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. 2007 Mar 7;26(5):1327-39.
doi: 10.1038/sj.emboj.7601585. Epub 2007 Feb 15.

Genome-wide Localization of pre-RC Sites and Identification of Replication Origins in Fission Yeast

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Free PMC article

Genome-wide Localization of pre-RC Sites and Identification of Replication Origins in Fission Yeast

Makoto Hayashi et al. EMBO J. .
Free PMC article

Erratum in

  • EMBO J. 2007 Jun 6;26(11):2821. Tazumi, Mitsutoshi [corrected to Tazumi, Atsutoshi]

Abstract

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.

Figures

Figure 1
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.
Figure 2
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.
Figure 3
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.
Figure 4
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.
Figure 5
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.
Figure 6
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 origin), non-ARS, ars2004 (early origin), cen2 and AT2080 (late origin) are shown. (B) Replication efficiency in the presence of HU. HM668 (h cdc25-22 nmt1-TK) and HM1347 (h cdc25-22 nmt1-TK ars2004AT2080 AT2080ars2004) synchronously released from G2/M block were labeled with BrdU in the presence of HU for 120 min and BrdU DNA was separated by CsCl centrifugation as described in Figure 1. The amounts of DNA in the light–light (LL) and heavy–light (HL) density fractions were measured by real-time PCR, and the replication efficiency (Re %) was obtained by the equation Re=[(HL/2)/(LL+HL/2)] × 100.
Figure 7
Figure 7
Locations of Orc1, Mcm6 and BrdU DNA in subtelomeric and centromeric heterochoromain regions. (A) Locations of Orc1 (orange bars, top), Mcm6 (blue bars, middle) and BrdU incorporation (green bars, bottom) in left and right subtelomeric regions on chromosome II in wild-type HM668 (h cdc25-22 nmt1-TK) are presented. White boxes above the panels denote open reading frames. Telomere repeats and telomere-associated sequence (TAS) are not included in the analysis. Signals are absent in gray-shaded regions because of the presence of homologous sequences. (B) Locations of Orc1, Mcm6 and BrdU incorporation at centromere on chromosome II are shown. The physical map of fission yeast cen2 above the panels denotes otr comprised of dg and dh elements, imr and cnt. Vertical lines indicate tRNA genes. Signals are not present in gray-shaded regions at imr2R and otr2R because of the presence of identical sequences with imr2L and otr2L, respectively. (C) Localization of Orc1 and Orc4 but not of Mcm6 and Mcm2 at cnt2. ChIP with Orc1-5Flag, Orc4, Mcm6 and Mcm2 was carried out from G1-arrested cells as described in Figure 1. DNA recovered in ChIP was analyzed by real-time PCR using primers amplifying non-ARS, ars2004, dg and cnt2 regions, and relative recovery normalized to non-ARS region is presented.

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