Correlation of the kinetics of finger domain mutants in RB69 DNA polymerase with its structure

We have estimated pre-steady-state kinetic parameters for the addition of a single nucleotide residue by a set of RB69 DNA polymerase mutants in which four highly conserved residues in the fingers domain have been replaced by Ala. The relationship between the kinetic constants exhibited by the mutants and the structure of the ternary complex [Franklin, M., Wang, J., and Steitz T. (2001) Cell 105, 657-667] was consistent with the following sets of interactions between the conserved residues and oxygen atoms in the triphosphate portion of the incoming dNTP: (i) the epsilon-amino group of K560 contacts oxygen atoms of the alpha- and gamma-phosphates, (ii) the amide side chain of Asn 564 forms a hydrogen bond via a water molecule with the nonbridging oxygen of the beta-phosphate, and (iii) the epsilon-amino and delta-guanidino groups of K486 and R482, respectively, contact the nonbridging oxygens of the gamma-phosphate. We have also determined the pre-steady-state kinetic parameters for the addition of both dCTP and dCDP onto a 13/20mer primer/template with an exo(-) derivative of RB69 DNA polymerase and have shown that the deoxynucleoside diphosphate can be incorporated, in contrast to the behavior of the Klenow fragment which cannot use dCDP as a substrate. We have shown that, with RB69 DNA polymerase, in contrast to the Klenow fragment, there is no inhibition of the primer-extension reaction by incoming NTPs having either noncomplementary bases or ribo- instead of a deoxyribose moieties. This implies that the mode of recognition of incoming dNTPs and triggering of the conformational change, which is thought to occur prior to the chemical step, differs between these two enzymes.     

DNA strand transfer catalyzed by the 5'-3' exonuclease domain of Escherichia coli DNA polymerase I.

A protein which promotes DNA strand transfer between linear double-stranded M13mp19 DNA and single-stranded viral M13mp19 DNA has been isolated from recA- E.coli. The protein is DNA polymerase I. Strand transfer activity residues in the small fragment encoding the 5'-3' exonuclease and can be detected using a recombinant protein comprising the first 324 amino acids encoded by polA. Either the recombinant 5'-3' exonuclease or intact DNA polymerase I can catalyze joint molecule formation, in reactions requiring only Mg2+ and homologous DNA substrates. Both kinds of reactions are unaffected by added ATP. Electron microscopy shows that the joint molecules formed in these reactions bear displaced single strands and therefore this reaction is not simply promoted by annealing of exonuclease-gapped molecules. The pairing reaction is also polar and displaces the 5'-end of the non-complementary strand, extending the heteroduplex joint in a 5'-3' direction relative to the displaced strand. Thus strand transfer occurs with the same polarity as nick translation. These results show that E.coli, like many eukaryotes, possesses a protein which can promote ATP-independent strand-transfer reactions and raises questions concerning the possible biological role of this function.     

A nuclear 3'-5' exonuclease proofreads for the exonuclease-deficient DNA polymerase alpha

DNA replication is a highly accurate process designed to duplicate the entire genome of a cell during each cell division. The accuracy of DNA replication is derived from the balance between three important components: base selectivity by the replicative DNA polymerases (pols), exonucleolytic proofreading, and post-replicative mismatch repair. Previously we identified a human 3'-5' exonuclease (exoN) whose properties suggested it may function as a proofreader for the exonuclease-deficient replicative DNA pol alpha. Purified exoN has no associated pol activity and catalyzes removal of mispaired nucleotides from DNA duplexes. Consistent with previous reports, it was found that mammalian pol alpha is inefficient at extending from mispaired DNA terminals. However, in similar reactions that included exoN, there was a 4.4-15.7-fold increase in pol alpha-catalyzed elongation from mispaired base pairs. In contrast, exoN did not have a dramatic impact on the ability of exonuclease-deficient variants of Klenow (K-) and T7 polymerase to catalyze extension from mispaired DNA. Continuous DNA replication catalyzed by either pol alpha or K- generated base substitutions at a frequency of 24.3x10(-4) and 38x10(-4), respectively. ExoN restored error-free DNA replication in reactions with pol alpha whereas it did not significantly improve the accuracy of K-. These results are consistent with a functional interaction between exoN and pol alpha to ensure accurate DNA replication.     

Mutational and pH studies of the 3' --> 5' exonuclease activity of bacteriophage T4 DNA polymerase.

The 3' --> 5' exonuclease activity of proofreading DNA polymerases requires two divalent metal ions, metal ions A and B. Mutational studies of the 3' --> 5' exonuclease active center of the bacteriophage T4 DNA polymerase indicate that residue Asp-324, which binds metal ion A, is the single most important residue for the hydrolysis reaction. In the absence of a nonenzymatic source of hydroxide ions, an alanine substitution for residue Asp-324 reduced exonuclease activity 10-100-fold more than alanine substitutions for the other metal-binding residues, Asp-112 and Asp-219. Thus, exonuclease activity is reduced 10(5)-fold for the D324A-DNA polymerase compared with the wild-type enzyme, while decreases of 10(3)- to 10(4)-fold are detected for the D219A- and D112A/E114A-DNA polymerases, respectively. Our results are consistent with the proposal that a water molecule, coordinated by metal ion A, forms a metal-hydroxide ion that is oriented to attack the phosphodiester bond at the site of cleavage. Residues Glu-114 and Lys-299 may assist the reaction by lowering the pK(a) of the metal ion-A coordinated water molecule, whereas residue Tyr-320 may help to reorient the DNA from the binding conformation to the catalytically active conformation.     

Editing of misaligned 3'-termini by an intrinsic 3'-5' exonuclease activity residing in the PHP domain of a family X DNA polymerase

Bacillus subtilis gene yshC encodes a family X DNA polymerase (PolX(Bs)), whose biochemical features suggest that it plays a role during DNA repair processes. Here, we show that, in addition to the polymerization activity, PolX(Bs) possesses an intrinsic 3'-5' exonuclease activity specialized in resecting unannealed 3'-termini in a gapped DNA substrate. Biochemical analysis of a PolX(Bs) deletion mutant lacking the C-terminal polymerase histidinol phosphatase (PHP) domain, present in most of the bacterial/archaeal PolXs, as well as of this separately expressed protein region, allow us to state that the 3'-5' exonuclease activity of PolX(Bs) resides in its PHP domain. Furthermore, site-directed mutagenesis of PolX(Bs) His339 and His341 residues, evolutionary conserved in the PHP superfamily members, demonstrated that the predicted metal binding site is directly involved in catalysis of the exonucleolytic reaction. The implications of the unannealed 3'-termini resection by the 3'-5' exonuclease activity of PolX(Bs) in the DNA repair context are discussed.     

Characterization of an endonuclease IV 3'-5' exonuclease activity

Previous characterization of Escherichia coli endonuclease IV has shown that the enzyme specifically cleaves the DNA backbone at apurinic/apyrimidinic sites and removes 3' DNA blocking groups. By contrast, and unlike the major apurinic/apyrimidinic endonuclease exonuclease III, negligible exonuclease activity has been associated with endonuclease IV. Here we report that endonuclease IV does possess an intrinsic 3'-5' exonuclease activity. The activity was detected in purified preparations of the endonuclease IV protein from E. coli and from the distantly related thermophile Thermotoga maritima; it co-eluted with both enzymes under different chromatographic conditions. Induction of either endonuclease IV in an E. coli overexpression system resulted in induction of the exonuclease activity, and the E. coli exonuclease activity had similar heat stability to the endonuclease IV AP endonuclease activity. Characterization of the exonuclease activity showed that its progression on substrate is sensitive to ionic strength, metal ions, EDTA, and reducing conditions. Substrates with 3' recessed ends were preferred substrates for the 3'-5' exonuclease activity. Comparison of the relative apurinic/apyrimidinic endonuclease and exonuclease activity of endonuclease IV shows that the relative exonuclease activity is high and is likely to be significant in vivo.     

3' to 5' exonuclease activity of herpes simplex virus type 1 DNA polymerase modulates its strand displacement activity

Using a minicircle DNA primer-template, the wild-type catalytic subunit of herpes simplex virus type 1 (HSV-1) DNA polymerase (pol) was shown to lack significant strand displacement activity with or without its processivity factor, UL42. However, an exonuclease-deficient (exo(-)) pol (D368A) was capable of slow strand displacement. Although UL42 increased the rate (2/s) and processivity of strand displacement by exo(-) pol, the rate was slower than that for gap-filling synthesis. High inherent excision rates on matched primer-templates and rapid idling-turnover (successive rounds of excision and polymerization) of exo-proficient polymerases correlated with poor strand displacement activity. The results suggest that the exo activity of HSV-1 pol modulates its ability to engage in strand displacement, a function that may be important to the viability and genome stability of the virus.     

A 3' to 5' exonuclease activity is associated with phage 029 DNA polymerase

Bacteriophage 029 produces its own DNA polymerase which is encoded by gene 2 [Watabe, K. and Ito, J. (1983) Nucleic Acid Res. 11, 8333]. This 029 DNA polymerase has been purified by phospho-cellulose, DEAE-cellulose, double-stranded DNA cellulose chromatography and glycerol gradient centrifugation. An exonuclease activity associated with the DNA polymerase was found through all the steps of the purification. This nuclease preferably degrades single-stranded DNA from the 3' to the 5' terminus direction, suggesting that the enzyme plays a role for proofreading during DNA replication. While DNA polymerase activity isolated from cells infected with temperature sensitive mutant of gene 2 is thermolabile, the nuclease activity is not significantly reduced at the restrictive temperature.     

A minimal exonuclease domain of WRN forms a hexamer on DNA and possesses both 3'- 5' exonuclease and 5'-protruding strand endonuclease activities

Werner syndrome is a rare autosomal recessive disease characterized by a premature aging phenotype, genomic instability, and a dramatically increased incidence of cancer and heart disease. Mutations in a single gene encoding a 1432-amino acid helicase/exonuclease (hWRN) have been shown to be responsible for the development of this disease. We have cloned, overexpressed, and purified a minimal, 171-amino acid fragment of hWRN that functions as an exonuclease. This fragment, encompassing residues 70-240 of hWRN (hWRN-N(70-240)), exhibits the same level of 3'-5' exonuclease activity as the previously described exonuclease fragment encompassing residues 1-333 of the full-length protein. The fragment also contains a 5'-protruding DNA strand endonuclease activity at a single-strand-double-strand DNA junction and within single-stranded DNA, as well as a 3'-5' exonuclease activity on single-stranded DNA. We find hWRN-N(70-240) is in a trimer-hexamer equilibrium in the absence of DNA when examined by gel filtration chromatography and atomic force microscopy. Upon addition of DNA substrate, hWRN-N(70-240) forms a hexamer and interacts with the recessed 3'-end of the DNA. Moreover, we find that the interaction of hWRN-N(70-240) with the replication protein PCNA also causes this minimal, 171-amino acid exonuclease region to form a hexamer. Thus, the active form of this minimal exonuclease fragment of human WRN appears to be a hexamer. The implications these results have on our understanding of hWRN's roles in DNA replication and repair are discussed.     

Molecular interactions of Escherichia coli ExoIX and identification of its associated 3'-5' exonuclease activity

The flap endonucleases (FENs) participate in a wide range of processes involving the structure-specific cleavage of branched nucleic acids. They are also able to hydrolyse DNA and RNA substrates from the 5′-end, liberating mono-, di- and polynucleotides terminating with a 5′ phosphate. Exonuclease IX is a paralogue of the small fragment of Escherichia coli DNA polymerase I, a FEN with which it shares 66% similarity. Here we show that both glutathione-S-transferase-tagged and native recombinant ExoIX are able to interact with the E. coli single-stranded DNA binding protein, SSB. Immobilized ExoIX was able to recover SSB from E. coli lysates both in the presence and absence of DNA. In vitro cross-linking studies carried out in the absence of DNA showed that the SSB tetramer appears to bind up to two molecules of ExoIX. Furthermore, we found that a 3′–5′ exodeoxyribonuclease activity previously associated with ExoIX can be separated from it by extensive liquid chromatography. The associated 3′–5′ exodeoxyribonuclease activity was excised from a 2D gel and identified as exonuclease III using matrix-assisted laser-desorption ionization mass spectrometry.     

The excision of AP sites by the 3'-5' exonuclease activity of the Klenow fragment of Escherichia coli DNA polymerase I

The 3' AP endonucleases (class I) are said to hydrolyze the phosphodiester bond 3' to AP sites yielding 3'-OH and 5'-phosphate ends; on the other hand, the resulting 3' terminal AP site is not removed by the 3'-5' exonuclease activity of the Klenow fragment [1]. We show that AP sites in DNA are easily removed by the 3'-5' exonuclease activity of the Klenow fragment and that they are excised as deoxyribose-5-phosphate. It is suggested that the 3' AP endonucleases are perhaps not the hydrolases they are supposed to be.     

Binding of captan to DNA polymerase I from Escherichia coli and the concomitant effect on 5'----3' exonuclease activity

Captan (N-[(trichloromethyl)thio]-4-cyclohexene-1,2-dicarboximide) was shown to bind to DNA polymerase I from Escherichia coli. The ratio of [14C] captan bound to DNA pol I was 1:1 as measured by filter binding studies and sucrose gradient analysis. Preincubation of enzyme with polynucleotide prevented the binding of captan, but preincubation of enzyme with dGTP did not. Conversely, when the enzyme was preincubated with captan, neither polynucleotide nor dGTP binding was blocked. The modification of the enzyme by captan was described by an irreversible second-order rate process with a rate of 68 +/- 0.7 M-1 s-1. The interaction of captan with DNA pol I altered each of the three catalytic functions. The 3'----5' exonuclease and polymerase activities were inhibited, and the 5'----3' exonuclease activity was enhanced. In order to study the 5'----3' exonuclease activity more closely, [3H]hpBR322 (DNA-[3H]RNA hybrid) was prepared from pBR322 plasmid DNA and used as a specific substrate for 5'----3' exonuclease activity. When either DNA pol I or polynucleotide was preincubated with 100 microM captan, 5'----3' exonuclease activity exhibited a doubling of reaction rate as compared to the untreated sample. When 100 microM captan was added to the reaction in progress, 5'----3' exonuclease activity was enhanced to 150% of the control value. Collectively, these data support the hypothesis that captan acts on DNA pol I by irreversibly binding in the template-primer binding site associated with polymerase and 3'----5' exonuclease activities. It is also shown that the chemical reaction between DNA pol I and a single captan molecule proceeds through a Michaelis complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Properties of the 3' to 5' exonuclease associated with calf DNA polymerase delta

The 3' to 5' exonuclease of calf thymus DNA polymerase delta has properties expected of a proofreading nuclease. It digests either single-stranded DNA or the single-stranded nucleotides of a mismatched primer on a DNA template by a nonprocessive mechanism. The distribution of oligonucleotide products suggests that a significant portion of the enzyme dissociates after the removal of one nucleotide. This mechanism is expected if the substrate in vivo is an incorrect nucleotide added by the polymerase. Digestion of single-stranded DNA does not proceed to completion, producing final products six to seven nucleotides long. Digestion of a long mismatched terminus accelerates when the mismatched region is reduced to less than six nucleotides. At the point of complementation, the digestion rate is greatly reduced. These results suggest that short mismatched regions are a preferred substrate. The use of a mismatched primer-template analogue, lacking the template single strand, greatly lowers digestion efficiency at the single-stranded 3'-terminus, suggesting that the template strand is important for substrate recognition. When oligonucleotides were examined for effectiveness as exonuclease inhibitors, (dG)8 was found to be the most potent inhibitor of single-stranded DNA digestion. (dG)8 was less effective at inhibiting digestion of mismatched primer termini, again suggesting that this DNA is a preferred substrate. Overall, these results indicate that the exonuclease of DNA polymerase delta efficiently removes short mismatched DNA, a structure formed from misincorporation during DNA synthesis.     

Coprecipitation of 3'----5' exonuclease and DNA polymerase gamma with anti-DNA polymerase gamma antibody

Highly purified preparations of chick embryo DNA polymerase gamma contained 3'----5' exonuclease activity which might be responsible for the exonucleolytic proofreading during DNA synthesis [Kunkel, T.A. & Soni, A. (1988) J. Biol. Chem. 262, 4450-4459]. A rabbit antibody produced against highly purified chick DNA polymerase gamma precipitated 3'----5' exonuclease activity to the same extent as DNA polymerase gamma activity. Furthermore, the antibody neutralized the two enzyme activities to an equal extent. However, the exonuclease activity was more resistant than DNA polymerase gamma activity to thermal treatment at 50 degrees C, although both activities were partially protected with polynucleotides. The results obtained suggest that these two enzymes are associated as a single enzyme complex or that the two activities reside in a single molecule, and the active site of DNA polymerase gamma and 3'----5' exonuclease are, although not identical, closely correlated.     

Inactivation of the 3'-5' exonuclease of the replicative T4 DNA polymerase allows translesion DNA synthesis at an abasic site

Here, we have investigated the consequences of the loss of proof-reading exonuclease function on the ability of the replicative T4 DNA polymerase (gp43) to elongate past a single abasic site located on model DNA substrates. Our results show that wild-type T4 DNA polymerase stopped at the base preceding the lesion on two linear substrates having different sequences, whereas the gp43 D219A exonuclease-deficient mutant was capable of efficient bypass when replicating the same substrates. The structure of the DNA template did not influence the behavior of the exonuclease-proficient or deficient T4 DNA polymerases. In fact, when replicating a damaged "minicircle" DNA substrate constructed by circularizing one of the linear DNA, elongation by wild-type enzyme was still completely blocked by the abasic site, while the D219A mutant was capable of bypass. During DNA replication, the T4 DNA polymerase associates with accessory factors whose combined action increases the polymerase-binding capacity and processivity, and could modulate the behavior of the enzyme towards an abasic site. We thus performed experiments measuring the ability of wild-type and exonuclease-deficient T4 DNA polymerases, in conjunction with these replicative accessory proteins, to perform translesion DNA replication on linear or circular damaged DNA substrates. We found no evidence of either stimulation or inhibition of the bypass activities of the wild-type and exonuclease-deficient forms of T4 DNA polymerase following addition of the accessory factors, indicating that the presence or absence of the proof-reading activity is the major determinant in dictating translesion synthesis of an abasic site by T4 DNA polymerase.     

Cleavage of double-stranded DNA by the intrinsic 3'-5' exonuclease activity of DNA polymerase B1 from the hyperthermophilic archaeon Sulfolobus solfataricus at high temperature

The substrate requirement of the intrinsic 3'-5' exonuclease of DNA polymerase B1 from the hyperthermophilic archaeon Sulfolobus solfataricus P2 (Sso polB1) was investigated. Sso polB1 degraded both single-stranded (ss) and double-stranded (ds) DNA at similar rates in vitro at temperatures of physiological relevance. No difference was found in the cleavage of 3'-recessive, 3'-protruding and blunt-ended DNA duplexes at these temperatures. However, a single-stranded nick in duplex DNA was less readily employed by the enzyme to initiate cleavage than a free 3' end. At lower temperatures, Sso polB1 cleaved ssDNA more efficiently than dsDNA. The strong 3'-5' exonuclease activity of polB1 was inhibited by 50% in the presence of 2 microM dNTPs, but remained measurable at up to 600 microM dNTPs. In view of the strong exonuclease activity of Sso polB1 on matched dsDNA, we suggest that S. solfataricus may have evolved mechanisms to regulate the exonuclease/polymerase ratio of the enzyme, thereby reducing the cost of proofreading at high temperature.     

Reduction of the potent DNA polymerase III holoenzyme 3'----5' exonuclease activity by template-primer analogues

The DNA polymerase III holoenzyme of Escherichia coli contains a potent 3'----5' exonuclease that removes the terminal nucleotide from a synthetic deoxyoligonucleotide primer with a half-life of approximately 2 s. Degradation of primers could not be effectively prevented by permitting the holoenzyme to "idle" at the primer terminus in the presence of limited deoxynucleoside triphosphates. To further characterize this exonuclease and to develop stable primers to facilitate experimental manipulations, we synthesized a series of twelve 25-mer oligonucleotides that differed only in the two 3'-terminal residues. The penultimate position contained either a CMP or a dCMP residue, while at the terminal position either AMP, dAMP, 2',3'-dideoxyAMP, cordycepin (3'-dAMP), dAMP alpha S, or 2',3'-dideoxyAMP alpha S was incorporated. No single change at either the 3'-penultimate or 3'-terminal positions resulted in a decrease in the exonuclease rate greater than 10-fold; however, combined changes at these two sites resulted in a strong synergistic effect. Placing a ribonucleotide at the penultimate position coupled by a phosphorothioate linkage to a terminal 2',3'-dideoxynucleotide reduced the rate of exonucleolytic activity almost 30,000-fold (half-life approximately 16 h). If only the ribonucleotide and phosphorothioate substitutions were made, a primer capable of being efficiently elongated was generated that exhibited a 500-fold increase in stability (half-life = 40 min). The elemental effect observed by substituting a nonbridging oxygen in the terminal phosphodiester bond for sulfur increased from 1.5 to 200 as other substitutions were made that decreased the exonuclease rate. This was consistent with a change in the rate-limiting step of the exonuclease reaction from a conformational change to the chemical step where the covalent bond is cleaved. At least part of this effect appears to be due to perturbations within the enzyme's active site and not solely due to changes in electrophilicity.