Phosphorylation and Localization of Replication Protein A during Oogenesis and Early Embryogenesis of Drosophila melanogaster

Abstract

The phosphorylation and localization of Drosophila melanogaster Replication Protein A (DRP-A) was examined during oogenesis and in single embryos during the syncytial nuclear divisions of embryogenesis. DRP-A from ovaries was separated by two-dimensional electrophoresis into multiple phosphorylated species that include a previously unresolved form of RP-A. These forms are developmentally regulated with a major phosphorylated form appearing at stage 11 of oogenesis and persisting into mature eggs. Actively cycling early embryos were examined to investigate DNA replication in the absence of repair synthesis due to perturbation by drugs or mutation. An oscillation of the two major forms of DRP-A was observed over multiple cell cycles. The phosphorylated form was most abundant at mitosis and the nonphosphorylated form at interphase. In contrast to other systems where a phosphorylated form of RPA has been correlated with S phase, only the nonphosphorylated form of Drosophila RP-A is observed in early Drosophila embryos during DNA replication. Consistent with this role in DNA metabolism, DRP-A was localized to the nucleus. Subsequently at mitosis, DRP-A becomes delocalized. Strikingly, in ovaries a relatively large amount of DRP-A was observed during the early mitotic stages of oogenesis.