The aim of this project is to understand, at the molecular level, the mechanism of initiation and termination of replication of a linear double-stranded DNA molecule: the 72 kb pair genome of coliphage N4. N4 DNA has at its ends short non-complementary 3' single-stranded tails. Preliminary evidence suggests that in vivo replication starts at or near the ends. We have recently developed an in vitro soluble replication system specific for N4 DNA where replication also starts at the ends of the DNA. Presently, available information, however, does not allow us to postulate a unique model for N4 DNA replication. In order to determine the mechanism of N4 DNA replication we will use biochemical and genetic approaches to: a) determine the exact structure at the ends of N4 DNA. b) investigate the role of the 3' single-stranded ends in DNA replication in vivo and in the in vitro system. c) determine the structure of in vivo and in vitro replication intermediates. d) determine the role of the terminally redundant sequences in replication. Several strategies are proposed in this application to explain how N4 DNA replicates. We expect that accomplishment of the above goals will allow us to formulate, test and confirm a unique model for N4 DNA replication. To understand the reactions and components involved in N4 DNA replication we propose to a) fractionate the in vitro replication system in order to identify additional host and phage components required for replication with specific emphasis on the role of the virion-associated RNA polymerase. b) clone and overproduce the proteins required in order to aid in their purification and characterization.
| Lindberg, G; Kowalczykowski, S C; Rist, J K et al. (1989) Purification and characterization of the coliphage N4-coded single-stranded DNA binding protein. J Biol Chem 264:12700-8 |
| Lindberg, G K; Rist, J K; Kunkel, T A et al. (1988) Purification and characterization of bacteriophage N4-induced DNA polymerase. J Biol Chem 263:11319-26 |
| Ohmori, H; Haynes, L L; Rothman-Denes, L B (1988) Structure of the ends of the coliphage N4 genome. J Mol Biol 202:1-10 |
| Malone, C; Spellman, S; Hyman, D et al. (1988) Cloning and generation of a genetic map of bacteriophage N4 DNA. Virology 162:328-36 |
| Rist, J K; Pearle, M; Sugino, A et al. (1986) Development of an in vitro bacteriophage N4 DNA replication system. J Biol Chem 261:10506-10 |
| Guinta, D; Lindberg, G; Rothman-Denes, L B (1986) Bacteriophage N4-coded 5'----3' exonuclease. Purification and characterization. J Biol Chem 261:10736-43 |
| Guinta, D; Stambouly, J; Falco, S C et al. (1986) Host and phage-coded functions required for coliphage N4 DNA replication. Virology 150:33-44 |
