The overall goal of this project is a complete understanding of the way in which DNA polymerase I of E. coli carries out accurate and processive template-directed DNA synthesis. Replication errors made by DNA polymerases are implicated in the causes of some human diseases; moreover the enzymatic properties of the polymerases themselves are frequently exploited in antiviral and chemotherapeutic strategies. Since evidence is accumulating that all polymerases share a similar active site layout and reaction mechanism, it is reasonable to assume that studies carried out on a relatively simple model system will have much wider relevance. DNA polymerase I provides a good model system since the high-resolution structure of the Klenow fragment portion of the molecule, containing the polymerase and 3'-5' (proofreading) exonuclease activities, provides an important foundation for relating polymerase function to the molecular structure using a combination of biochemistry and molecular genetics. The polymerase and 3'-5' exonuclease active sites of Klenow fragment catalyze analogous phosphoryl transfer reactions, and are thought to do so using a pair of divalent metal ions appropriately positioned by coordination to a cluster of carboxylate side chains. For both active sites, a variety of substrate analogs will be used to test she proposed reaction mechanism and to investigate interactions between the substrate molecules and active site side chains. Experiments are proposed to characterize the polymerase-DNA interactions specifically addressing the location and orientation of the DNA, the extent to which interactions change between polymerase and 3'-5' exonuclease modes, and the identification of specific contacts between protein and DNA. Experiments will also be carried out to analyze the way in which the protein maintains accuracy in the polymerase reaction, discriminating against both base substitution errors and DNA misalignments that can give rise to frameshifts. DNA polymerase I also contains a third activity, the 5'-3' exonuclease which plays an important role in """"""""nick translation' activities in vivo. Site-directed mutagenesis will be used to locate active-site residues, complementing ongoing structural studies. A variety of biochemical approaches will be used to determine the way in which the 5'-3' exonuclease activity is coordinated with the polymerase to accomplish the in vivo functions of DNA polymerase I in replication and repair.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Microbial Physiology and Genetics Subcommittee 2 (MBC)
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Yale University
Schools of Medicine
New Haven
United States
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Hohlbein, Johannes; Aigrain, Louise; Craggs, Timothy D et al. (2013) Conformational landscapes of DNA polymerase I and mutator derivatives establish fidelity checkpoints for nucleotide insertion. Nat Commun 4:2131
Bermek, Oya; Grindley, Nigel D F; Joyce, Catherine M (2013) Prechemistry nucleotide selection checkpoints in the reaction pathway of DNA polymerase I and roles of glu710 and tyr766. Biochemistry 52:6258-74
Bermek, Oya; Grindley, Nigel D F; Joyce, Catherine M (2011) Distinct roles of the active-site Mg2+ ligands, Asp882 and Asp705, of DNA polymerase I (Klenow fragment) during the prechemistry conformational transitions. J Biol Chem 286:3755-66
Foti, James J; Delucia, Angela M; Joyce, Catherine M et al. (2010) UmuD(2) inhibits a non-covalent step during DinB-mediated template slippage on homopolymeric nucleotide runs. J Biol Chem 285:23086-95
Santoso, Yusdi; Joyce, Catherine M; Potapova, Olga et al. (2010) Conformational transitions in DNA polymerase I revealed by single-molecule FRET. Proc Natl Acad Sci U S A 107:715-20
Joyce, Catherine M (2010) Techniques used to study the DNA polymerase reaction pathway. Biochim Biophys Acta 1804:1032-40
Joyce, Catherine M; Potapova, Olga; Delucia, Angela M et al. (2008) Fingers-closing and other rapid conformational changes in DNA polymerase I (Klenow fragment) and their role in nucleotide selectivity. Biochemistry 47:6103-16
DeLucia, Angela M; Grindley, Nigel D F; Joyce, Catherine M (2007) Conformational changes during normal and error-prone incorporation of nucleotides by a Y-family DNA polymerase detected by 2-aminopurine fluorescence. Biochemistry 46:10790-803
DeLucia, Angela M; Chaudhuri, Santanov; Potapova, Olga et al. (2006) The properties of steric gate mutants reveal different constraints within the active sites of Y-family and A-family DNA polymerases. J Biol Chem 281:27286-91
Potapova, Olga; Chan, Chikio; DeLucia, Angela M et al. (2006) DNA polymerase catalysis in the absence of Watson-Crick hydrogen bonds: analysis by single-turnover kinetics. Biochemistry 45:890-8

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