In this proposal we outline the next stages of a continuing physical biochemical study of the structural and functional interactions of the protein and nucleic acid components of the bacteriophage T4 DNA replication system. Building on the results of Alberts and Nossal and their coworkers, who have defined this in vitro system, as well as on our own earlier studies, we will continue to focus on a step-wise """"""""building-up"""""""" of the complex to gain further insight into the functional role of each component in the integrated system. To this end we plan to continue to use our newly developed UV laser cross-linking methodology to determine contacts (and affinities) between the various proteins of the system and the DNA of the primer-template junction, as well as the DNA strand displaced by the advancing replication fork in asymmetric (leading-strand-only) DNA synthesis. We will also continue to study the assembly and molecular activities of functional subsets of the replication system, using enzymatic and physical chemical """"""""probes"""""""". These subsets include the DNA polymerase (gene 43 protein) itself, the polymerase accessory-helicase complex (genes 44/62 + 45 proteins) and the lagging strand primase-helicase complex (genes 41 and 61 protein), each of which also interacts with the T4 single-strand DNA binding (gene 32) protein. We will also continue to study the processivity of the polymerase as a measure of the functional integration of the various components of the system (working both """"""""forward"""""""" in DNA synthesis and """"""""backward"""""""" as an exonuclease """"""""editor"""""""" of the growing primer strand), and plan to use this approach to begin to study the issue of the fidelity of DNA synthesis as well. We hope that these studies will help to provide at least a partial molecular understanding of the various sets of protein-protein and protein-nucleic acid interactions that control the function of this DNA elongation complex, and of how these interactions work together to form a coupled system that conduct leading- and lagging-strand DNA synthesis at physiological rates and with physiological fidelities.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM029158-07
Application #
3276662
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1981-04-01
Project End
1991-03-31
Budget Start
1987-04-01
Budget End
1988-03-31
Support Year
7
Fiscal Year
1987
Total Cost
Indirect Cost
Name
University of Oregon
Department
Type
Graduate Schools
DUNS #
948117312
City
Eugene
State
OR
Country
United States
Zip Code
97403
Zhao, Huaying; Ghirlando, Rodolfo; Alfonso, Carlos et al. (2015) A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation. PLoS One 10:e0126420
von Hippel, Peter H; Johnson, Neil P; Marcus, Andrew H (2013) Fifty years of DNA ""breathing"": Reflections on old and new approaches. Biopolymers 99:923-54
Pietroni, Paola; von Hippel, Peter H (2008) Multiple ATP binding is required to stabilize the ""activated"" (clamp open) clamp loader of the T4 DNA replication complex. J Biol Chem 283:28338-53
Conant, Clarke R; Goodarzi, Jim P; Weitzel, Steven E et al. (2008) The antitermination activity of bacteriophage lambda N protein is controlled by the kinetics of an RNA-looping-facilitated interaction with the transcription complex. J Mol Biol 384:87-108
Datta, Kausiki; von Hippel, Peter H (2008) Direct spectroscopic study of reconstituted transcription complexes reveals that intrinsic termination is driven primarily by thermodynamic destabilization of the nucleic acid framework. J Biol Chem 283:3537-49
von Hippel, Peter H (2007) From ""simple"" DNA-protein interactions to the macromolecular machines of gene expression. Annu Rev Biophys Biomol Struct 36:79-105
Datta, Kausiki; Johnson, Neil P; von Hippel, Peter H (2006) Mapping the conformation of the nucleic acid framework of the T7 RNA polymerase elongation complex in solution using low-energy CD and fluorescence spectroscopy. J Mol Biol 360:800-13
Conant, Clarke R; Van Gilst, Marc R; Weitzel, Stephen E et al. (2005) A quantitative description of the binding states and in vitro function of antitermination protein N of bacteriophage lambda. J Mol Biol 348:1039-57
Delagoutte, Emmanuelle; von Hippel, Peter H (2005) Mechanistic studies of the T4 DNA (gp41) replication helicase: functional interactions of the C-terminal Tails of the helicase subunits with the T4 (gp59) helicase loader protein. J Mol Biol 347:257-75
Johnson, Neil P; Baase, Walter A; von Hippel, Peter H (2005) Low energy CD of RNA hairpin unveils a loop conformation required for lambdaN antitermination activity. J Biol Chem 280:32177-83

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