Biochemical and molecular analyses of DNA replication in Drosophila melanogaster

Abstract

Since regulation of DNA replication in Drosophila melanogaster resides in the initiation phase of DNA replication, it was reasoned that accessory factors must exist that would act in collaboration with DNA polymerase alpha in the co-ordination of these events. The approach undertaken in this work was to apply biochemical methods as a means to identify and purify protein(s) in Drosophila melanogaster that may act in aiding DNA polymerase alpha during replication of the chromosome. An activity in Drosophila embryo extracts that stimulates the rate of in vitro DNA replication initiation events was identified and purified to homogeneity. Biochemical and physical characterizations of the stimulatory protein were applied to establish the mechanism of stimulation of DNA polymerase alpha taking into consideration interactions involving the DNA substrate and protein-protein interactions as well. The results of this work suggest that the properties of the protein are consistent with a role in aiding polymerase in the completion of gaps normally found during discontinuous synthesis at the fork. In addition, the unusual substrate recognition properties of the protein further suggest a possible involvement of the protein during replication reactions associated with DNA repair. Molecular approaches were utilized to obtain a cDNA containing the putative coding sequence of the stimulatory protein. This resulted in the generation of the complete nucleotide sequence of the cDNA, the determination of the developmental profile of accumulation of the RNA transcript, as well as the localization of the gene to a cytological interval on a Drosophila chromosome. Furthermore, the derived amino acid sequence was used in the search of Genebank and EMBL data bases for comparisons with previously identified homologous proteins and provided independent support of the predictions based on the in vitro characterizations in this work.