DNA polymerases α, δ, and ɛ localize and function together at replication forks inSaccharomyces cerevisiae

Abstract

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 α, ‐δ, and ‐ɛ. This study examines cell cycle‐dependent localization of pol α, ‐δ and ‐ɛ in Saccharomyces cerevisiae using immuno‐histochemical and chromatin immuno‐precipitation methods. The results show that pol α, ‐δ, or ‐ɛ 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 α, ‐δ, and ‐ɛ 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 α, ‐δ, and ‐ɛ localize and function together at multiple replication forks during S phase.