PRE-STEADY-STATE KINETIC-ANALYSIS OF SEQUENCE-DEPENDENT NUCLEOTIDE EXCISION BY THE 3'-EXONUCLEASE ACTIVITY OF BACTERIOPHAGE-T4 DNA-POLYMERASE

The effects of local DNA sequence on the proofreading efficiency of wi ld-type T4 DNA polymerase were examined by measuring the kinetics of r emoval of the fluorescent nucleotide analog 2-aminopurine deoxynucleos ide monophosphate (dAPMP) from primer/templates of defined sequences. The effects of (1) interactions with the 5'-neighboring bases, (2) bas e pair stability, and (3) G.C content of the surrounding sequences on the pre-steady-state kinetics of dAPMP excision were measured. Rates o f excision dAPMP from a primer 3'-terminus located opposite a template T (AP.T base pair) increased, over a 3-fold range, with the 5'-neighb or to AP in the order C < G < T < A. Rates of removal of dAPMP from AP .X base pairs located in the same surrounding Sequence increased as AP .T < AP.A < AP.C < AP.G, which correlates with the decrease in the sta bilities of these base pairs predicted by T-m measurements. A key find ing was that AP was excised at a slower rate when mispaired opposite C located next to four G.C base pairs than when correctly paired opposi te T next to four A.T base pairs, suggesting that exonuclease mismatch removal specificities may be enhanced to a much greater extent by ins tabilities of local primer termini than by specific recognition of inc orrect base pairs. In polymerase-initiated reactions, biphasic reactio n kinetics were observed for the excision of AP within most but not al l sequence contexts. Rates of the rapid phases (30-40 s(-1)) were rela tively insensitive to sequence context. Rapid-phase rates reflect the rate constants for exonucleolytic excision of dAPMP from melted primer termini for both correct and incorrect base pairs and were roughly co mparable to rates of removal of dAPMP from single-stranded DNA (65-80 s(-1)). Rates of the slow phases (3-13 s(-1)) were dependent on sequen ce context; the slow phase may reflect the rate of switching from the polymerase to the exonuclease active site, or perhaps the conversion o f a primer/template terminus from an annealed to a melted state in the exonuclease active site. These data, using wild-type T4 DNA polymeras e and two exonuclease-deficient T4 polymerases, support a model in whi ch exonuclease excision occurs on melted primer 3'-termini for both mi smatched and correctly matched primer termini, and where specificity f avoring removal of terminally mismatched base pairs is determined by t he much larger fraction of melted-out primer 3'-termini for mispairs c ompared to that for correct pairs.