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; (iii) analysis of the hot gene of bacteriophage P1, encoding a homolog (Hot) of theta subunit. These studies have revealed that Hot is not essential for phage growth. On the other hand, Hot can compete efficiently with theta and can be incorporated in the Pol III HE instead of theta. This incorporation affects the fidelity of replication by HE, generally leading to an increased mutation rate. We have also demonstrated that the hot gene is expressed during various stages of the viral life cycle, including the lysogenic (plasmid) stage. Competition experiments between wild-type P1 and phage with a deleted hot gene revealed that the wild-type phage outcompetes the deletion mutant, indicating that the hot gene plays a beneficial role in the phage replication cycle. (iv) NMR and X-ray structural studies of epsilon and theta (Hot), individually and in complex. These studies have lead to a structural model for epsilon, consistent with the reported structure of other proofreading exonucleases, and to definition of the theta interaction site on epsilon. Finally, the crystal structure of the epsilon-Hot complex has been obtained and has provided direct insight into the stabilizing function of Hot (theta) on the epsilon proofreading function.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Intramural Research (Z01)
Project #
1Z01ES065088-13
Application #
7734484
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
13
Fiscal Year
2008
Total Cost
$315,214
Indirect Cost
City
State
Country
United States
Zip Code
Kozmin, Stanislav G; Schaaper, Roel M (2007) Molybdenum cofactor-dependent resistance to N-hydroxylated base analogs in Escherichia coli is independent of MobA function. Mutat Res 619:9-15
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
Chikova, Anna K; Schaaper, Roel M (2006) Mutator and antimutator effects of the bacteriophage P1 hot gene product. J Bacteriol 188:5831-8
Kirby, Thomas W; Harvey, Scott; DeRose, Eugene F et al. (2006) Structure of the Escherichia coli DNA polymerase III epsilon-HOT proofreading complex. J Biol Chem 281:38466-71
Chikova, Anna K; Schaaper, Roel M (2005) The bacteriophage P1 hot gene product can substitute for the Escherichia coli DNA polymerase III {theta} subunit. J Bacteriol 187:5528-36
Mueller, Geoffrey A; Kirby, Thomas W; DeRose, Eugene F et al. (2005) Nuclear magnetic resonance solution structure of the Escherichia coli DNA polymerase III theta subunit. J Bacteriol 187:7081-9
Taft-Benz, Sharon A; Schaaper, Roel M (2004) The theta subunit of Escherichia coli DNA polymerase III: a role in stabilizing the epsilon proofreading subunit. J Bacteriol 186:2774-80
Derose, Eugene F; Kirby, Thomas W; Mueller, Geoffrey A et al. (2004) Phage like it HOT: solution structure of the bacteriophage P1-encoded HOT protein, a homolog of the theta subunit of E. coli DNA polymerase III. Structure 12:2221-31
DeRose, Eugene F; Darden, Thomas; Harvey, Scott et al. (2003) Elucidation of the epsilon-theta subunit interface of Escherichia coli DNA polymerase III by NMR spectroscopy. Biochemistry 42:3635-44
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