beta,gamma-Fluoromethylene analogues of nucleotides are generally considered to be useful mimics of the natural substrates for DNA polymerases, but direct structural evidence defining their active site interactions has not been available. In addition, the effect of introducing a new chiral center (the CHF carbon) has been unexplored. We report here structural studies of the diastereomeric beta,gamma- CHF analogues (R, 3; S, 4) of dGTP interacting with the active site of DNA pol beta, a repair enzyme that plays an important role in base excision repair (BER) and oncogenesis. The conjugation of dGMP 5'-morpholidate with a tetrabutylammonium salt of (fluoromethylene)bisphosphonic acid (6b, prepared like its difluoro analogue 7b via fluorination of tetraisopropyl methylenebisphosphonate carbanion with Selectfluor) gives a 1:1 mixture of 3 and 4 (by F-19 NMR, pH 10). The beta,gamma-CF2 (2) and beta,gamma-CH2 (1) dGTP analogues were also synthesized. Crystallization from a solution containing 3 + 4 together with a preformed DNA pol beta complex of a 16-mer template DNA and a one-nucleotide gapped primer produced crystals containing the (R)-analogue 3, as shown by the X-ray structure (2.1 angstrom), which revealed a 3.0 angstrom (bonding) distance between a guanidine N of Arg 183 and the CHF fluorine atom. Ligand docking simulations of 3 vs 4 using Autodock 3.0 predicted that both 3 and 4 can adopt an overall orientation closely overlaying that of dGTP itself in the active site; however, a polar C-F center dot center dot Arg183 bonding interaction is favored only with 3. A similar orientation of one fluorine atom in 2 is observed. The results suggest that introduction of a single fluorine atom at the bridging carbon atom of a beta,gamma-methylene-dNTP analogue may enable a new, stereospecific interaction within the pre-organized active site complex.