Mechanism of DNA replication fidelity for three mutants of DNA polymerase I: Klenow fragment KF(exo+), KF(polA5), and KF(exo-)

Inhibition of the pre-steady-state burst of nucleotide incorporation by a single incorrect nucleotide (nucleotide discrimination) was measured with the Klenow fragment of DNA polymerase I [KF(exo+)]. For the eight mispairs studied on three DNA sequences, only low levels of discrimination ranging from none to 23-fold were found. The kinetics of dNTP incorporation into the 9/20-mer at low nucleotide concentrations was also determined. A limit of greater than or equal to 250 s-1 was placed on the nucleotide off-rate from the KF(exo+)-9/20-dTTP complex in accord with nucleotide binding being at equilibrium in the overall kinetic sequence. The influence of the relatively short length of the 9/20-mer on the mechanism of DNA replication fidelity was determined by remeasuring important kinetic parameters on a 30/M13-mer with high homology to the 9/20-mer. Pre-steady-state data on the nucleotide turnover rates, the dATP(alpha S) elemental effect, and the burst of dAMP misincorporation into the 30/M13-mer demonstrated that the kinetics were not affected by the length of the DNA primer/template. The effects on fidelity of two site-specific mutations, KF(polA5) and KF(exo-), were also examined. KF(polA5) showed an increased rate of DNA dissociation and a decreased rate of polymerization resulting in less processive DNA synthesis. Nevertheless, with at least one misincorporation event, that of dAMP into the 9/20-mer, KF(polA5) displays an increased replication fidelity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic analysis of nucleotide incorporation by mammalian DNA polymerase delta

The kinetics of nucleotide incorporation into 24/36-mer primer/template DNA by purified fetal calf thymus DNA polymerase (pol) delta was examined using steady-state and pre-steady-state kinetics. The role of the pol delta accessory protein, proliferating cell nuclear antigen (PCNA), on DNA replication by pol delta was also examined by kinetic analysis. The steady-state parameter k(cat) was similar for pol delta in the presence and absence of PCNA (0.36 and 0.30 min(-1), respectively); however, the K(m) for dNTP was 20-fold higher in the absence of PCNA (0.067 versus 1.2 microm), decreasing the efficiency of nucleotide insertion. Pre-steady-state bursts of nucleotide incorporation were observed for pol delta in the presence and absence of PCNA (rates of polymerization (k(pol)) of 1260 and 400 min(-1), respectively). The reduction in polymerization rate in the absence of PCNA was also accompanied by a 2-fold decrease in burst amplitude. The steady-state exonuclease rate of pol delta was 0.56 min(-1) (no burst, 10(3)-fold lower than the rate of polymerization). The small phosphorothioate effect of 2 for correct nucleotide incorporation into DNA by pol delta.PCNA indicated that the rate-limiting step in the polymerization cycle occurs prior to phosphodiester bond formation. A K(d)(dNTP) value of 0.93 microm for poldelta.dNTP binding was determined by pre-steady-state kinetics. A 5-fold increase in K(d)(DNA) for the pol delta.DNA complex was measured in the absence of PCNA. We conclude that the major replicative mammalian polymerase, pol delta, exhibits kinetic behavior generally similar to that observed for several prokaryotic model polymerases, particularly a rate-limiting step following product formation in the steady state (dissociation of oligonucleotides) and a rate-limiting step (probably conformational change) preceding phosphodiester bond formation. PCNA appears to affect pol delta replication in this model mainly by decreasing the dissociation of the polymerase from the DNA.     

Analysis of non-template-directed nucleotide addition and template switching by DNA polymerase

DNA polymerases use an uninterrupted template strand to direct synthesis of DNA. However, some DNA polymerases can synthesize DNA across two discontinuous templates by binding and juxtaposing them, resulting in synthesis across the junction. Primer/template duplexes with 3' overhangs are especially efficient substrates, suggesting that DNA polymerases use the overhangs as regions of microhomology for template synapsis. The formation of these overhangs may be the result of non-template-directed nucleotide addition by DNA polymerases. To examine the relative magnitude and mechanism of template switching, we studied the in vitro enzyme kinetics of template switching and non-template-directed nucleotide addition by the 3'-5' exonuclease-deficient large fragment of Escherichia coli DNA polymerase I. Non-template-directed nucleotide addition and template switching were compared to that of standard primer extension. We found that non-template-directed nucleotide addition and template switching showed similar rates and were approximately 100-fold slower than normal template-directed DNA synthesis. Furthermore, non-template-directed nucleotide addition showed a 10-fold preference for adding dAMP to the ends of DNA over that of the other three nucleotides. For template switching, kinetic analysis revealed that the two template substrates acted as a random bireactant system with mixed-type inhibition of substrate binding by one substrate over the other. These data are the first to establish the binding kinetics of two discontinuous DNA substrates to a single DNA polymerase. Our results suggest that although the activities are relatively weak, non-template-directed nucleotide addition and template switching allow DNA polymerases to overcome breaks in the template strand in an error-prone manner.     

Influence of DNA structure on DNA polymerase beta active site function: extension of mutagenic DNA intermediates

In the ternary substrate complex of DNA polymerase (pol) beta, the nascent base pair (templating and incoming nucleotides) is sandwiched between the duplex DNA terminus and polymerase. To probe molecular interactions in the dNTP-binding pocket, we analyzed the kinetic behavior of wild-type pol beta on modified DNA substrates that alter the structure of the DNA terminus and represent mutagenic intermediates. The DNA substrates were modified to 1) alter the sequence of the duplex terminus (matched and mismatched), 2) introduce abasic sites near the nascent base pair, and 3) insert extra bases in the primer or template strands to mimic frameshift intermediates. The results indicate that the nucleotide insertion efficiency (k(cat)/K(m), dGTP-dC) is highly dependent on the sequence identity of the matched (i.e. Watson-Crick base pair) DNA terminus (template/primer, G/C approximately A/T > T/A approximately C/G). Mismatches at the primer terminus strongly diminish correct nucleotide insertion efficiency but do not affect DNA binding affinity. Transition intermediates are generally extended more easily than transversions. Most mismatched primer termini decrease the rate of insertion and binding affinity of the incoming nucleotide. In contrast, the loss of catalytic efficiency with homopurine mismatches at the duplex DNA terminus is entirely due to the inability to insert the incoming nucleotide, since K(d)((dGTP)) is not affected. Abasic sites and extra nucleotides in and around the duplex terminus decrease catalytic efficiency and are more detrimental to the nascent base pair binding pocket when situated in the primer strand than the equivalent position in the template strand.     

Kinetic characterization of the polymerase and exonuclease activities of the gene 43 protein of bacteriophage T4.

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 microM) followed by rapid conversion (400 s-1) of the T4.13/20-mer.dATP complex to the T4.14/20-mer.PPi product species. A slow step (2 s-1) following PPi release 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 > 10(3) against 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 3'-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, 5 s-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 (100s-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.