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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM028550-23
Application #
6519025
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Program Officer
Lewis, Catherine D
Project Start
1980-05-01
Project End
2003-04-30
Budget Start
2002-05-01
Budget End
2003-04-30
Support Year
23
Fiscal Year
2002
Total Cost
$447,079
Indirect Cost
Name
Yale University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
082359691
City
New Haven
State
CT
Country
United States
Zip Code
06520
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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