INFLUENCE OF 5'-NEAREST NEIGHBORS ON THE INSERTION KINETICS OF THE FLUORESCENT NUCLEOTIDE ANALOG 2-AMINOPURINE BY KLENOW FRAGMENT

The effects of nearest neighbor interactions between a nucleotide base at the primer 3'-terminus and an incoming deoxyribonucleoside triphos phate on DNA polymerase catalyzed insertion were examined. Kinetics of inserting the fluorescent nucleotide analog 2-aminopurine deoxyribonu cleotide (dAPMP) and dAMP opposite a template T by 3'-->5' exonuclease -deficient mutants of Klenow fragment (KF-) were measured on primer/te mplates of identical sequence except for the base pair at the 3'-prime r terminus. In addition to its fluorescence properties, 2-aminopurine (AP) is an attractive probe because it is misinserted opposite T by po lymerases at much higher frequencies than natural nucleotides. Misinse rtion frequencies for AP are on the same order of magnitude as variati ons in misinsertion frequencies due to changes in local DNA sequence, which makes the statistical significance of these variations easier to document. We have established that changes in the fluorescence of AP can be used to follow the insertion of dAPMP on both steady-state and pre-steady-state time scales. Rates of insertion of dAPMP measured by fluorescence and by a polyacrylamide gel assay were similar and are se nsitive to the identity of the base at the 3'-primer terminus. The rat e of inserting dAPMP following a primer terminus G, C, or A was twice as fast as insertion following a primer terminus T. The difference in rates arises primarily from differences in k(cat) values, which were f astest next to G and slowest next to T, while apparent K(m) values wer e similar next to each of the 4 different nearest neighbors. The gel a ssay was used to measure AP misinsertion efficiencies by two methods: (1) by having dAPTP and dATP directly compete for insertion opposite T in the same reaction and (2) by measuring V(max)/K(m) values for each substrate in separate reactions. The results from the direct competit ion and separate kinetics measurements are similar. The misinsertion e fficiency of dAPMP relative to dAMP opposite a template T was signific antly higher next to a 3'-primer terminus G (f(ins) = 0.31 +/- 0.06) t han next to T (f(ins) = 0.15 +/- 0.03) for the KF- single mutant (D424 A). The corresponding misinsertion efficiencies next to a 3'-primer te rminus G and T were 0.20 +/- 0.02 and 0.16, respectively, for the KF- double mutant (D355A, E357A). Relative rates of insertion of dAPMP and dAMP correlate with melting temperatures calculated for nearest neigh bor doublets which reflect the relative base-stacking energies. In add ition to changes in insertion kinetics, polymerase-DNA dissociation ra tes varied with the identity of the 3'-primer terminus, differing by a s much as 7-20-fold depending on the polymerase and the primer/templat e.