The long term objectives of this proposal are to understand the basis for the high fidelity exhibited by replicative DNA polymerases (DNA pols). Specifically we want to: (i) understand how the dynamics of conformational changes exhibited by DNA pols affect base discrimination;(ii) follow structural changes in DNA pols and their "fidelity" mutants during DNA synthesis with correct (R) and incorrect (W) incoming dNTPs;(iii) identify transient intermediates in the reaction pathway for primer extension with R and W dNTPs using wild type (wt) and "fidelity" mutants of DNA pols;(iv) identify structural features of the RB69 pol "fidelity" mutants that are responsible for the high level of misincorporation exhibited by these mutants;(v) identify the chemical features of a nascent base-pair that allow the incoming dNTP to be inserted with high efficiency. To reach these goals we will determine rates of conformational changes when the wt and mutant pols encounter dNTPs that harbor either non-complementary bases or nucleobase analogs. For this purpose we will employ;(i) stopped-flow fluorescence with dye labeled polymerases and with fluorescent base analogs in the template strand of primer-templates (P/Ts) in ensemble-averaged and in single-molecule experiments;(ii) Single-Molecule Fluorescence Energy Transfer (smFRET) to investigate the dynamics of the nucleotide addition cycle with the aim of capturing transients in the reaction pathway;(iii) rapid chemical quench experiments to obtain rates of product formation;(iv) X-ray crystallography to determine structural changes that occur when wt dNTPs or dNTPs containing base analogs are bound in ternary complexes (v) polychromatic, time-resolved X-ray crystallography using Laue diffraction to visualize changes in the structure of intermediates during the nucleotidyl transfer reaction. This can be accomplished by using "caged" dNTPs that can be converted to dNTP substrates by photolysis. We are using RB69 pol, a member of the B family (which includes two human DNA pols, pol alpha and pol delta) so that information acquired from RB69 pol and its mutants should be relevant to human DNA pols as well. Taken together, the results of these investigations will contribute to a better understanding of diseases that involve malfunctioning of DNA pols required for replication and repair. They will also provide the basis for devising therapeutic strategies to curtail or halt DNA replication and/or repair which could cripple malignant cells. This information could also be helpful in combating infections caused by viruses, bacteria or parasites.

Public Health Relevance

Narrative DNA polymerases are responsible for copying and repairing the genomes of all living organisms. If they malfunction disease and even death may follow. Thus it is important to understand how they function to faithfully replicate DNA so that therapeutic intervention, when necessary can be effective.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM063276-12
Application #
8442339
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Barski, Oleg
Project Start
2001-04-01
Project End
2014-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
12
Fiscal Year
2013
Total Cost
$347,843
Indirect Cost
$137,666
Name
Yale University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
Xia, Shuangluo; Konigsberg, William H (2014) Mispairs with Watson-Crick base-pair geometry observed in ternary complexes of an RB69 DNA polymerase variant. Protein Sci 23:508-13
Xia, Shuangluo; Konigsberg, William H (2014) RB69 DNA polymerase structure, kinetics, and fidelity. Biochemistry 53:2752-67
Xia, Shuangluo; Wood, Marcus; Bradley, Michael J et al. (2013) Alteration in the cavity size adjacent to the active site of RB69 DNA polymerase changes its conformational dynamics. Nucleic Acids Res 41:9077-89
Xia, Shuangluo; Wang, Jimin; Konigsberg, William H (2013) DNA mismatch synthesis complexes provide insights into base selectivity of a B family DNA polymerase. J Am Chem Soc 135:193-202
Xia, Shuangluo; Eom, Soo Hyun; Konigsberg, William H et al. (2012) Bidentate and tridentate metal-ion coordination states within ternary complexes of RB69 DNA polymerase. Protein Sci 21:447-51
Xia, Shuangluo; Eom, Soo Hyun; Konigsberg, William H et al. (2012) Structural basis for differential insertion kinetics of dNMPs opposite a difluorotoluene nucleotide residue. Biochemistry 51:1476-85
Xia, Shuangluo; Wang, Mina; Lee, Harold R et al. (2011) Variation in mutation rates caused by RB69pol fidelity mutants can be rationalized on the basis of their kinetic behavior and crystal structures. J Mol Biol 406:558-70
Wang, Mina; Xia, Shuangluo; Blaha, Gregor et al. (2011) Insights into base selectivity from the 1.8 A resolution structure of an RB69 DNA polymerase ternary complex. Biochemistry 50:581-90
Xia, Shuangluo; Wang, Mina; Blaha, Gregor et al. (2011) Structural insights into complete metal ion coordination from ternary complexes of B family RB69 DNA polymerase. Biochemistry 50:9114-24
Xia, Shuangluo; Konigsberg, William H; Wang, Jimin (2011) Hydrogen-bonding capability of a templating difluorotoluene nucleotide residue in an RB69 DNA polymerase ternary complex. J Am Chem Soc 133:10003-5

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