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Shin-Ichiro Hiraga^,a, Aki Hagihara-Hayashi^,b, Tomoko Ohya^c and Akio Sugino^*

Laboratories for Biomolecular Networks, Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamada-oka, Suita, Osaka 565-0871, Japan

Early in eukaryotic cell cycle, a pre-RC is assembled at each replication origin with ORC, Cdc6, Cdt1 and Mcm2-7 proteins to license the origin for use in the subsequent S phase. Licensed origin must then be activated by S-Cdk and Ddk. At the onset of S phase, RPA is loaded on to the ARS in a reaction stimulated by S-Cdk and Ddk, followed by Cdc45-dependent loading of pol , -, and -. This study examines cell cycle-dependent localization of pol , - and - in Saccharomyces cerevisiae using immuno-histochemical and chromatin immuno-precipitation methods. The results show that pol , -, or - localizes on chromatin as punctate foci at all stages of the cell cycle. However, some foci overlap with or are adjacent to foci pulse-labeled with bromodeoxyuridine during S phase, indicating these are replicating foci. DNA microarray analysis localized pol , -, and - to early firing ARSs on yeast chromosome III and VI at the beginning of S phase. These data collectively suggest that bidirectional replication occurs at specific foci in yeast chromosomes and that pol , -, and - localize and function together at multiple replication forks during S phase.

^aPresent address: Department of Molecular and Cell Biology, Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, UK.

^bPresent address: Communications Research Laboratory, Kansai Advanced Research Center, 588-2 Iwaoka, Iwaoka-cho, Nishi-ku, Kobe, Hyogo 651–2492, Japan.

^cPresent address: Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan.

These authors equally contributed to this work.

^* Correspondence: E-mail: asugino{at}fbs.osaka-u.ac.jp

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