This project is concerned with the structural and functional aspects of the core assembly of DNA polymerase III, the enzyme responsible for replicating the bacterial chromosome. Pol III core is composed of three tightly-bound subunits: alpha, epsilon, theta (in this linear order). Alpha (dnaE gene product) is the 135 kD DNA polymerase, epsilon (28 kD, dnaQ gene product) is the 3' exonucleolytic proofreader, and theta (8kD, holE gene product) has an as yet unknown function. In addition to its catalytic proofreading function, epsilon also has an important structural function within pol III core, as evidenced by the conditional lethality of dnaQ deletion mutants. Our experiments have entailed: (i) a structure-function analysis of epsilon by detailed analysis of a series of dnaQ mutator mutants isolated previously in our laboratory. This has provided new insight in the functional organization of epsilon, including the function of the three conserved Exo motifs and of the C-terminal domain, which we showed responsible for epsilon-alpha interaction; (ii) analysis of the cellular function of theta, using a strain deleted for the holE gene. These studies have revealed a role for theta in stabilizing epsilon; and (iii) NMR structural studies of epsilon and theta, individually and in complex. These studies have lead to a structural model for epsilon, consistent with the reported str

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Intramural Research (Z01)
Project #
1Z01ES065088-09
Application #
7007445
Study Section
(LMG)
Project Start
Project End
Budget Start
Budget End
Support Year
9
Fiscal Year
2004
Total Cost
Indirect Cost
Name
U.S. National Inst of Environ Hlth Scis
Department
Type
DUNS #
City
State
Country
United States
Zip Code
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Chikova, Anna K; Schaaper, Roel M (2007) The bacteriophage P1 hot gene, encoding a homolog of the E. coli DNA polymerase III theta subunit, is expressed during both lysogenic and lytic growth stages. Mutat Res 624:1-8
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DeRose, Eugene F; Li, Dawei; Darden, Thomas et al. (2002) Model for the catalytic domain of the proofreading epsilon subunit of Escherichia coli DNA polymerase III based on NMR structural data. Biochemistry 41:94-110