DNA polymerase catalysis and fidelity studies typically compare incorporation of "right" versus "wrong" nucleotide bases where the leaving group is pyrophosphate. Here we use dGTP analogues replacing the beta,gamma-bridging O with CH2, CHF, CF2, or CCl2 to explore leaving-group effects on the nucleotidyl transfer mechanism and fidelity of DNA polymerase (pol) beta. T.G mismatches occur with fidelities similar to dGTP with the exception of the CH2 analogue, which is incorporated with 5-fold higher fidelity. All analogues are observed to bind opposite template C with Kds between 1 and 4 microM, and structural evidence suggests that the analogues bind in essentially the native conformation, making them suitable substrates for probing linear free energy relationships (LFERs) in transient-kinetics experiments. Importantly, Brnsted correlations of log(kpol) versus leaving-group pKa for both right and wrong base incorporation reveal similar sensitivities (betalg approximately -0.8) followed by departures from linearity, suggesting that a chemical step rather than enzyme conformational change is rate-limiting for either process. The location of the breaks relative to pKas of CF2, O, and the sterically bulky CCl2-bridging compounds suggests a modification-induced change in the mechanism by stabilization of leaving-group elimination. The results are addressed theoretically in terms of the energetics of successive primer 3'-O addition (bond forming) and pyrophosphate analogue elimination (bond breaking) reaction energy barriers.

The mechanism of DNA polymerase beta-catalyzed nucleotidyl transfer consists of chemical steps involving primer 3' OH deprotonation, nucleophilic attack, and pyrophosphate leaving-group elimination, preceded by dNTP binding which induces a large-amplitude conformational change for Watson-Crick nascent base pairs. Ambiguity in the nature of the rate-limiting step and active-site structural differences between correct and incorrect base-paired transition states remain obstacles to understanding DNA replication fidelity. Analogues of dGTP where the beta-gamma bridging oxygen is replaced with fluorine-substituted methylene groups have been shown to probe the contribution of leaving-group elimination to the overall catalytic rate (Biochemistry 46, 461-471). Here, the analysis is expanded substantially to include a broad range of halogen substituents with disparate steric and electronic properties. Evaluation of linear free energy relationships for incorporation of dGTP analogues opposite either template base C or T reveals a strong correlation of log(kpol) to leaving group pKa. Significantly different kpol behavior is observed with a subset of the analogues, with magnitude dependent on the identity of the nascent base pair. This observation, and the absence of an analogous effect on ground state analogue binding (Kd values), points to active-site structural differences at the chemical transition state. Reduced catalysis with bulky halo-containing substrates is manifested in the fidelity of T-G incorporation, where the CCl2-bridging analogue shows a 27-fold increase in fidelity over the natural dGTP. Solvent pH and deuterium isotope-effect data are also used to evaluate mechanistic differences between correct and mispaired incorporation.

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Background: Apo3G, an ssDNA-dependent C deaminase, inactivates HIV-1 in T cells by C to T hypermutation. Results: smFRET is used to detect Apo3G scanning and C-deamination on ssDNA. Conclusion: Apo3G scans ssDNA randomly and bidirectionally, favoring nonrandom 3Ј to 5Ј deamination. Significance: This smFRET study describes a broadly applicable approach to visualize motion and catalysis in real time by an enzyme that scans ssDNA.

Abstractβ,γ-Fluoromethylene analogues of nucleotides are considered to be useful mimics of the natural substrates, but direct structural evidence defining their active site interactions has not been available, including the influence of the new chiral center introduced at the CHF carbon, as in β,γ-fluoromethylene-dGTP, which forms a active site complex with DNA polymerase β, a repair enzyme that plays an important role in base excision repair (BER) and oncogenesis. We report X-ray crystallographic results for a series of β,γ-CXY dGTP analogues, where X,Y = H, F, Cl, Br, and/or CH 3 . For all three monofluorinated analogues examined (CHF, 3/4; CCH 3 F , 13/14; CClF 15/16), a single CXF-diastereomer (3, 13, 15) is observed in the active site complex, with the CXF fluorine atom at a ~3 Å (bonding) distance to a guanidinium N of Arg183. In contrast, for the CHCl, CHBr and CHCH 3 analogues, both diasteromers (6/7, 8/9, 10/11) populate the dGTP site in the enzyme complex about equally. The structures of the bound dichloro (5) and dimethyl (12) analogue complexes indicate little to no steric effect on the placement of the bound nucleotide backbone. The results suggest that introduction of a single fluorine atom at the β,γ-bridging carbon atom of these dNTP analogues enables a new, stereospecific interaction within the pre-organized active site complex that is unique to fluorine. The results also provide the first diverse structural dataset permitting an assessment of how closely this class of dNTP analogues mimics the conformation of the parent nucleotide within the active site complex.

Abstractα,β-Difluoromethylene deoxynucleoside 5'-triphosphates (dNTPs, N = A or C) are advantageously obtained via phosphorylation of corresponding dNDP analogues using catalytic ATP, PEP, nucleoside diphosphate kinase (NDPK) and pyruvate kinase (PK). DNA pol β K d values for the α,β-CF 2 and unmodified dNTPs, α,β-NH dUTP, and the a,β-CH 2 analogues of dATP and dGTP are discussed in relation to the conformations of α,β-CF 2 dTTP v. α,β-NH dUTP bound into the enzyme active site.In an ongoing multidisciplinary study of structure and function of DNA polymerase β, a eukaryotic enzyme primarily involved in filling short DNA gaps 1 , we required a series of α,β-methylene-substituted analogues of deoxynucleoside 5'-triphosphates (dNTPs). When the *Email Address: mckenna@usc.edu. Supporting Information Available General methods used; detailed synthetic procedures and characterization data for 1 -9; analytical and preparative HPLC methods; HPLC studies of conversions of 1 to 2, 2 to 3, 7 to 4, 4 to 5, 5 to 6; 1 H NMR spectra of 1-7 and 9; 31 P and 19 F NMR spectra of 2, 3, 5, 6, 9; 13 C NMR spectrum of 9; analytical HPLC traces for 3, 6 and 9; HRMS spectra for 3 and 6, and LRMS spectrum for 9; DNA synthesis gel analysis of 9 before and after HPLC purification; protocol and inhibition plot for inhibition of pol β dCTP incorporation by 6; crystallographic methods and statistics for the ternary pol β complex with 9; and supplementary literature references are available online at http://pubs.acs.org. P α -O-P β bridging oxygen in a natural mononucleotide substrate is replaced by an imido (NH) 2-4 or methylene (CXY) 4,5 group, the P-N or P-C bond should resist cleavage in the nucleotidyl transfer reaction catalyzed by the enzyme. As a result, these analogues will remain intact in stable ternary DNA complexes with the polymerase and therefore should be useful to probe pre-chemistry enzyme-complex function and structure, as recently shown with in an X-ray crystallographic study of α,β-NH dUTP with DNA pol β. 6 Information about such complexes provides a reference point for theoretical analysis of the chemical mechanism 7 for the complete transfer of a monophosphate nucleoside donor to the sugar acceptor in the active site. As probes for the mechanism of polymerase catalysis and its relationship to polymerase fidelity, α,β-methylene dNTP analogues permit exploration of stereoelectronic effects on active site interactions, by making appropriate substitutions X,Y on the adjacent P α CXY bridging carbon. The largest obtainable electron-withdrawing effect with minimal steric perturbation can be achieved using X,Y = F, resulting in analogues in which the bisphosphonate group is expected to be less basic than the pyrophosphate moiety in the natural dNTPs. 8,9 In this article, we describe the first synthesis of α,β-CF 2 dCTP 6, using a modified chemicalenzymological approach that also can be applied to synthesis of α,β-CF 2 dATP 3, affording these compounds in sufficient purity to virtually eliminate detectable contaminating substrate ...

It is difficult to overestimate the importance of nucleoside triphosphates in cellular chemistry: They are the building blocks for DNA and RNA and important sources of energy. Modifications of biologically important organic molecules with fluorine are of great interest to chemists and biologists because the size and electronegativity of the fluorine atom can be used to make defined structural alterations to biologically important molecules. Although the concept of nonhydrolyzable nucleotides has been around for some time, the progress in the area of modified triphosphates was limited by the lack of synthetic methods allowing to access bisCF 2 -substituted nucleotide analogs-one of the most interesting classes of nonhydrolyzable nucleotides. These compounds have "correct" polarity and the smallest possible steric perturbation compared to natural nucleotides. No other known nucleotides have these advantages, making bisCF 2 -substituted analogs unique. Herein, we report a concise route for the preparation of hitherto unknown highly acidic and polybasic bis(difluoromethylene)triphosphoric acid 1 using a phosphorous(III)/phosphorous(V) interconversion approach. The analog 1 compared to triphosphoric acid is enzymatically nonhydrolyzable due to substitution of two bridging oxygen atoms with CF 2 groups, maintaining minimal perturbations in steric bulkiness and overall polarity of the triphosphate polyanion. The fluorinated triphosphoric acid 1 was used for the preparation of the corresponding fluorinated deoxynucleotides (dNTPs). One of these dNTP analogs (dT) was demonstrated to fit into DNA polymerase beta (DNA pol β) binding pocket by obtaining a 2.5 Å resolution crystal structure of a ternary complex with the enzyme. Unexpected dominating effect of triphosphate∕Mg 2þ interaction over Watson-Crick hydrogen bonding was found and discussed.DNA polymerase beta | nonhydrolyzable nucleotides | fluorinated triphosphate | pentabasic acid | isopolarity and bioisotericity

A deuterated cavitand host was examined for its affinity to a series of guests; for halogenated, preorganized guests binding was significantly stronger than the corresponding protium host.