DNA polymerase mechanisms of base selection and catalysis are explored using a tool-kit of dNTP analogs that have bisphosphonates in place of the , ? -bridge oxygen. These analogs are Pol substrates that have leaving groups with widely dispirate electronic properties, pKa4 values ranging from 7.8 to 12.3, enabling us to use presteady state kinetic measurements to determine the selection of right and wrong deoxynucleotides occuring at the chemical transition state. Especially important members of the toolkit include all four individually synthesized (R)- and (S)- , ? -CHF and , ? -CHCI diastereomers. Our recent observation of a pronounced stereoselection for (R)-CHF in Pol , involving an electrostatic interaction of F with Arg183, unique to family X pols such as Pol , serves as the impetus for a "scaffold" strategy for the selective inhibition of Pol relative to the cellular replication Pols ?, ?, ?, and mitochondrial Pol ?.
In Aim 1, Study "a" explores stereoselection in the cellular and mitochondrial pols. Studies "b" and "c" make use of the entire toolkit to explore chemistry vs. conformational change as a rate-limiting step In the transition-state of family X Pol ?, family Y error-prone Pol r , and for a series of cancer-associated Pol variants. In Study "d", newly synthesized bisphosphonate PPI analog leaving groups are used to reverse the polymerase reaction to attain a free energy reaction profile for Pol . Study "e" addresses an entirely new area of polymerase mechanistics, namely the use of EPR and ENDOR to elucidate the structure of the metal coordination site and Its role in fidelity.
Aim 2, containing three interrelated studies, spells out a detailed scaffold strategy for the design and synthesis of selective Inhibitors of Pol and BER by the dual targeting of the dNTP binding site and active site Arg183 (Study "a"). In Study "b", we develop a new approach, using pamoic acid analogs, to inhibit BER by selectively interfering with the Pol -assocated lyase. In Study "c", we take an important step along the path toward translation, by evaluating the ability of cell permeabilized scaffold compounds to inhibit cultured cancer cells.
The proposed research will apply innovative experimental strategies to elucidate the mechanisms of fidelity occurring in the active site of DNA polymerase. We will focus on studies of human DNA polymerase p, an exceptionally important repair enzyme. Mutants of Pol have been associated with numerous different human cancers. We have devised a logical strategy using a new class of polymerase substrate analogs to selectively inhibit Pol in cell free systems and in cultured cancer cells.
|Kirby, Thomas W; Derose, Eugene F; Beard, William A et al. (2014) Substrate rescue of DNA polymerase ? containing a catastrophic L22P mutation. Biochemistry 53:2413-22|
|Wu, Sangwook; Beard, William A; Pedersen, Lee G et al. (2014) Structural comparison of DNA polymerase architecture suggests a nucleotide gateway to the polymerase active site. Chem Rev 114:2759-74|
|Prasad, Rajendra; Horton, Julie K; Chastain 2nd, Paul D et al. (2014) Suicidal cross-linking of PARP-1 to AP site intermediates in cells undergoing base excision repair. Nucleic Acids Res 42:6337-51|
|Freudenthal, Bret D; Beard, William A; Wilson, Samuel H (2014) Watching a DNA polymerase in action. Cell Cycle 13:691-2|
|Towle-Weicksel, Jamie B; Dalal, Shibani; Sohl, Christal D et al. (2014) Fluorescence resonance energy transfer studies of DNA polymerase ?: the critical role of fingers domain movements and a novel non-covalent step during nucleotide selection. J Biol Chem 289:16541-50|
|Beard, William A; Wilson, Samuel H (2014) Structure and mechanism of DNA polymerase ?. Biochemistry 53:2768-80|
|Oertell, Keriann; Chamberlain, Brian T; Wu, Yue et al. (2014) Transition state in DNA polymerase ? catalysis: rate-limiting chemistry altered by base-pair configuration. Biochemistry 53:1842-8|
|Hwang, Candy S; Kashemirov, Boris A; McKenna, Charles E (2014) On the observation of discrete fluorine NMR spectra for uridine 5'-?,?-fluoromethylenetriphosphate diastereomers at basic pH. J Org Chem 79:5315-9|
|Seamon, Kyle J; Hansen, Erik C; Kadina, Anastasia P et al. (2014) Small molecule inhibition of SAMHD1 dNTPase by tetramer destabilization. J Am Chem Soc 136:9822-5|
|Sassa, Akira; Ça?layan, Melike; Dyrkheeva, Nadezhda S et al. (2014) Base excision repair of tandem modifications in a methylated CpG dinucleotide. J Biol Chem 289:13996-4008|