Pre-steady-state kinetics of RB69 DNA polymerase and its exo domain mutants: effect of pH and thiophosphoryl linkages on 3'-5' exonuclease activity

DNA polymerases from the A and B families with 3'-5' exonucleolytic activity have exonuclease domains with similar three-dimensional structures that require two divalent metal ions for catalysis. B family DNA polymerases that are part of a replicase generally have a more potent 3'-5' exonuclease (exo) activity than A family DNA polymerases that mainly function in DNA repair. To investigate the basis for these differences, we determined pH-activity profiles for the exonuclease reactions of T4, RB69, and phi29 DNA polymerases as representatives of B family replicative DNA polymerases and the Klenow fragment (KF) as an example of a repair DNA polymerase in the A family. We performed exo assays under single-turnover conditions and found that excision rates exhibited by the B family DNA polymerases were essentially independent of pH between pH 6.5 and 8.5, whereas the exo activity of KF increased 10-fold for each unit increase in pH. Three exo domain mutants of RB69 polymerase had much lower exo activities than the wild-type enzyme and exhibited pH-activity profiles similar to that of KF. On the basis of pH versus activity data and elemental effects obtained using short double-stranded DNA substrates terminating in phosphorothioate linkages, we suggest that the rate of the chemical step is reduced to the point where it becomes limiting with RB69 pol mutants K302A, Y323F, and E116A, in contrast to the wild-type enzyme where chemistry is faster than the rate-determining step that precedes it.     

The common A467T mutation in the human mitochondrial DNA polymerase (POLG) compromises catalytic efficiency and interaction with the accessory subunit

Among the nearly 50 disease mutations in the gene for the catalytic subunit of human DNA polymerase gamma, POLG, the A467T substitution is the most common and has been found in 0.6% of the Belgian population. The A467T mutation is associated with a wide range of mitochondrial disorders, including Alpers syndrome, juvenile spinocerebellar ataxia-epilepsy syndrome, and progressive external ophthalmoplegia, each with vastly different clinical presentations, tissue specificities, and ages of onset. The A467T mutant enzyme possesses only 4% of wild-type DNA polymerase activity, and the catalytic defect is manifest primarily through a 6-fold reduction in kcat with minimal effect on exonuclease function. Human DNA polymerase gamma (pol gamma) requires association of a 55-kDa accessory subunit for enhanced DNA binding and highly processive DNA synthesis. However, the A467T mutant enzyme failed to interact with and was not stimulated by the accessory subunit, as judged by processivity, heat inactivation, and N-ethylmaleimide protection assays in vitro. Thermolysin digestion and immunoprecipitation experiments further indicate weak association of the subunits for A467T pol gamma. This is the first example of a mutation in POLG that disrupts physical association of the pol gamma subunits. We propose that reduced polymerase activity and loss of accessory subunit interaction are responsible for the depletion and deletion of mitochondrial DNA observed in patients with this POLG mutation.     

Klenow exo-, as opposed to exo+, traverses through G-G:C triplex by melting G-G base pairs

G-G base-paired hairpin DNA structures on template strands offer potential "road-blocks" to a traversing polymerase. Klenow polymerase (exo+) pauses while replicating through G-G base-paired hairpin DNA due to the generation of G-G:C triplex. However, exonuclease-deficient Klenow traverses through de novo generated G-G:C triplexes leading to full-length C:G duplexes. Alleviation of such road-blocks by exo- Klenow ensues faster at lower Mg2+, a kinetic effect consistent with the role of Mg2+ in stabilizing G-G:C triplex fold. The ability of exonuclease-deficient polymerase to go past the de novo generated G-G:C triplexes suggests that the "idling" of exo+ polymerase at G-G road-block is due to the reiterative polymerase/exonuclease action. The full-length replication product carrying a C(n)-G(n) duplex at one end is further "expanded" by exo- Klenow through C-strand "slippage" leading to the generation of C+-G:C triplex, which is exemplified by the premature arrest of the same at low pH that further stabilizes the C+-G:C triplex.     

Optimal conditions to use Pfu exo(-) DNA polymerase for highly efficient ligation-mediated polymerase chain reaction protocols

Ligation-Mediated Polymerase Chain Reaction (LMPCR) is the most sensitive sequencing technique available to map single-stranded DNA breaks at the nucleotide level of resolution using genomic DNA. LMPCR has been adapted to map DNA damage and reveal DNA-protein interactions inside living cells. However, the sequence context (GC content), the global break frequency and the current combination of DNA polymerases used in LMPCR affect the quality of the results. In this study, we developed and optimized an LMPCR protocol adapted for Pyrococcus furiosus exo(-) DNA polymerase (Pfu exo(-)). The relative efficiency of Pfu exo(-) was compared to T7-modified DNA polymerase (Sequenase 2.0) at the primer extension step and to Thermus aquaticus DNA polymerase (Taq) at the PCR amplification step of LMPCR. At all break frequencies tested, Pfu exo(-) proved to be more efficient than Sequenase 2.0. During both primer extension and PCR amplification steps, the ratio of DNA molecules per unit of DNA polymerase was the main determinant of the efficiency of Pfu exo(-), while the efficiency of Taq was less affected by this ratio. Substitution of NaCl for KCl in the PCR reaction buffer of Taq strikingly improved the efficiency of the DNA polymerase. Pfu exo(-) was clearly more efficient than Taq to specifically amplify extremely GC-rich genomic DNA sequences. Our results show that a combination of Pfu exo(-) at the primer extension step and Taq at the PCR amplification step is ideal for in vivo DNA analysis and DNA damage mapping using LMPCR.     

A unique region in bacteriophage t7 DNA polymerase important for exonucleolytic hydrolysis of DNA

A crystal structure of the bacteriophage T7 gene 5 protein/Escherichia coli thioredoxin complex reveals a region in the exonuclease domain (residues 144-157) that is not present in other members of the E. coli DNA polymerase I family. To examine the role of this region, a genetically altered enzyme that lacked residues 144-157 (T7 polymerase (pol) Delta144-157) was purified and characterized biochemically. The polymerase activity and processivity of T7 pol Delta144-157 on primed M13 DNA are similar to that of wild-type T7 DNA polymerase implying that these residues are not important for DNA synthesis. The ability of T7 pol Delta144-157 to catalyze the hydrolysis of a phosphodiester bond, as judged from the rate of hydrolysis of a p-nitrophenyl ester of thymidine monophosphate, also remains unaffected. However, the 3'-5' exonuclease activity on polynucleotide substrates is drastically reduced; exonuclease activity on single-stranded DNA is 10-fold lower and that on double-stranded DNA is 20-fold lower as compared with wild-type T7 DNA polymerase. Taken together, our results suggest that residues 144-157 of gene 5 protein, although not crucial for polymerase activity, are important for DNA binding during hydrolysis of polynucleotides.     

Replication slippage of different DNA polymerases is inversely related to their strand displacement efficiency.

Replication slippage is a particular type of error caused by DNA polymerases believed to occur both in bacterial and eukaryotic cells. Previous studies have shown that deletion events can occur in Escherichia coli by replication slippage between short duplications and that the main E. coli polymerase, DNA polymerase III holoenzyme is prone to such slippage. In this work, we present evidence that the two other DNA polymerases of E. coli, DNA polymerase I and DNA polymerase II, as well as polymerases of two phages, T4 (T4 pol) and T7 (T7 pol), undergo slippage in vitro, whereas DNA polymerase from another phage, Phi29, does not. Furthermore, we have measured the strand displacement activity of the different polymerases tested for slippage in the absence and in the presence of the E. coli single-stranded DNA-binding protein (SSB), and we show that: (i) polymerases having a strong strand displacement activity cannot slip (DNA polymerase from Phi29); (ii) polymerases devoid of any strand displacement activity slip very efficiently (DNA polymerase II and T4 pol); and (iii) stimulation of the strand displacement activity by E. coli SSB (DNA polymerase I and T7 pol), by phagic SSB (T4 pol), or by a mutation that affects the 3' --> 5' exonuclease domain (DNA polymerase II exo(-) and T7 pol exo(-)) is correlated with the inhibition of slippage. We propose that these observations can be interpreted in terms of a model, for which we have shown that high strand displacement activity of a polymerase diminishes its propensity to slip.     

On-off regulation of 3' exonuclease excision to DNA polymerization by Exo+ polymerase

The role of 3' exonuclease excision in DNA polymerization was evaluated in primer extensions using 3' allele-specific primers that had exonuclease-digestible and exonuclease-resistant 3' termini. With exonuclease-digestible unmodified 3' mismatched primers, the exo+ polymerase yielded template-dependent products. Using exonuclease-resistant 3' mismatched primers, no primer-extended product resulted from exo+ polymerase. As a control, polymerase without proofreading activity yielded primer-dependent products from 3' mismatched primers. These data indicated that a successful removal of the mismatch is required for DNA polymerization from the 3' mismatched primers by exo+ polymerase. In addition to the well-known proofreading from this mismatch removal, the premature termination in DNA polymerization, due to the failure of the efficient removal of the mismatched nucleotides, worked as an off-switch in maintaining the high fidelity in DNA replication from exo+ polymerase.     

A DNA binding motif coordinating synthesis and degradation in proofreading DNA polymerases.

The functional significance of the conserved motif ''YxGG/A'', located between the 3''-5'' exonuclease and polymerization domains of eukaryotic-type DNA polymerases, has been studied by site-directed mutagenesis in phi29 DNA polymerase. Single substitutions at this region were obtained, and 11 phi29 DNA polymerase mutant derivatives were overproduced in Escherichia coli and purified to homogeneity. Nine mutants showed an altered polymerase/3''-5'' exonuclease balance on a template/primer DNA structure, giving rise to three different mutant phenotypes: (i) favored polymerization (high pol/exo ratio); (ii) favored exonucleolysis (low pol/exo ratio); and (iii) favored exonucleolysis and null polymerization. Interestingly, these three different phenotypes could be obtained by mutating a single amino acid at the ''YxGG/A'' motif. All different phenotypes could be directly related to defects in DNA binding at a particular active site. Thus, a high pol/exo ratio was related to a poor stability at the 3''-5'' exonuclease active site. On the contrary, a low pol/exo ratio or null polymerization capacity was related to a poor stability at the polymerization active site and either a normal or an increased accessibility to the exonuclease active site. These results allow us to propose that this motif, located in the connecting region between the N-terminal and C-terminal domains, has a primary role in DNA binding, playing a critical role in the coordination or cross-talk between synthesis and degradation.     

Pausing of DNA polymerases on duplex DNA templates due to ligand binding in vitro

Using the recently developed peptide nucleic acid (PNA)-assisted assay, which makes it possible to extend a primer on duplex DNA, we study the sequence-specific inhibition of the DNA polymerase movement along double-stranded DNA templates imposed by DNA-binding ligands. To this end, a plasmid vector has been prepared featuring the polylinker with two flanking priming sites to bi-directionally initiate the primer-extension reactions towards each other. Within this plasmid, we have cloned a set of random DNA sequences and analyzed the products of these reactions with several phage and bacterial DNA polymerases capable of strand-displacement synthesis. Two of them, ?29 and modified T7 (Sequenase 2.0) enzymes, were found to be most potent for primer extension in the presence of DNA-binding ligands. We used these enzymes for a detailed study of ligand-induced pausing effects with four ligands differing in modes of binding to the DNA double-helix. GC-specific intercalator actinomycin D and three minor groove-binders, chromomycin A(3) (GC-specific), distamycin A and netropsin (both AT-specific), have been chosen. In the presence of each ligand both selected DNA polymerases experienced multiple clear-cut pauses. Each ligand yielded its own characteristic pausing pattern for a particular DNA sequence. The majority of pausing sites could be located with a single-nucleotide resolution and corresponded to the preferred binding sites known from the literature for the ligands under study. Besides, DNA polymerases stalled exactly at the positions occupied by PNA oligomers that were employed to initiate the primer extension. These findings provide an important insight into the DNA polymerase performance. In addition, the high-resolution ligand-induced pausing patterns we obtained for the first time for DNA polymerase elongation on duplex DNA may become a valuable addition to the existing arsenal of methods used to monitor duplex DNA interactions with various DNA-binding ligands, including drugs.     

The highly processive DNA polymerase of bacteriophage T5. Role of the unique N and C termini

The DNA polymerase encoded by bacteriophage T5 has been reported previously to be processive and to catalyze extensive strand displacement synthesis. The enzyme, purified from phage-infected cells, did not require accessory proteins for these activities. Although T5 DNA polymerase shares extensive sequence homology with Escherichia coli DNA polymerase I and T7 DNA polymerase, it contains unique regions of 130 and 71 residues at its N and C termini, respectively. We cloned the gene encoding wild-type T5 DNA polymerase and characterized the overproduced protein. We also examined the effect of N- and C-terminal deletions on processivity and strand displacement synthesis. T5 DNA polymerase lacking its N-terminal 30 residues resembled the wild-type enzyme albeit with a 2-fold reduction in polymerase activity. Deletion of 24 residues at the C terminus resulted in a 30-fold reduction in polymerase activity on primed circular DNA, had dramatically reduced processivity, and was unable to carry out strand displacement synthesis. Deletion of 63 residues at the C terminus resulted in a 20,000-fold reduction in polymerase activity. The 3' to 5' double-stranded DNA exonuclease activity associated with T5 DNA polymerase was reduced by a factor of 5 in the polymerase truncated at the N terminus but was stimulated by a factor of 7 in the polymerase truncated at the C terminus. We propose a model in which the C terminus increases the affinity of the DNA for the polymerase active site, thus increasing processivity and decreasing the accessibility of the DNA to the exonuclease active site.     

Exonuclease proofreading by human mitochondrial DNA polymerase

We have examined the ability of the human mitochondrial DNA polymerase to correct errors in DNA sequence using single turnover kinetic methods. The rate of excision of single-stranded DNA ranged from 0.07 to 0.17 x s(-1), depending on the identity of the 3'-base. Excision of the 3'-terminal base from correctly base paired DNA occurred at a rate of 0.05 x s(-1), indicating that the cost of proofreading is minimal, as defined by the ratio of the k(exo) for correctly base-paired DNA divided by the rate of forward polymerization (0.05/37 = 0.14%). Excision of duplex DNA containing 1-7 mismatches was biphasic, and the rate and amplitude of the fast phase increased with the number of mismatches, reaching a maximum of 9 x s(-1). We showed that transfer of DNA from the polymerase to the exonuclease active site and back again occurs through an intramolecular reaction, allowing for a complete cycle of reactions for error correction. For DNA containing a buried mismatch (T:T followed by C:G base pairs), the 3' base was removed at a rate of 3 x s(-1). The addition of nucleotide to the reaction that is identical to the 3' base increased the rate of excision 7-fold to 21 x s(-1). We propose that the free nucleotide enhances the rate of transfer of the DNA to the exonuclease active site by interrupting the correct 3' base pair through interaction with the template base. The exonuclease contribution to fidelity is minimal if the calculation is based on hydrolysis of a single mismatch: (k(exo) + k(pol,over))/(k(pol,over)) = 10, but this value increases to approximately 200 when examining error correction in the presence of nucleotides.     

Molecular and biochemical characterisation of a novel mutation in <Emphasis Type="Italic">POLG</Emphasis>associated with Alpers syndrome

Background  

DNA polymerase γ (POLG) is the only known mitochondrial DNA (mtDNA) polymerase. It mediates mtDNA replication and base excision repair. Mutations in the POLG gene lead to reduction of functional mtDNA (mtDNA depletion and/or deletions) and are therefore predicted to result in defective oxidative phosphorylation (OXPHOS). Many mutations map to the polymerase and exonuclease domains of the enzyme and produce a broad clinical spectrum. The most frequent mutation p.A467T is localised in the linker region between these domains. In compound heterozygote patients the p.A467T mutation has been described to be associated amongst others with fatal childhood encephalopathy. These patients have a poorer survival rate compared to homozygotes.     

Rapid high-efficiency site-directed mutagenesis by the phosphorothioate approach.

Several improvements to the existing phosphorothioate-based site-directed mutagenesis methodology are reported, and here it is demonstrated that the new procedure is able to produce large deletions, insertions and point mutations rapidly and with very high efficiency. The time required for the polymerization step has been reduced by using T7 DNA polymerase to extend the mutant oligonucleotide primer-template. The reaction produces good yields of double-stranded closed-circular DNA and some partially polymerized template. The reaction was treated with T5 D15 exonuclease to selectively destroy partially polymerized single-stranded phage DNA that may otherwise contribute to an increased background of wild-type transformants. The use of these enzymes greatly facilitates the implementation of the phosphorothioate-based site-directed mutagenesis method by requiring less template DNA and by allowing all the in vitro manipulations to be completed in a day. In its present form the method may easily be automated, enabling large systematic site-directed mutagenesis projects to be undertaken.