The DNA polymerase from the bacteriophage T4 is part of a multienzyme complex required for the synthesis of DNA. As a first step in understanding the contributions of individual proteins to the dynamic properties of the complex, e.g., turnover, processivity, and fidelity of replication, the minimal kinetic schemes for the polymerase and exonuclease activities of the gene 43 protein have been determined by pre-steady-state kinetic methods and fit by computer simulation. A DNA primer/template (13/20-mer) was used as substrate; duplexes that contained more single-strand DNA resulted in nonproductive binding of the polymerase. The reaction sequence features an ordered addition of 13/20-mer followed by dATP to the T4 enzyme (dissociation constants of 70 nM and 20 μM) followed by rapid conversion (400 s−1) of the T4-13/20-mer-dATP complex to the T4-14/20-mer-PPiproduct species. A slow step (2 s−1) following PPirelease limits a single turnover, although this step is bypassed in multiple incorporations (13/20-mer → 17/20-mer) which occur at rates 400 s−1. Competition between correct versus incorrect nucleotides relative to the template strand indicates that the dissociation constants for the incorrect nucleotides are at millimolar values, thus providing evidence that the T4 polymerase, like the T7 but unlike the Klenow fragment polymerases, discriminates by factors 103against misincorporation in the nucleotide binding step. The exonuclease activity of the T4 enzyme requires an activation step, i.e., T4-DNA → T4-(DNA)*, whose rate constants reflect whether the 3ʹ-terminus of the primer is matched or mismatched; for matched 13/20-mer the constant is 1 s_1, and for mismatched 13T/20-mer, 5s−1. Evidence is presented from crossover experiments that this step may represent a melting of the terminus of the duplex, which is followed by rapid exonucleolytic cleavage (100 s−1). In the presence of the correct dNTP, primer extension is the rate-limiting step rather than a step involving travel of the duplex between separated exonuclease and polymerase sites. Since the rate constant for 13/20-mer or 13T/20-mer dissociation from the enzyme is 6 or 8 s−1 and competes with that for activation, the exonucleolytic editing by the enzyme alone in a single pass is somewhat inefficient (5 s−1/(8 s−1+ 5 s−1)), ca. 40%. Consequently, a major role for the accessory proteins may be to slow the rate of enzyme-substrate dissociation, thereby increasing overall fidelity and processivity.
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