Visualization of replication initiation and elongation in Drosophila.
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.
Claycomb
Julie M
JM
Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge, MA 02142, USA.
MacAlpine
David M
DM
Evans
James G
JG
Bell
Stephen P
SP
Orr-Weaver
Terry L
TL
D000818:Animals; D001973:Bromodeoxyuridine / Q000032:analysis; D018797:Cell Cycle Proteins / Q000032:analysis; D002823:Chorion / Q000737:chemistry / Q000502:physiology; D002875:Chromosomes / Q000502:physiology; D004261:DNA Replication / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000032:analysis; D029721:Drosophila Proteins / Q000032:analysis; D004331:Drosophila melanogaster / Q000502:physiology*; D005260:Female; D018628:Gene Dosage; D019476:Insect Proteins / Q000032:analysis; D018613:Microscopy, Confocal / Q000379:methods; D064151:Minichromosome Maintenance Complex Component 6; D064168:Minichromosome Maintenance Complex Component 7; D009687:Nuclear Proteins / Q000032:analysis; D051738:Origin Recognition Complex; D010053:Ovary / Q000737:chemistry / Q000502:physiology; D016133:Polymerase Chain Reaction / Q000379:methods; D018809:Proliferating Cell Nuclear Antigen / Q000032:analysis; D029702:Schizosaccharomyces pombe Proteins
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C411983:DUP protein, Drosophila; D029721:Drosophila Proteins; D019476:Insect Proteins; C491087:Mcm7 protein, Drosophila; D009687:Nuclear Proteins; D051738:Origin Recognition Complex; D018809:Proliferating Cell Nuclear Antigen; D029702:Schizosaccharomyces pombe Proteins; C085711:mcm5 protein, S pombe; C491204:Mcm6 protein, Drosophila; D064151:Minichromosome Maintenance Complex Component 6; D064168:Minichromosome Maintenance Complex Component 7; D001973:Bromodeoxyuridine