4Center for Biological Resources and Informatics, Division of Gene Research, and Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Midori-ku, Yokohama, Japan *Corresponding author. Department of Biology, Graduate School of Science, Osaka University, 1-1, Machikaneyama-cho, Toyonaka, Osaka 560-0043, Japan. Tel.: +81 6 6850 5432; Fax: +81 6 6850 5440; E-mail: [email protected] The EMBO Journal (2007)26:1327-1339https://doi.org/10.1038/sj.emboj.7601585 Correction(s) for this article Genome-wide localization of pre-RC sites and identification of replication origins in fission yeast06 June 2007 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info DNA replication of eukaryotic chromosomes initiates at a number of discrete loci, called replication origins. Distribution and regulation of origins are important for complete duplication of the genome. Here, we determined locations of Orc1 and Mcm6, components of pre-replicative complex (pre-RC), on the whole genome of Schizosaccharomyces pombe using a high-resolution tiling array. Pre-RC sites were identified in 460 intergenic regions, where Orc1 and Mcm6 colocalized. By mapping of 5-bromo-2′-deoxyuridine (BrdU)-incorporated DNA in the presence of hydroxyurea (HU), 307 pre-RC sites were identified as early-firing origins. In contrast, 153 pre-RC sites without BrdU incorporation were considered to be late and/or inefficient origins. Inactivation of replication checkpoint by Cds1 deletion resulted in BrdU incorporation with HU specifically at the late origins. Early and late origins tend to distribute separately in large chromosome regions. Interestingly, pericentromeric heterochromatin and the silent mating-type locus replicated in the presence of HU, whereas the inner centromere or subtelomeric heterochromatin did not. Notably, MCM did not bind to inner centromeres where origin recognition complex was located. Thus, replication is differentially regulated in chromosome domains. Introduction To ensure that a complete set of the eukaryotic genome is precisely duplicated during the limited period of S phase in every cell cycle, DNA replication initiates at a number of replication origins on chromosomes (Gilbert, 2001; Bell and Dutta, 2002). As each chromosome region replicates in a specific period within S phase, timing of origin activation must be regulated. Although we have a growing understanding of protein factors involved in initiation and elongation of replication, the mechanisms of origin activation at the chromosome level are yet to be clarified in detail. Thus, it is important to determine locations of all replication origins on chromosomes. However, only small numbers of replication origins have so far been identified in most organisms other than budding yeast Saccharomyces cerevisiae (MacAlpine and Bell, 2005). The process of initiation of replication at individual replication origins is composed of two major steps, licensing of replication origins in G1 phase and activation of the origins in S phase. In G1 phase, pre-replicative complexes (pre-RCs) are formed at replication origins (Bell and Dutta, 2002; Kearsey and Cotterill, 2003). This requires binding of the origin recognition complex (ORC) to a replication origin, followed by assembly of the minichromosome maintenance (MCM) complex, depending on the loading factors, Cdc6/Cdc18 and Cdt1 (Diffley et al, 1994; Bell and Dutta, 2002). Although pre-RC formation is essential for initiation of replication, it is not in itself sufficient. Origin activation in S phase is regulated by two conserved protein kinases, cyclin-dependent protein kinase (CDK) and Cdc7–Dbf4 protein kinase (Dbf4-dependent kinase, DDK). These kinases are required for assembly of several other protein factors, including Cdc45 and GINS onto pre-RCs. This may lead to activation of MCM helicase and origin DNA unwinding, and the replication machinery is established through assembly of RPA and DNA polymerases onto the single-stranded DNA (Bell and Dutta, 2002). Although proteins involved in initiation of replication are conserved among eukaryotes, the nucleotide sequences of replication origins are very diverse among organisms (Gilbert, 2001), mainly because of differences in DNA-binding properties of ORCs. In budding yeast, ORC recognizes the specific sequence called the ARS consensus sequence (ACS). In contrast, no clear consensus sequence has been found in origins in fission yeast, Schizosaccharomyces pombe (Clyne and Kelly, 1995; Dubey et al, 1996; Okuno et al, 1999), although AT-rich sequences to which ORC preferentially binds are required (Chuang and Kelly, 1999). Requirements for specific sequences become less clear in multicellular organisms such as metazoans, and ORC exhibits little sequence specificity in DNA binding in vitro (Vashee et al, 2003; Remus et al, 2004). Therefore, it is important to determine the locations of ORC binding and DNA synthesis experimentally. Genome-wide analyses of replication kinetics and distribution of ORC and MCM proteins using DNA microarrays have been performed in budding yeast (Raghuraman et al, 2001; Wyrick et al, 2001). The majority of proposed ARS (pro-ARS) sites identified by ChIP-based analysis exhibit ARS activity, and the correlation with actual initiation sites has been demonstrated (Feng et al, 2006). On the other hand, replication timing analyses using microarrays with human, mouse and Drosophila chromosomes have suggested links between early replication timing and active transcription in large chromatin domains (MacAlpine and Bell, 2005). However, because of difficulties in genome-wide analysis of replication factor binding sites in metazoans, it has not been possible to clarify the relationship between pre-RC sites and selection of active origins. Fission yeast is a suitable model organism to study genome-wide regulation of chromosome replication, because both the structures of replication origins and chromatin configuration have similarities with those in metazoan organisms. In fission yeast, owing to preferred binding of ORC to AT-rich sequences (Chuang and Kelly, 1999), and the requirement of multiple ORC binding sites for origin activity (Takahashi et al, 2003), replication origins have been predicted to be ‘A+T-rich islands’ located preferentially in intergenic regions (Segurado et al, 2003). Locations of single-stranded DNA regions under nucleotide-depleting conditions are consistent with this prediction (Feng et al, 2006). Because the ARS activity of intergenic regions correlates with the AT content and the length, it has been proposed that replication origins fire stochastically in fission yeast (Dai et al, 2005). Single-molecule analyses using DNA combing also support the stochastic model (Patel et al, 2006). However, owing to the lack of information on genome-wide distribution of pre-RC sites, it has remained an open question whether all the pre-RCs are activated or only a subset is selected to fire. In this study, we conducted high-resolution mapping of Orc1 and Mcm6 binding sites in G1 phase, using a tiling array covering almost the entire genome of fission yeast, resulting in identification of pre-RC sites precisely on the whole genome. Mapping of nascent DNA in the presence of HU in wild-type and checkpoint-deficient cds1Δ cells allowed us to identify early-firing replication origins and late and/or inefficient origins. Replication timing is not intrinsic to origin function but dependent on its surrounding regions. The centromeric and subtelomeric heterochromatin regions behaved differently, suggesting distinct regulation in chromatin domains. Results Mapping of pre-RCs on fission yeast chromosomes Initiation of DNA replication in eukaryotic cells requires the ordered assembly of replication factors at specific sites on the chromosome. To determine the site of pre-RC formation on fission yeast chromosomes, DNA immunoprecipitated with Orc1 and Mcm6 in G1-arrested cells (Ogawa et al, 1999; Takahashi et al, 2003) was analyzed with a tiling array that covers almost the entire genome of fission yeast at 250 base pair (bp) resolution, except for telomeres and rDNA repeats. Both Orc1 and Mcm6 were located exclusively at intergenic regions, and 84% of Orc1-binding sites colocalized with Mcm6 (Figure 1 and Supplementary Figures S1, S2 and Supplementary Table S1). A total of 460 pre-RC sites, where peaks of Orc1 and Mcm6 shown in Supplementary Figures S1 and S2 colocalized, were identified (black triangles in Figure 1, Supplementary Table S1). The pre-RC sites were distributed throughout the chromosomes, with an average separation of 26.7 kb and enriched at the centromeres and the subtelomeric regions. Enrichment at the subtelomeric regions was not observed on chromosome III, where both ends of the array were flanked with rDNA repeats. Orc4, another ORC complex component, was highly colocalized with Orc1 (>90%; Supplementary Figure S3 and Supplementary Table S1), suggesting that ORC complex was localized at pre-RC sites. Figure 1.Locations of Orc1- and Mcm6-binding sites and BrdU incorporation sites on fission yeast chromosomes. For mapping of Orc1 and Mcm6 localization sites, HM568 (h− nda3-KM311 cdc10-129 ura4-D18 leu1-32 orp1-5flag/pREP82-cdc18 pREP81-cdt1) cells expressing Cdc18 and Cdt1 were arrested at the cdc10 arrest point in G1 phase and used for ChIP. The orange and blue vertical bars represent the binding ratios of loci showing enrichment of ChIP fractions with anti-Flag-Orc1 (orange bars in top panels) and anti-Mcm6 (blue bars in middle panels) antibodies, respectively, for regions 1000–1100 kb on chromosome I, 1500–1600 kb on chromosme II and 1800–1900 kb on chromosome III. For mapping of nascent DNA synthesis, HM668 (h− cdc25-22 nmt1-TK) cells arrested at the G2/M boundary were released at 25°C for 90 min in the presence of 10 mM HU and 200 μM BrdU. Cellular DNA was digested with HaeIII and centrifuged in a CsCl gradient. The experimental scheme is shown in Figure 2A. Relative enrichment of BrdU-labeled DNA compared with the control whole cell DNA is presented (green bars in bottom panels). Black triangles indicate pre-RC sites identified as colocalization sites of Orc1 and Mcm6, which were programmatically picked up (Supplementary Figure S1, S2 and Supplementary Table S1). Names of known replication origins colocalized with pre-RCs are shown. Horizontal bars show open reading frames. The scale of the vertical axis is log2. Download figure Download PowerPoint Identification of early-firing replication origins that incorporate BrdU in the presence of HU For identification of active replication origins, it is crucial to label newly synthesized DNA around replication origins. We labeled newly synthesized DNA by incorporation of 5-bromo-2′-deoxyuridine (BrdU), a heavy-density nucleotide analogue. Because fission yeast cells do not normally intake BrdU, owing to lack of thymidine kinase activity, the herpes simplex virus thymidine kinase gene was expressed from the inducible promoter. The experimental scheme is shown in Figure 2A. Fission yeast cells expressing thymidine kinase were synchronously released from the G2/M boundary in the presence of BrdU and hydroxyurea (HU) that depletes dNTPs. BrdU-labeled DNA was separated in an equilibrium gradient of CsCl by centrifugation. To examine whether BrdU was selectively incorporated around replication origins, the amounts of BrdU–DNA for the ars2004 locus, an early-firing replication origin (Okuno et al, 1999), and a non-ARS (non-origin) region were analyzed by real-time PCR. After 90 min of BrdU labeling in the presence of HU, about 50% of the ars2004 region was recovered in the heavy–light (HL) density fractions, whereas non-ARS fragment, about 30 kb distant from the origin, remained in the light–light density fractions (Figure 2B), indicating selective incorporation of BrdU around the origin. We also confirmed that both ars2004 and non-ARS regions were fully substituted with BrdU under HU-free conditions (Supplementary Figure S4), excluding the possibility that selective incorporation of BrdU around the ars2004 was due to a shortage of BrdU. As recovery of the nascent DNA by the method was verified, the HL DNA fractions were pooled and subjected to the whole-genome analysis. Figure 2.Incorporation of BrdU preferentially into origin proximal regions. (A) A scheme of the experiment is shown. HM668 (h− cdc25-22 nmt1-TK) cells were synchronized and labeled with BrdU in the presence of HU as described in Figure 1. Cellular DNA at 0 and 90 min after release from G2/M block was digested with HaeIII and centrifuged in a CsCl gradient. (B) DNA in each fraction at 0 (blue open circles) and 90 min (red filled circles) was analyzed by real-time PCR using primers for ars2004 (left) and non-ARS (right) regions. Relative recovery (%) among total DNA recovered is presented together with the refractive index (green triangles). For DNA microarray analysis, the heavy–light density fractions 8–12 of 90 min (BrdU–DNA) and light fractions 1–6 of 0 min (whole DNA) were pooled and used for comparative analysis with tiling array. Download figure Download PowerPoint The results of microarray analysis showed that BrdU-labeled DNA was colocalized with Orc1 and Mcm6 at a very high frequency (Figure 1, green bars in bottom panels; Supplementary Figure S5 and Supplementary Table S1). At the ars2004 origin locus, BrdU-labeled DNA spanned about 10 kb around the intergenic region, where Orc1 and Mcm6 were confined (Figure 1, middle set of panels). This is consistent with bidirectional DNA synthesis initiated from the origin in early S phase (Okuno et al, 1997; Takahashi et al, 2003). The colocalization sites of Orc1, Mcm6 and BrdU, a total of 307 loci, 119, 107 and 81, on chromosomes I, II and III, respectively, were defined as early-firing replication origins (called as early origins) that initiated replication in the presence of HU (red diamonds in Figure 3 and Supplementary Table S1). In contrast, 153 Orc1-Mcm6 colocalization sites, 88, 62 and 3, on chromosomes I, II and III, respectively, did not incorporate BrdU in the presence of HU. Because these BrdU-negative origins are composed of late-firing origins and inefficient origins, they are collectively designated as late origins below (blue diamonds in Figure 3 and Supplementary Table S1). We show that some late origins are repressed by replication checkpoint in the later section. Figure 3.Distributions of early and late origins. Locations of the early origins (red diamonds) and the late and/or inefficient origins (blue diamonds) are shown on chromosomes I, II and III. Positions of known replication origins are shown. Positions of centromeres are shown by green ellipses. Download figure Download PowerPoint Among 36 origins previously identified by two-dimensional (2D) gel electrophoresis, 28 coincide with the early origins identified here, whereas two match to the late origins (Figure 3 and Supplementary Table S1) (Dubey et al, 1994; Wohlgemuth et al, 1994; Smith et al, 1995; Okuno et al, 1997; Sanchez et al, 1998; Gomez and Antequera, 1999; Segurado et al, 2002, 2003). At the remaining six known origin loci, Orc1 signals were below the standard, although at least either BrdU or Mcm6 signals were detected. When the replication origins identified in this study were compared with those obtained in the previous genome-wide analyses, 189 out of 307 early origins (62%) and 69 out of 153 late origins (45%) coincide precisely with the origins (A+T-rich islands) predicted from AT content calculation (Segurado et al, 2003) (Supplementary Table S1). On the other hand, the early origins are colocalized at a high frequency (239 origins, 78%) with the origins identified as peaks of DNA content increase, whereas the late origins coincide at a lower frequency (34 origins, 22%) (Heichinger et al, 2006). In comparison with the origins identified as the center of single-stranded DNA formed in the presence of HU (Feng et al, 2006), 50% (154 loci) of the early origins and 16% (24 loci) of the late origins match with the previously identified origins (Supplementary Table S1). Early origins clustered in narrow regions It should be noted that Orc1-Mcm6 colocalization sites are frequently clustered within a broad BrdU-labeled region extending 20–30 kb, such as at positions 510–530 of chromosome II (Figure 4A). The presence of multiple peaks of BrdU-labeled DNA corresponding to Orc1-Mcm6-binding sites is consistent with the initiation from closely located several origins, although the possibility that DNA synthesis extended from a unique origin remains. To distinguish these possibilities, we first examined whether each of Orc1-Mcm6 colocalization sites exhibited the ARS activity. Among 11 fragments derived from the region, five generated transformants at a high frequency (Figure 4B), indicating that the ARS fragments are clustered. Figure 4.ARS activity and two-dimensional gel electrophoresis analysis of clustered early-firing origins. (A) Locations of Orc1 (orange, top panel), Mcm6 (blue, middle panel) and BrdU-labeled DNA (green, bottom panel) in the 500–540 kb region of chromosome II are presented. (B) Eleven fragments containing intergenic regions, shown by horizontal lines in (A), were cloned into the pYC11 vector and used for transformation of HM123 (h− leu1-32). Transformants formed on minimal media plates after 4 days at 30°C are presented. Vector alone and the ars2004 plasmid were used as controls. Plus signs, (+++, ++ and +) below panels represent large, middle and small colony size, respectively, whereas a minus sign shows the absence of any visible colony. (C) HM668 (h− cdc25-22 nmt1-TK) cells released from the G2/M block were cultured at 25°C for 90 min in the presence of 10 mM HU, and replication intermediates were analyzed by 2D gel electrophoresis. Locations of the restriction fragments analyzed by 2D gel methods are shown above the map of open reading frames and the relevant restriction enzyme sites: B, BamHI; Xb, XbaI; N, NdeI; H, HindIII; C, ClaI; S, SpeI; E, EcoRI. Positions of the hybridization probes, which correspond to the fragments used for the ARS assay in (B), are shown as gray bars. Download figure Download PowerPoint Next, we examined whether replication initiated from these ARSs on the chromosome. Chromosomal DNA of cells synchronously released from G2/M in the presence of HU was analyzed by 2D gel electrophoresis. The results presented in Figure 4C show that bubble arcs, which are indicative of initiation of replication, were detected for fragments 3, 7, 8 and 11 (black triangles in Figure 4C). Fragments 7, 8 and 11 correspond to ori2031E, 2032E and 2033E, respectively (Supplementary Table S1). Another early origin exists in fragment 3, although this site was not identified as the origin by genome-wide analysis owing to weak Orc1 signal (Supplementary Figure S1). These results demonstrate that clustered pre-RC sites act as early origins. Initiation of replication from closely located origins has been reported for the ura4+ locus on chromosome III by 2D gel and DNA combing analyses (Dubey et al, 1994; Patel et al, 2006), and it seems to be common on fission yeast chromosomes. Repression of late origins by checkpoint kinase Cds1 Late-firing origins in budding yeast are repressed by checkpoint pathway under replication stress such as depletion of nucleotides by HU (Santocanale and Diffley, 1998; Feng et al, 2006). Mapping of single-stranded DNA in the HU-arrested fission yeast cells has suggested that similar regulation exists in fission yeast (Feng et al, 2006). On the other hand, deletion of Rad3, the ATR homologue in fission yeast, affects initiation from a small number of origins (Heichinger et al, 2006). We tested whether the late origins identified in this study might be activated in the absence of replication checkpoint kinase Cds1/Chk2. Wild-type and cds1Δ cells were synchronously released from G2/M block and labeled with BrdU in the presence of HU for 150 min. The BrdU DNA purified by CsCl centrifugation was analyzed by DNA microarray. The results of wild type at 150 min were very similar to those at 90 min, except that BrdU DNA extended further than those at 90 min, which is consistent with slow DNA synthesis in the presence of HU (top panels in Figure 5; Supplementary Figure S6). In contrast, small but significant BrdU incorporation was detected in the subtelomeric regions of chromosomes I and II specifically in cds1Δ cells, although BrdU DNA did not form peaks at most of late origins (middle panels in Figure 5; Supplementary Figure S7). These results suggested that the majority of late origins did not fire at a comparable efficiency to the early origins even in cds1Δ mutant. However, when the ratio of BrdU DNA in cds1Δ to that in wild type was calculated, cds1Δ-specific BrdU incorporation was observed at subtelomeric regions and at the late-origin loci, but not at the early-origin loci (brown bars in bottom panels of Figure 5; Supplementary Figure S8). BrdU incorporation was increased at 90 late-origin loci (59% of the late origins) and at 10 early-origin loci (3% of the early origins), showing specific firing of late origins in cds1Δ cells. We also examined locations of BrdU-labeled DNA prepared at 210 min after release in the presence of HU, and the results were very similar to those at 150 min (data not shown). Furthermore, the results of real-time PCR analysis showed that BrdU incorporation increased in cds1Δ cells compared with wild type at the late origin AT2080 and at the subtelomere locus but not at the early origin or non-ARS locus (Supplementary Figure S9). These results suggest that subsets of late origins in the arm and the subtelomeric regions are repressed in part by replication checkpoint regulation. Figure 5.Incorporation of BrdU at late origins in subtelomere and chromosome arm in cds1Δ cells. HM668 (h− cdc25-22 nmt1-TK) and HM1405 (h− cdc25-22 nmt1-TK cds1Δ∷kanMX6) were released from G2/M block and labeled with BrdU for 150 min at 25°C in the presence of HU and the genomic DNA was analyzed with DNA microarray, as described in Figure 1. Green vertical bars represent relative enrichment of BrdU-incorporated DNA in wild type (top panels) and in cds1Δ (middle panels) in 300 kb region from the left end of chromosome I. The bottom panels show ratios of enrichment of BrdU DNA in cds1Δ to that in wild type (brown vertical bars). Red and blue triangles above panels show locations of the early and late origins identified in this work, respectively. Horizontal bars show open reading frames. The scale of the vertical axis is log2. BrdU signals were detected in spite of the absence of replication origins in gray-shaded regions, where localization of Orc1 or Mcm6 was not analyzed (in Supplementary Figures S1 and S2) because of the presence of homologous sequences. Download figure Download PowerPoint Replication timing of early and late origins is affected by their surrounding regions To examine whether replication timing of the early and late origins is intrinsic to replication origins or affected by their surrounding regions, we constructed a strain in which an early origin, ars2004, and a late origin, AT2080, were mutually replaced (Figure 6A). These origins were chosen because initiation of replication from the loci were confirmed by 2D gel analysis (Okuno et al, 1997; Segurado et al, 2003) and because two flanking genes are not essential for the viability, allowing manipulation of the loci. Fragments retaining ARS activity in plasmid transformation assay were used for replacement (data not shown). Wild type and ars2004/AT2080 cells were synchronized and labeled with BrdU in the presence of HU, and the HL (replicated) and light–light (unreplicated) DNA was separated in a CsCl gradient. The replication efficiencies of ars2004 and AT2080 as well as the early origin and non-ARS controls were measured by real-time PCR analyses. In the case of wild type, replication efficiencies of AT2024 and ars2004 were about 20–35% in the presence of HU, whereas the efficiency of AT2080 was about 1%, similar to the level of non-ARS locus, consistent with early firing at ars2004 but not at AT2080 (Figure 6B). In the origin-replaced strain ars2004/AT2080, however, the efficiency of ars2004 fragment integrated at AT2080 locus was about 3%, which was similar to the non-ARS locus, whereas the efficiency of AT2080 placed at the ars2004 locus was 17%, similar to the value of the early origin AT2024 (24%) (Figure 6B). These results show that replication timing of the origin is affected by its surrounding regions. Figure 6.Replication of the early and late origin fragments at the mutually exchanged loci. (A) Schematic drawing of chromosome II in wild type and the origin-exchange strains. Positions of AT2024 (early o
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