The overall goal of this project is a full understanding, at the molecular level, of the reactions catalyzed by DMA polymerases, with particular emphasis on how polymerases ensure substrate specificity and accuracy in copying DMA. The question of polymerase accuracy has important health implications because the errors made by DMA polymerases can result in mutations leading to human disease. Moreover, DMA polymerases are frequently targeted in chemotherapeutic and antiviral strategies, as well as being important in a variety of diagnostic biotechnology applications, so an understanding of their reaction mechanisms is crucial. Our investigations focus on two model DMA polymerases that have contrasting enzymatic properties: the highly accurate DMA polymerase I (Klenow fragment) of E. coli, and the much less accurate Dbh bypass polymerase from the archaeon S. solfataricus. Structural data are available for both these enzymes and several close homologues, and serve as the basis for many of the planned experiments. Moreover, because the important features of the polymerase active site and reaction mechanism are conserved throughout the polymerase family, the results obtained with these simple model systems will have much wider relevance. A major priority will be the investigation of noncovalent steps in the polymerase reaction pathway because these conformational transitions are likely to be involved in distinguishing between correctly paired substrates and the mispairs that result in polymerase errors. We will use fluorescence assays in combination with rapid single-turnover kinetics to investigate the rates of conformational changes and the effect on the reaction pathway of mispaired substrates, active site mutations and damaged DMA. DMA damage and errors in DMA synthesis can cause mutations that lead to diseases such as cancer, making it important to understand how DMA polymerases function to avoid these outcomes. Antiviral drugs frequently target viral polymerases so research into polymerase structures and mechanism is relevant in designing effective drugs and understanding how they work. DNA polymerases are also a crucial part of many of the diagnostic tools used in modern medicine and will be pivotal in the development of new DNA sequencing technologies for diagnostic purposes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM028550-30
Application #
7616682
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Preusch, Peter C
Project Start
1980-05-01
Project End
2011-04-30
Budget Start
2009-05-01
Budget End
2010-04-30
Support Year
30
Fiscal Year
2009
Total Cost
$589,458
Indirect Cost
Name
Yale University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
043207562
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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