DNA polymerases alpha, delta, and epsilon localize and function together at replication forks in Saccharomyces cerevisiae.
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 alpha, -delta, and -epsilon. This study examines cell cycle-dependent localization of pol alpha, -delta and -epsilon in Saccharomyces cerevisiae using immuno-histochemical and chromatin immuno-precipitation methods. The results show that pol alpha, -delta, or -epsilon 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 alpha, -delta, and -epsilon 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 alpha, -delta, and -epsilon localize and function together at multiple replication forks during S phase.
Hiraga
Shin-Ichiro
S
Laboratories for Biomolecular Networks, Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamada-oka, Suita, Osaka 565-0871, Japan.
Hagihara-Hayashi
Aki
A
Ohya
Tomoko
T
Sugino
Akio
A
D002453:Cell Cycle / Q000235:genetics / Q000502:physiology; D047369:Chromatin Immunoprecipitation; D015825:Chromosomes, Fungal / Q000201:enzymology / Q000235:genetics; D004256:DNA Polymerase I / Q000235:genetics / Q000378:metabolism*; D004257:DNA Polymerase II / Q000235:genetics / Q000378:metabolism*; D019951:DNA Polymerase beta / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication / Q000235:genetics / Q000502:physiology*; D007150:Immunohistochemistry; D020411:Oligonucleotide Array Sequence Analysis; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D004256:DNA Polymerase I; D004257:DNA Polymerase II; D019951:DNA Polymerase beta