Halogenated β,γ-Methylene- and Ethylidene-dGTP-DNA Ternary Complexes with DNA Polymerase β: Structural Evidence for Stereospecific Binding of the Fluoromethylene Analogues

摘要

Abstract: u0001,γ-Fluoromethylene analogues of nucleotides are considered to be useful mimics of the naturalnsubstrates, but direct structural evidence defining their active site interactions has not been available,nincluding the influence of the new chiral center introduced at the CHF carbon, as in u0001,γ-fluoromethylene-ndGTP, which forms an active site complex with DNA polymerase u0001, a repair enzyme that plays an importantnrole in base excision repair (BER) and oncogenesis. We report X-ray crystallographic results for a seriesnof u0001,γ-CXY dGTP analogues, where X,Y H, F, Cl, Br, and/or CH3. For all three R/S monofluorinatednanalogues examined (CHF, 3/4; CCH3F, 13/14; CClF 15/16), a single CXF-diastereomer (3, 13, 16)isnobserved in the active site complex, with the CXF fluorine atom at a ∼3 Å (bonding) distance to a guanidiniumnN of Arg183. In contrast, for the CHCl, CHBr, and CHCH3 analogues, both diasteromers (6/7, 8/9, 10/11)npopulate the dGTP site in the enzyme complex about equally. The structures of the bound dichloro (5) andndimethyl (12) analogue complexes indicate little to no steric effect on the placement of the bound nucleotidenbackbone. The results suggest that introduction of a single fluorine atom at the u0001,γ-bridging carbon atomnof these dNTP analogues enables a new, stereospecific interaction within the preorganized active sitencomplex that is unique to fluorine. The results also provide the first diverse structural data set permittingnan assessment of how closely this class of dNTP analogues mimics the conformation of the parent nucleotidenwithin the active site complex.