The overall goal of this project is a complete description, at the molecular level, of the three reactions carried out by DNA polymerase I of E. coli: polymerase, 3'-5' exonuclease, and 5'-3' exonuclease. Together the polymerase and 3'-5' exonuclease (proofreading) functions ensure the accuracy of DNA synthesis, while the 5'-3' exonuclease coordinates with the polymerase activity in DNA repair and lagging strand replication. The enzymatic properties of polymerases are frequently exploited therapeutically in antiviral and chemotherapeutic strategies; moreover, replication errors made by DNA polymerases are the likely causes of a variety of human diseases. The 5'-3' exonuclease is a relative of the eukaryotic """"""""flap endonucleases"""""""" which play an essential role in various aspects of DNA replication, recombination and repair, and whose absence increases genome instability. Structural studies of a substantial number of polymerases, of several different types, show that all polymerases share a similar active site layout and reaction mechanism. Therefore, the comparatively simple E. coli DNA polymerase I (Pol I) serves as a valuable model for addressing issues relevant to all polymerases, many of which would be much less tractable as experimental systems. The structural data available for Pol I and its close relatives (including several cocrystal structures) provides a wealth of detail about the layout of the active sites, but poses many additional questions. Taking the structural information as a starting point, experiments are planned which will address the functional significance of the interactions seen in the cocrystal structures. Photo-crosslinking will be used to determine the path of the uncopied template strand of DNA, and a combination of mutagenesis and biochemical studies will be used to identify functionally important protein-DNA contacts. Moving beyond the static picture provided by the cocrystal complexes, a variety of fluorescence methods will be employed to probe several aspects of Pol I enzymology that involve movement: conformational transitions that take place within the polymerase reaction pathway, translocation during processive DNA synthesis, and shuttling of a DNA substrate between polymerase and 3'-5' exonuclease sites during proofreading. Many of the experiments in this proposal relate the biochemical properties of Pol I to its function in vivo: investigating how fidelity is achieved in the polymerase reaction, what distinguishes a DNA polymerase from an RNA polymerase, and how polymerase and 5'-3' exonuclease activities are coordinated so as to produce the correct biological end point.

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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Lewis, Catherine D
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Yale University
Schools of Medicine
New Haven
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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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