Abstract

The ultimate aims of this research program are to understand in molecular detail the mechanism of DNA replication in a model eukaryote, Drosophila melanogaster, and the mechanism of homologous recombination in a prokaryote, Escherichia coli. The investigation will be organized along the following lines: 1. Mechanism of DNA replication in embryos of Drosophila melanogaster a. DNA chain elongation, i.e., initiation and elongation of Okazaki fragments (1) Assembly of a DNA polymerase-primase holoenzyme (a) Isolation of DNA polymerase core enzyme (b) Isolation of DNA polymerase-primase accessory proteins (2) Direct isolation of a DNA polymerase-primase holoenzyme b. Initiation of DNA synthesis at a replication origin c. Regulation of DNA replication (1) Investigation of factors that control rate of DNA replication (2) Identification of DNA polymerase-primase mutants 2. Mechanism of DNA strand exchange promoted by recA protein of Escherichia coli a. Formation of non-dissociable recA protein-single-stranded DNA complex b. Mechanism of initial pairing between single-stranded and duplex DNAs (1) Processive versus distributive search for homology (2) Initiation of strand assimilation at a nick in duplex DNA (3) Strand assimilation in the presence of nonhydrolyzable ATP analogues c. Role of ATP hydrolysis in the branch migration phase of DNA strand exchange (1) Search for factors that increase efficiency of branch migration (2) Search for factors that increase rate of branch migration d. Recycling of recA protein following strand exchange e. Mechanism of recA protein-catalyzed annealing of complementary single strands.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM006196-30
Application #
3267904
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1974-09-01
Project End
1989-08-31
Budget Start
1988-09-01
Budget End
1989-08-31
Support Year
30
Fiscal Year
1988
Total Cost
Indirect Cost

  Institution

Name
Stanford University
Department
Type
Schools of Medicine
DUNS #
800771545
City
Stanford
State
CA
Country
United States
Zip Code
94305

  Publications

Mitsis, P G; Kowalczykowski, S C; Lehman, I R (1993) A single-stranded DNA binding protein from Drosophila melanogaster: characterization of the heterotrimeric protein and its interaction with single-stranded DNA. Biochemistry 32:5257-66
Chiang, C S; Mitsis, P G; Lehman, I R (1993) DNA polymerase delta from embryos of Drosophila melanogaster. Proc Natl Acad Sci U S A 90:9105-9
Crute, J J; Tsurumi, T; Zhu, L A et al. (1989) Herpes simplex virus 1 helicase-primase: a complex of three herpes-encoded gene products. Proc Natl Acad Sci U S A 86:2186-9
Lehman, I R; Kaguni, L S (1989) DNA polymerase alpha. J Biol Chem 264:4265-8
Reyland, M E; Lehman, I R; Loeb, L A (1988) Specificity of proofreading by the 3'----5' exonuclease of the DNA polymerase-primase of Drosophila melanogaster. J Biol Chem 263:6518-24
Kaguni, L S; Lehman, I R (1988) Eukaryotic DNA polymerase-primase: structure, mechanism and function. Biochim Biophys Acta 950:87-101
Crute, J J; Mocarski, E S; Lehman, I R (1988) A DNA helicase induced by herpes simplex virus type 1. Nucleic Acids Res 16:6585-96
Elias, P; Lehman, I R (1988) Interaction of origin binding protein with an origin of replication of herpes simplex virus 1. Proc Natl Acad Sci U S A 85:2959-63
Cotterill, S M; Reyland, M E; Loeb, L A et al. (1987) A cryptic proofreading 3'----5' exonuclease associated with the polymerase subunit of the DNA polymerase-primase from Drosophila melanogaster. Proc Natl Acad Sci U S A 84:5635-9
Cox, M M; Lehman, I R (1987) Enzymes of general recombination. Annu Rev Biochem 56:229-62

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