© Blackwell Science Limited Genes to Cells (2002) 7 , 781–789 781 Blackwell Science, LtdOxford, UKGTCGenes to Cells1356-9597© Blackwell Science LtdAugust 200278Original ArticleGenome-wide mapping of early firing originsN Yabuki et al. Mapping of early firing origins on a replication profile of budding yeast Nami Yabuki†, Hiromichi Terashima† and Kunio Kitada* Department of Genome Science, Nippon Roche Research Center, Kamakura, Kanagawa 247-8530, Japan Abstract Background : Understanding of the firing time deter-mination of replication origins in the entire genomewill require a genome-wide survey of replicationorigins and their mapping on chromosomes. A micro-array technology was applied to obtain a genome-wide profile of DNA replication and to classify earlyfiring origins. Results : A total of 260 potential replication origins(PROs) were identified in the entire budding yeastgenome: 247 as defined peaks on the replicationprofile and 13 as regions located in the chromosomaltermini. Based on the firing time, the 247 PROswere classified into 143 early PROs and 104 latePROs, that were not randomly distributed on chro-mosomes but formed separated clusters. Most ofthe early PROs were found to fire in the presence ofhydroxyurea, indicating that they were free from thecontrol of the intra-S-checkpoint mediated by Mec1and Rad53. Conclusions : The monitoring method of DNA repli-cation and the analysis method of microarray dataused in this study proved powerful for obtaining agenome-wide view of the initiation and progressionof DNA replication. Introduction The budding yeast genome, which comprises 16 chro-mosomes, replicates from a number of replication originsin the S phase. The S phase lasts 25–30 min under normalculture conditions and DNA strands of 13.5M bp in totalare duplicated. The mechanism of both initiation andregulation of DNA replication has been extensivelystudied (Campbell & Newlon 1991; Sugino 1995; Kelly& Brown 2000; Diffley 2001). Replication origins havespecific nucleotide sequences, to which several proteinsbind to form an initiation/replication complex. Theseorigins have their own time of firing in the S phase andare classified into early origins and late origins. The earlyorigins fire in the presence of hydroxyurea (HU), whilethe late origins do not. HU is an inhibitor of ribonu-cleotide reductase, which is required for the synthesis ofdNTPs. The nucleotide depletion caused by HU wouldactivate the intra-S-checkpoint and block the firing ofthe late origins (Santocanale & Diffley 1998; Shirahige et al . 1998; Desany et al . 1998). The molecular mechanismdetermining the time of origin firing has not been wellunderstood. A genome-wide fractionation of earlyand late origins and their positioning on the chromo-somes would be necessary for a more complete under-standing of the timing mechanism of origin firing inrelation to the replication of the entire genome. Here,we show a genome-wide replication profile on whichearly origins, being able to fire in the presence of HU,are mapped. Results Copy number changes detected by a microarray technology DNA microarray technology was used to obtain a repli-cation profile of the yeast genome. Recently, Fangman’sgroup analysed the replication profile by combining adensity-separation method and a DNA microarraytechnology (Raghuraman et al . 2001). Their strategywas based on isotopically labelling of DNA in order todifferentiate newly synthesized DNA from not-yet-synthesized DNA in the progression of DNA replication.We took a more direct approach: monitoring the changeof copy number from one to two during DNA replication Communicated by : Fumio Hanaoka* Correspondence : E-mail: [email protected]†These authors contributed equally to this work. GTC_559.fm Page 781 Tuesday, July 30, 2002 1:49 PMN Yabuki et al. Genes to Cells (2002) 7 , 781–789 © Blackwell Science Limited 782 using DNA microarray technology. As a beginning, thesensitivity of the microarray technology was examined.Genomic DNA was extracted from a mutant strainwhich had an extra minichromosome constructedfrom the entire right arm of chromosome IX, labelledwith a biotinyl nucleotide and used for hybridization tooligonucleotide microarrays. Hybridization signals of251 probes on chromosome IX from the mutant strain werecompared with those from the original wild-type. Onlythe probes in the right arm of chromosome IX exhibitedhigher signals and their ratios to the wild-type strainreached around 2.0 (Fig. 1). Signal ratios in the probeson the left arm of chromosome IX and all the otherchromosomes remained around 1.0. The use of meanratios, calculated from three neighbouring ratio values, cansmooth the data. These results were reproducible inrepeated experiments. We concluded that the microarraytechnology we used allowed us to monitor small changesin copy number of the yeast genome. DNA replication profile of the whole yeast genome The cell cycle of yeast cells was synchronized by treat-ment with α -factor. After release from the G1 block, thecells were harvested in every 2.5 min and their genomicDNAs were hybridized to microarrays. A data set ofhybridization signals from the 8105 probes was obtainedat each sampling time, from 0 to 55 min after the release.The relative DNA content of the cells was measured byflow cytometry and a logistic curve was obtained (Fig. 2).The DNA content on the curve was used for scaling ofthe hybridization signals. The signals were convertedinto ratios relative to the signals of the G1 arrested cells.To smooth the data, each ratio value was replaced withthe mean of nine neighbouring ratio values in a 3 × 3Figure 1 Identification of two-copy regionson chromosome IX. Genomic DNA wasisolated from the mutant strain SH5752which has an extra minichromosome originatingfrom the entire right arm of chromosome IXand its wild-type strain W303-1A and separatelyhybridized to oligonucleotide microarrays.Hybridization signals from 251 probes fromthe mutant strain are shown as ratios to thewild-type strain. Two-repeated experimentalresults are shown: (A) and (B) from the firstexperiment and (C) and (D) from the secondexperiment. In (B) and (D), the signal ratioswere converted into means of the neighbouringthree ratios. Numbers in (A–D) on the hori-zontal axes indicate probes placed in