Study of DNA replication in Drosophila using cell free in vitro system.
Using Drosophila early egg extracts we have developed an optimized cell free system to study DNA replication. The efficiency of replication depends on a cold treatment of Drosophila embryos before the extract preparation and a formation of nuclei facilitated by the addition of membrane fractions to the extracts. In vitro DNA replication is ORC and CDC6 dependent, as a removal of these proteins from the extracts abolishes DNA replication. The N-terminal part of Orc1 protein, which is important for non-replicative functions of ORC, is dispensable for the replication in vitro. We also show that the conserved ATP ase motif of CDC6 is crucial for the replication. Our studies indicate that a Drosophila cell free system proves to be an extremely useful tool for a functional dissection of the processes and factors involved in DNA replication in metazoans.
Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, School of Medicine, Birmingham, AL, USA.
Anton
A
Svitin
Igor
I
Chesnokov
D000818:Animals; D018797:Cell Cycle Proteins / Q000378:metabolism; D003080:Cold Temperature; D004261:DNA Replication / Q000502:physiology*; D004330:Drosophila / Q000254:growth & development / Q000378:metabolism*; D029721:Drosophila Proteins / Q000378:metabolism; D051738:Origin Recognition Complex / Q000378:metabolism; D010063:Ovum / Q000378:metabolism
C504976:Cdc6 protein, Drosophila; D018797:Cell Cycle Proteins; D029721:Drosophila Proteins; C497213:Orc1 protein, Drosophila; D051738:Origin Recognition Complex