Proofreading Function Research Articles

Hyper-processive and slower DNA chain elongation catalysed by DNA polymerase III holoenzyme purified from the dnaE173 mutator mutant of Escherichia coli.

A strong mutator mutation, dnaE173, leads to a Glu612 --> Lys amino acid change in the alpha subunit of Escherichia coli DNA polymerase III (PolIII) holoenzyme and abolishes the proofreading function of the replicative enzyme without affecting the 3' --> 5' exonuclease activity of the epsilon subunit. The dnaE173 mutator is unique in its ability to induce sequence-substitution mutations, suggesting that an unknown function of the alpha subunit is hampered by the dnaE173 mutation. A PolIII holoenzyme reconstituted from dnaE173 PolIII* (DNA polymerase III holoenzyme lacking the beta clamp subunit) and the beta subunit showed a strong resistance to replication-pausing on the template DNA and readily promoted strand-displacement DNA synthesis. Unlike wild-type PolIII*, dnaE173 PolIII* was able to catalyse highly processive DNA synthesis without the aid of the beta-clamp subunit. The rate of chain elongation by the dnaE173 holoenzyme was reduced to one-third of that determined for the wild-type enzyme. In contrast, an exonuclease-deficient PolIII holoenzyme was vastly prone to pausing, but had the same rate of chain elongation as the wild-type. The hyper-processivity and slower DNA chain elongation rate of the dnaE173 holoenzyme are distinct effects caused by the dnaE173 mutation and are likely to be involved in the sequence-substitution mutagenesis. A link between the proofreading and chain elongation processes was suggested.

The TREX2 3'-->5' exonuclease physically interacts with DNA polymerase delta and increases its accuracy.

Proofreading function by the 3′→ 5′ exonuclease of DNA polymerase δ (pol δ) is consistent with the observation that deficiency of the associated exonuclease can lead to a strong mutation phenotype, high error rates during DNA replication, and ultimately cancer. We have isolated pol δd from isotonic (pol δi) and detergent (pol δd) calf thymus extracts. Pol δd had a 20-fold higher ratio of exonuclease to DNA polymerase than pol δi. This was due to the physical association of the TREX2 exonuclease to pol δd, which was missing from pol δi. Pol δd was fivefold more accurate than pol δi under error-prone conditions (1 μM dGTP and 20 dATP, dCTP, and dTTP) in a M13mp2 DNA forward mutation assay, and fourfold more accurate in an M13mp2T90 reversion assay. Under error-free conditions (20 μM each of the four dNTPs), however, both polymerases showed equal fidelity. Our data suggested that autonomous 3′→ 5′ exonucleases, such as TREX2, through its association with pol I can guarantee high fidelity under difficult conditions in the cell (e.g., imbalance of dNTPs) and can add to the accuracy of the DNA replication machinery, thus preventing mutagenesis.

Observation of positive selection within hypervariable regions of a newly identified DNA virus (SEN virus)

To elucidate the evolution of SEN virus (SEN-V), serial sequences of chronically SEN-V-infected patients were analyzed. In the hypervariable regions, non-synonymous substitutions significantly predominated. This could be attributed to positive selection in evading immune surveillance of the hosts and to establish a persistent infection. On the basis of the sequences in the two open reading frames of SEN-V DNA, the rate of synonymous substitutions was 7.32×10 −4 per site per year. Since this rate is close to RNA viruses and higher than other DNA viruses, the SEN-V might be replicated by machinery with poor or no proofreading function.

P53 enhances the fidelity of DNA synthesis by human immunodeficiency virus type 1 reverse transcriptase.

The tumor suppressor protein p53 plays a critical role in the maintenance of genetic integrity. p53 possesses 3'-->5' exonuclease activity, however, the significance of this function in DNA replication process remains elusive. It was suggested that 3'-->5' exonuclease activity of p53 may provide a proofreading function for DNA polymerases. In order to better understand the significance of this activity, the purified wild-type recombinant p53 was further evaluated for substrate specificity and for contribution to the accuracy of DNA synthesis. p53-associated 3'-->5' exonuclease displays 3' terminal nucleotide excision from RNA/DNA template-primer using ribosomal RNA as a template. The data demonstrate that p53 is highly efficient in removing a terminal mispair. Analysis of mispair excision opposite the template adenine residue shows that p53 catalyzes 3' terminal mismatch excision with a specificity of A : G>A : A>A : C. Hence, the observed specificity of mismatch excision indicates that p53 exonucleolytic proofreading preferentially repairs transversion mutations. The influence of the p53 on the accuracy of DNA synthesis was determined with exonuclease-deficient human immunodeficiency virus-1 (HIV-1) reverse transcriptase (RT), a key enzyme in the life cycle of the virus, that contributes significantly to the low accuracy of proviral DNA synthesis. Using an in vitro biochemical assay with recombinant purified HIV-1 RT, p53 and defined RNA/DNA or DNA/DNA template-primers, two basic features related to fidelity of DNA synthesis were studied: the misinsertion and mispair extension. The misincorporation of non-complementary deoxynucleotides into nascent DNA and subsequent mispair extension by HIV-1 RT were substantially decreased in the presence of p53 with both RNA/DNA and DNA/DNA template-primers. In addition, the productive interaction between polymerization (by HIV-1 RT) and exonuclease (by p53) activities was observed; p53 preferentially hydrolyzes mispaired 3'-termini, permitting subsequent extension of the correctly paired 3'-terminus by HIV-1 RT. Taken together the data demonstrate that preferential excision of mismatched nucleotides by 3'-->5' exonuclease activity of wild-type p53 enhances the fidelity of DNA synthesis by HIV-1 RT in vitro, thus providing a biochemical mechanism to reduce mutations caused by incorporation of mismatched nucleotides. The fact that p53 is reactive with both RNA/DNA and DNA/DNA template-primers raises an interesting possibility of the existence of functional cooperation between p53 and HIV-1 RT in cytoplasm during the reverse transcription process, which may be important for maintaining HIV genomic integrity.

Molecular mechanisms of DNA mismatch repair

DNA mismatch repair (MMR) safeguards the integrity of the genome. In its role in postreplicative repair, this repair pathway corrects base–base and insertion/deletion (I/D) mismatches that have escaped the proofreading function of replicative polymerases. In its absence, cells assume a mutator phenotype in which the rate of spontaneous mutation is greatly elevated. The discovery that defects in mismatch repair segregate with certain cancer predisposition syndromes highlights its essential role in mutation avoidance. Recently, three-dimensional structures of MutS, a key repair protein that recognizes mismatches, have been determined by X-ray crystallography. This article provides an overview of the structural features of MutS proteins and discusses how the structural data together with biochemical and genetic studies reveal new insights into the molecular mechanisms of mismatch repair.

Interacting Fidelity Defects in the Replicative DNA Polymerase of Bacteriophage RB69

The DNA polymerases (gp43s) of the related bacteriophages T4 and RB69 are B family (polymerase alpha class) enzymes that determine the fidelity of phage DNA replication. A T4 whose gene 43 has been mutationally inactivated can be replicated by a cognate RB69 gp43 encoded by a recombinant plasmid in T4-infected Escherichia coli. We used this phage-plasmid complementation assay to obtain rapid and sensitive measurements of the mutational specificities of mutator derivatives of the RB69 enzyme. RB69 gp43s lacking proofreading function (Exo(-) enzymes) and/or substituted with alanine, serine, or threonine at the conserved polymerase function residue Tyr(567) (Pol(Y567(A/S/T)) enzymes) were examined for their effects on the reversion of specific mutations in the T4 rII gene and on forward mutation in the T4 rI gene. The results reveal that Tyr(567) is a key determinant of the fidelity of base selection and that the Pol and Exo functions are strongly coupled in this B family enzyme. In vitro assays show that the Pol(Y567A) Exo(-) enzyme generates mispairs more frequently but extends them less efficiently than does a Pol(+) Exo(-) enzyme. Other replicative DNA polymerases may control fidelity by strategies similar to those used by RB69 gp43.

A nucleoside analogue of 2, 4-difluoropyridine has potential as an antiretroviral agent with multiple and unique mechanisms of action, and may be effective against the HIV organism

The high rate of mutation which is inherent in reverse transcription of the HIV genome is a result of the lack of proof-reading function of the reverse transcriptase enzyme. This has allowed the HIV virus to develop resistance to multiple antiviral agents. It may be possible to use this viral property to advantage by treatment with an antiviral nucleoside analogue which is a close structural isostere of uridine and deoxyuridine. The drug is unable to form hydrogen bonds with adenine and will be excluded from host cell DNA by its 3′ to 5′ proof-reading exonuclease activity. However, reverse transcriptase, which has no such mechanism, will allow incorporation of the drug into proviral DNA.The drug will have an inhibitory effect on RNase H function. It will also be expected to cause delay in elongation at those sites in the template strand that contain two or more adjacent adenine bases, because two drug molecules will, for practical purposes, never be inserted in the same strand next to each other. The length of the delay in strand elongation will therefore be a function of the availability of the natural NTP or dNTP. Both the rate and fidelity of protein synthesis will be affected by the drug. There will be decreased stability of the proviral double stranded DNA and if the proviral DNA is able to integrate into the host cell chromosome, double stranded breaks may be produced by the host cells’ DNA repair mechanisms. Finally there will be a specific ‘strand trade’ mutation that the drug will induce specifically into viral but not into cellular genetic material.

Domain exchange: chimeras of Thermus aquaticus DNA polymerase, Escherichia coli DNA polymerase I and Thermotoga neapolitana DNA polymerase

The intervening domain of the thermostable Thermus aquaticus DNA polymerase (TAQ: polymerase), which has no catalytic activity, has been exchanged for the 3'-5' exonuclease domain of the homologous mesophile Escherichia coli DNA polymerase I (E.coli pol I) and the homologous thermostable Thermotoga neapolitana DNA polymerase (TNE: polymerase). Three chimeric DNA polymerases have been constructed using the three-dimensional (3D) structure of the Klenow fragment of the E.coli pol I and 3D models of the intervening and polymerase domains of the TAQ: polymerase and the TNE: polymerase: chimera TaqEc1 (exchange of residues 292-423 from TAQ: polymerase for residues 327-519 of E.coli pol I), chimera TaqTne1 (exchange of residues 292-423 of TAQ: polymerase for residues 295-485 of TNE: polymerase) and chimera TaqTne2 (exchange of residues 292-448 of TAQ: polymerase for residues 295-510 of TNE: polymerase). The chimera TaqEc1 showed characteristics from both parental polymerases at an intermediate temperature of 50 degrees C: high polymerase activity, processivity, 3'-5' exonuclease activity and proof-reading function. In comparison, the chimeras TaqTne1 and TaqTne2 showed no significant 3'-5' exonuclease activity and no proof-reading function. The chimera TaqTne1 showed an optimum temperature at 60 degrees C, decreased polymerase activity compared with the TAQ: polymerase and reduced processivity. The chimera TaqTne2 showed high polymerase activity at 72 degrees C, processivity and less reduced thermostability compared with the chimera TaqTne1.

The effect of DNA replication mutations on CAG tract stability in yeast.

CAG repeat tracts are unstable in yeast, leading to frequent contractions and infrequent expansions in repeat tract length. To compare CAG repeats to other simple repeats and palindromic sequences, we examined the effect of DNA replication mutations, including alleles of pol alpha, pol delta, pol epsilon, and PCNA (proliferating cell nuclear antigen), on tract stability. Among the polymerase mutations, the pol delta mutation (pol3-14) destabilizes tracts with either CAG or CTG as the lagging strand template. One pol alpha mutation, pol1-1, destabilizes the orientation with CAG as the lagging strand template, but it has little effect on the CTG orientation. In contrast, the pol1-17 mutation has no effect on either orientation. Similarly, mutations in the proofreading functions of pol delta and pol epsilon, as well as a temperature-sensitive pol epsilon mutation, pol2-18, have no effect on tract stability. Three PCNA mutations, pol30-52, pol30-79, and pol30-90, all have drastic effects on tract stability. Of the three, pol30-52 is unique in yielding small tract changes that are indicative of an impairment in mismatch repair. These results show that while CAG repeats are destabilized by many of the same mutations that destabilize other simple repeats, they also have some behaviors that are suggestive of their potential to form hairpin structures.

Efficient translesion replication in the absence of Escherichia coli Umu proteins and 3'-5' exonuclease proofreading function.

Translesion replication (TR) past a cyclobutane pyrimidine dimer in Escherichia coli normally requires the UmuD'2C complex, RecA protein, and DNA polymerase III holoenzyme (pol III). However, we find that efficient TR can occur in the absence of the Umu proteins if the 3'-5' exonuclease proofreading activity of the pol III epsilon-subunit also is disabled. TR was measured in isogenic uvrA6 DeltaumuDC strains carrying the dominant negative dnaQ allele, mutD5, or DeltadnaQ spq-2 mutations by transfecting them with single-stranded M13-based vectors containing a specifically located cis-syn T-T dimer. As expected, little TR was observed in the DeltaumuDC dnaQ+ strain. Surprisingly, 26% TR occurred in UV-irradiated DeltaumuDC mutD5 cells, one-half the frequency found in a uvrA6 umuDC+mutD5 strain. lexA3 (Ind-) derivatives of the strains showed that this TR was contingent on two inducible functions, one LexA-dependent, responsible for approximately 70% of the TR, and another LexA-independent, responsible for the remaining approximately 30%. Curiously, the DeltaumuDC DeltadnaQ spq-2 strain exhibited only the LexA-independent level of TR. The cause of this result appears to be the spq-2 allele, a dnaE mutation required for viability in DeltadnaQ strains, since introduction of spq-2 into the DeltaumuDC mutD5 strain also reduces the frequency of TR to the LexA-independent level. The molecular mechanism responsible for the LexA-independent TR is unknown but may be related to the UVM phenomenon [Palejwala, V. A., Wang, G. E., Murphy, H. S. & Humayun, M. Z. (1995) J. Bacteriol. 177, 6041-6048]. LexA-dependent TR does not result from the induction of pol II, since TR in the DeltaumuDC mutD5 strain is unchanged by introduction of a DeltapolB mutation.

Discrimination of 5′-terminal start codons by translation initiation factor 3 is mediated by ribosomal protein S1

The interrelation between ribosomal protein S1 and IF3 in recognition/discrimination of 5′-terminal start codons by 30S ribosomes has been studied using in vitro toeprinting. The study has been performed with two naturally occurring leaderless mRNAs, λ cI and phage r1t rro mRNA, as well as with an artificial leaderless mRNA derived from the E. coli ompA gene. We show that in the absence of S1, IF3 does not discriminate against the authentic 5′-terminal start codon of both cI and rro mRNA. Since IF3 was able to exert its proofreading function for initiator tRNA Met f on 30S ribosomes lacking S1, this observation cannot be attributed to a lack of binding to or action of IF3 on 30S(−S1) ribosomes. In contrast to leaderless mRNAs, ternary complex formation occurs in the presence of IF3 with 30S ribosomes when the start codon is preceded by a short 20-nucleotide 5′-untranslated region containing a canonical Shine and Dalgarno sequence. This suggests that 5′-terminal start codons are recognised by IF3 as non-standard because of the lack of 16S rRNA-mRNA contacts.

Amino acid residues involved in determining the processivity of the 3'-5' exonuclease activity in a family B DNA polymerase from the thermoacidophilic archaeon Sulfolobus solfataricus.

Herein, we report on the mutational analysis of a 70-amino acid segment (region 1, residues 438-508) of family B DNA polymerase from the thermoacidophilic archaeon Sulfolobus solfataricus (Sso DNA pol). Region 1, which lies between the Exo III sequence and the similarity motif D- -SLYP, connects the exonuclease and polymerase domains of Sso DNA pol. Two C-terminally deleted forms of the enzyme, proteins N438 (residues 1-438) and N508 (residues 1-508), were overproduced in the recombinant form and biochemically characterized. They contain the three evolutionarily conserved Exo motifs, but differ in the extent of the C-terminal deletion, since only N508 includes region 1. Both have been found to retain a Mn2+-dependent 3'-5' exonuclease activity, whose thermal stability appears to be increased in comparison to that of the full-sized enzyme. Assays for processive 3'-5' exonuclease activity, carried out with the heparin trap method on a 24-base oligonucleotide, have revealed that protein N508, as well as the full-length Sso DNA pol, retains a level of processivity of the degradative function substantially higher than that for protein N438. In addition, six site-specific mutations have been introduced at the highly conserved Y-GG/A motif, which has been found within Sso DNA pol region 1. All mutant proteins (Lys491Ile, Tyr495Ser, Lys496Ile, Gly497Ala, and Ala498Val) display increased processivity of their 3'-5' exonuclease activity, with the exception of protein Tyr495Phe. By a steady-state kinetic analysis of the exonucleolytic reaction on a 24-base oligonucleotide, the above site-specific mutations have been found to affect Km values consistently with the observed differences in the processivity values, whereas the effect on the kcat values seems to be less important. The results from this mutational analysis indicate that region 1 is involved in determining the processivity of the proofreading function, directly interacting with the nucleic acid substrate.

Excision of mismatched nucleotides from DNA: a potential mechanism for enhancing DNA replication fidelity by the wild-type p53 protein.

The tumor suppressor p53 plays a critical role in the regulation of the cell cycle and the maintenance of genetic stability. The 3'-->5' exonuclease activity of p53 has recently been recognized as a novel biochemical function of this molecule, but the biological significance of this activity remains elusive. Using an in vitro DNA replication assay with purified human DNA polymerases, p53 protein, and defined DNA primer/templates, we demonstrated that the wild-type (wt) p53 protein, but not the mutant p53 protein, specifically enhanced the DNA replication fidelity of polymerase (pol) alpha, an enzyme that lacks 3'-->5' exonuclease activity. The misincorporation of non-complementary deoxynucleotides into DNA by pol alpha was substantially decreased by p53. In contrast, wt p53 showed no significant effect on replication fidelity or the rates of DNA synthesis by human pol epsilon or the bacterial enzyme pol I. Inhibition of 3'-->5' exonuclease activity by guanosine monophosphate (GMP) abolished the ability of p53 to enhance the replication fidelity of pol alpha. Quantitative analyses revealed that the 3'-->5' exonuclease activity of p53 effectively removed mismatched nucleotides from DNA in preference over matched nucleotides. Furthermore, study in intact cells revealed that the wt p53 protein was co-localized with DNA synthesis activity in S phase cells. These results suggest a possibility that the 3'-->5' exonuclease of the wt p53 protein might provide the proof-reading function for DNA pol alpha. The preferential excision of mismatched nucleotides from the replicating DNA strand appears to be a potential biochemical mechanism by which p53 enhances DNA replication fidelity and thereby helps to maintain genomic integrity.

Two-way trafficking with Ran

The small Ras-related GTPase Ran is directly involved in nuclear protein import and export. However, the question of how Ran functions in transport is highly controversial. Here, we suggest that Ran is important for the formation, vectorial movement and disassembly of many different classes of transport complexes that traverse the nuclear pore complex during import and export processes. Comparison of Ran with the translation elongation factor Ef-Tu raises the possibility that Ran might also be involved in a proofreading function related to the assembly of import complexes. Although aspects of this model are hypothetical and challenge some current dogma in the field, we believe that it can integrate most of the current data into a coherent picture of the import process.

Flexible positioning of the telomerase-associated nuclease leads to preferential elimination of nontelomeric DNA.

In addition to a reverse transcriptase activity, telomerase is associated with a DNA endonuclease that removes nucleotides from a primer 3' terminus prior to telomere repeat addition. Here we examine the DNA specificity of the primer cleavage-elongation reaction carried out by the Euplotes crassus telomerase. We show that the primer cleavage activity copurified with the E. crassus telomerase polymerase, indicating that it either is an intrinsic property of telomerase or is catalyzed by a tightly associated factor. Using chimeric primers containing stretches of telomeric DNA that could be precisely positioned on the RNA template, we found that the cleavage site is more flexible than originally proposed. Primers harboring mismatches in dT tracts that aligned opposite nucleotides 37 to 40 in the RNA template were cleaved to eliminate the mismatched residues along with the adjacent 3' sequence. The cleaved product was then elongated to generate perfect telomeric repeats. Mismatches in dG tracts were not removed, implying that the nuclease does not track coordinately with the polymerase active site. Our data indicate that the telomerase-associated nuclease could provide a rudimentary proofreading function in telomere synthesis by eliminating mismatches between the DNA primer and the 5' region of the telomerase RNA template.

Effect of a 3′→5′ exonuclease with a proofreading function on the fidelity of error-prone DNA polymerase α from regenerating liver of aged rats

A nuclease that releases noncomplementary nucleotides from the 3′-end of DNA was isolated and highly purified from rat liver extract. The d(T 9-C) priming activities for DNA synthesis in vitro by DNA polymerases α and β were recovered by the addition of this enzyme, which itself does not contain a DNA polymerase activity. This nuclease hydrolysed nucleotides from the 3′-end, but did not remove [ 32P]-labeled nucleotides from the 5′-terminus of specifically labeled DNA. Also, the reaction products released from the 3′-end of DNA were all mononucleotides. These results indicate that the exonuclease is a 3′→5′ exonuclease with properties the same as those of DNase VII from human placenta. Rat DNase VII requires 4 mM MgCl 2 or 0.125 mM MnCl 2 for maximum activity, and shows a pH optimum of 7.5. These optimal conditions are similar to those of DNA polymerases, and indicate that both rat DNase VII and DNA polymerases are able to act under same conditions. Non-complementary nucleotide incorporation by DNA polymerase α from aged rat has been observed during in vitro DNA synthesis on poly dA–dT 10. The amount of this mis-incorporation is decreased by the coexistence of the 3′→5′ exonuclease, but not all errors are edited out. Thus, this rat DNase VII is suggested to play an important role in proofreading during DNA synthesis.

MutT prevents leakiness.

Reading the genetic code into RNA and then protein is never 100% accurate, and cells have several proofreading and editing functions to correct for this. In his Perspective Bridges discusses the mechanisms used to cleanse the RNA and DNA precursor pools of nucleotides damaged by oxidation, including one requiring the MutT protein as reported by Taddei on p. 128 of this issue.

ACV Synthetase: Expression of Amino Acid Activating Domains of thePenicillium chrysogenumEnzyme inAspergillus nidulans

Fragments of ACV synthetase ofPenicillium chrysogenumcarrying partial activities of amino acid activation were expressed under thealcApromoter in anacvA-deletion mutant ofAspergillus nidulans.The 210 kDa domain A-β-galactosidase fusion protein was partially cleaved to fragments of 200 and 97 kDa. The domain A fragment and the 312 kDa domain BC construct were identified by peptide specific antibodies and shown to catalyze α-aminoadipate-, cysteine-, and valine-dependent ATP/[32P]PPi exchange activity. Substrate specificities were investigated using amino acid analogues. Unexpectedly neither α-aminoadipate nor valine activation was exclusive, implying possible misactivations and proof reading functions. Both fragments were only expressed in limited amounts and found to be unstable.

Retention of replication fidelity by a DNA polymerase functioning in a distantly related environment.

The primary structures of the replicative DNA polymerases (gp43s) of bacteriophage T4 and its distant phylogenetic relative RB69 are diverged, retaining only 61% identity and 74% similarity. Nevertheless, RB69 gp43 substitutes effectively for T4 gp43 in T4 DNA replication in vivo. We show here that RB69 gp43 replicates T4 genomes in vivo with a fidelity similar to that achieved by T4 gp43. Furthermore, replication by RB69 gp43 in the distantly related environment does not enhance the mutator activities of mutations in T4 genes that encode other components of the multienzyme DNA replicase. We also show that the fidelities of RB69 gp43 and T4 gp43 are both high in vitro and that they are similarly and sharply reduced in vivo by mutations that eliminate the 3'-exonucleolytic proofreading function. We conclude that gp43 interactions with the other replication proteins are probably nonessential for polymerase fidelity.

An invariant lysine residue is involved in catalysis at the 3′-5′ exonuclease active site of eukaryotic-type DNA polymerases

A lysine residue, contained in the motif “ Kx 2 h”, has been invariantly found in the eukaryotic-type (family B) class of DNA-dependent DNA polymerases with a proofreading function. The importance of this lysine has been assessed by site-directed mutagenesis in the corresponding residue (Lys143) of ø29 DNA polymerase. Substitution of this residue either by arginine or isoleucine severely impaired the catalytic efficiency of the 3′-5′ exonuclease activity, giving a characteristic distributive pattern that contrasts with the processive pattern displayed by the wild-type ø29 DNA polymerase. Exonuclease assays carried out in the presence of a DNA trap, together with direct analysis of enzyme/ssDNA interaction, allowed us to conclude that this altered pattern was due to a reduction in the catalytic rate of these mutants, but not to a weakened association with ssDNA. These phenotypes indicate that the lysine residue of motif Kx 2 h plays an auxiliary role in catalysis of the exonuclease reaction, in very good agreement with recent crystallographic data showing that the lysine homologue of T4 DNA polymerase is indirectly involved in metal binding at the 3′-5′ exonuclease active site. In agreement with a critical role in proofreading, substitution of Lys143 of ø29 DNA polymerase by arginine or isoleucine produced mutator enzymes that displayed a high frequency of misincorporation. Mutants at Lys143 also showed a reduced DNA polymerization capacity, but only when DNA synthesis was coupled to strand-displacement, an intrinsic property of ø29 DNA polymerase that is specifically affected by mutations at residues directly or indirectly involved in metal binding at the 3′-5′ exonuclease active site.