Visualization of replication initiation and elongation in Drosophila.

Abstract

Chorion gene amplification in the ovaries of Drosophila melanogaster is a powerful system for the study of metazoan DNA replication in vivo. Using a combination of high-resolution confocal and deconvolution microscopy and quantitative realtime PCR, we found that initiation and elongation occur during separate developmental stages, thus permitting analysis of these two phases of replication in vivo. Bromodeoxyuridine, origin recognition complex, and the elongation factors minichromosome maintenance proteins (MCM)2–7 and proliferating cell nuclear antigen were precisely localized, and the DNA copy number along the third chromosome chorion amplicon was quantified during multiple developmental stages. These studies revealed that initiation takes place during stages 10B and 11 of egg chamber development, whereas only elongation of existing replication forks occurs during egg chamber stages 12 and 13. The ability to distinguish initiation from elongation makes this an outstanding model to decipher the roles of various replication factors during metazoan DNA replication. We utilized this system to demonstrate that the pre–replication complex component, double-parked protein/cell division cycle 10–dependent transcript 1, is not only necessary for proper MCM2–7 localization, but, unexpectedly, is present during elongation.