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. We have also obtained 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. Quantum mechanics/molecular mechanics calculations on the epsilon proofreading subunit have provided further insight into the exonuclease reaction mechanism, including active site metal coordination and the role of catalytically relevant amino acid residues.
| Cisneros, G Andrés; Perera, Lalith; Schaaper, Roel M et al. (2009) Reaction mechanism of the epsilon subunit of E. coli DNA polymerase III: insights into active site metal coordination and catalytically significant residues. J Am Chem Soc 131:1550-6 |
