Conformation of Deoxynucleoside Triphosphate Substrates on DNA Polymerase I from Escherichia coZi as Determined by Nuclear Magnetic Relaxation*

A unique conformation of deoxynucleoside triphosphate substrates bound to Escherichia coli DNA polymerase I has been determined by nuclear magnetic resonance techniques. The effects of Mn(I1) bound at the active site of the enzyme on the longitudinal (Ti,‘) and transverse (T;;,‘) relaxation rates of the 01, /3, and y phosphorus atoms and 5 protons of enzyme-bound thymidine 5’.triphosphate (dTTP) were measured at 40.5 MHz ($lP), 100 and 220 MHz ( ‘H). From the frequency dependence of Tt’, a correlation time of 7 x 10-l” s and Mn(II) to proton distances of 10.4, 9.9, 10.3, 10.8, and 8.4 8, were calculated for the -CH,, H,, H’,, H’,, and H’, protons. The calculated Mn(I1) to phosphorus distances of 4.2, 4.8, and 3.2 A for the u, /3, and y phosphorus atoms indicates that Mn(I1) coordinates directly only with the yphosphoryl group and that a puckered triphosphate conformation exists for the enzyme-bound dTTP. This differs from the binary Mn(I1) .dTTP complex in which (Y, 8, and y phosphoryl coordination occurs, and a thymine-deoxyribose ttirsion angle (x) about the glycosidic bond of 40” is detected. The eight manganese-substrate distances on the enzyme are fit by a unique Mn .dTTP conformation, with a torsion angle equal to 90”, indistinguishable from that found for a deoxynucleotidyl unit in double helical DNA-B. Hence, binding to DNA polymerase appears to adjust the conformation of dTTP for Watson-Crick basepairing. Similarly, the binding of Mn .dATP to DNA polymerase I increased the distances from Mn(I1) to the H, H,, H’,, and H’, protons of dATP but the adenine-deoxyribose torsion angle of 90” was preserved. Such preorientation of substrates could facilitate incorporation of the complementary nucleotide. When positioned within the DNA structure, the conformation of enzyme-bound Mn -dTTP requires an inline nucleophilic attack on the (Y phosphorus with Mn(I1) promoting pyrophosphate departure.