The objective of the proposed work is the elucidation of the mechanism(s) of DNA replication in the animal Drosophila melanogaster. This may be accomplished when purified enzymes are reconstituted in vitro to yield replication complexes which initiate and sustain DNA synthesis on specific Drosophila DNA sequences. A combined biochemical and genetic approach will be employed to obtain, characterize, fractionate, and reconstitute cell-free extracts of embryos of Drosophila melanogaster that specifically replicate recombinant DNAs containing Drosophila chromosomal and mitochondrial DNA sequences. This will be accomplished by: (i) molecular cloning and characterization of origins of replication from Drosophila melanogaster; (ii) preparing and examining embryo extracts for their ability to initiate and sustain DNA replication; and (iii) purifying enzymes implicated in the DNA synthesis reactions in order to study their mechanisms of action. The control of animal cell reproduction during normal development, and the loss of control during cancerous development, is of central importance in the processes of human growth, aging, and disease. Prior to every cell division, the DNA complement of each cell is duplicated. Because DNA replication and cell division are tightly coupled, an understanding of the mechanism of DNA replication in animal cells is essential to our understanding of these basic processes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM034042-03
Application #
3284460
Study Section
Molecular Biology Study Section (MBY)
Project Start
1985-04-01
Project End
1989-03-31
Budget Start
1987-04-01
Budget End
1989-03-31
Support Year
3
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Michigan State University
Department
Type
Schools of Medicine
DUNS #
193247145
City
East Lansing
State
MI
Country
United States
Zip Code
48824
Kaguni, Laurie S (2004) DNA polymerase gamma, the mitochondrial replicase. Annu Rev Biochem 73:293-320
Kaguni, L S; Olson, M W (1989) Mismatch-specific 3'----5' exonuclease associated with the mitochondrial DNA polymerase from Drosophila embryos. Proc Natl Acad Sci U S A 86:6469-73
Lehman, I R; Kaguni, L S (1989) DNA polymerase alpha. J Biol Chem 264:4265-8
Kaguni, L S; Lehman, I R (1988) Eukaryotic DNA polymerase-primase: structure, mechanism and function. Biochim Biophys Acta 950:87-101
Wernette, C M; Conway, M C; Kaguni, L S (1988) Mitochondrial DNA polymerase from Drosophila melanogaster embryos: kinetics, processivity, and fidelity of DNA polymerization. Biochemistry 27:6046-54
Wernette, C M; Kaguni, L S (1986) A mitochondrial DNA polymerase from embryos of Drosophila melanogaster. Purification, subunit structure, and partial characterization. J Biol Chem 261:14764-70