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.     

DNA polymerase gamma from Xenopus laevis. II. A 3'----5' exonuclease is tightly associated with the DNA polymerase activity

Xenopus laevis DNA polymerase gamma co-purifies with a tightly associated 3'----5' exonuclease. The purified enzyme lacks 5'----3' exonuclease and endonuclease activity. The ratio of the 3'----5' exonuclease activity to DNA polymerase gamma activity remains constant over the final three chromatographic procedures. In addition, these activities co-sediment under partially denaturing conditions in the presence of ethylene glycol. The associated 3'----5' exonuclease activity removes a terminally mismatched nucleotide more rapidly than a correctly base-paired 3'-terminal residue, as expected if this exonuclease has a proofreading function. The 3'----5' exonuclease has the ability to release a terminal phosphorothioated nucleotide, a property shared with T4 DNA polymerase, but not with Escherichia coli DNA polymerase I.     

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.     

Identification and characterization of a 3' to 5' exonuclease associated with spinach chloroplast DNA polymerase.

Spinach chloroplast DNA polymerase was shown to copurify with a 3' to 5' exonuclease activity during DEAE-cellulose, hydroxylapatite, and heparin-agarose column chromatography. In addition, both activities comigrated during nondenaturing polyacrylamide gel electrophoresis and cosedimented through a glycerol gradient with an apparent molecular weight of 105,000. However, two forms of exonuclease activity were detected following velocity sedimentation analysis. Form I constituted approximately 35% of the exonuclease activity and was associated with the DNA polymerase, whereas the remaining activity (form II) was free of DNA polymerase and exhibited a molecular weight of approximately 26,500. Resedimentation of form I exonuclease generated both DNA polymerase associated and DNA polymerase unassociated forms of the exonuclease, suggesting that polymerase/exonuclease dissociation occurred. The exonuclease activity (form I) was somewhat resistant to inhibition by N-ethylmaleimide, whereas the DNA polymerase activity was extremely sensitive. Using in situ detection following SDS-polyacrylamide activity gel electrophoresis, both form I and II exonucleases were shown to reside in a similar, if not identical, polypeptide of approximately 20,000 molecular weight. Both form I and II exonucleases were equally inhibited by NaCl and required 7.5 mM MgCl2 for optimal activity. The 3' to 5' exonuclease excised deoxyribonucleoside 5'-monophosphates from both 3'-terminally matched and 3'-terminally mismatched primer termini. In general, the exonuclease preferred to hydrolyze mismatched 3'-terminal nucleotides as determined from the Vmax/Km ratios for all 16 possible combinations of matched and mismatched terminal base pairs. These results suggest that the 3' to 5' exonuclease may be involved in proofreading errors made by chloroplast DNA polymerase.     

3'-->5' exonuclease in Drosophila mitochondrial DNA polymerase. Substrate specificity and functional coordination of nucleotide polymerization and mispair hydrolysis.

A mispair-specific 3'-->5' exonuclease copurifies quantitatively with the near-homogeneous Drosophila gamma polymerase (Kaguni, L.S., and Olson, M.W. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6469-6473). The exonuclease and polymerase exhibit similar reaction requirements and optima, suggesting functional coordination of their activities. Under nonpolymerization conditions, the 3'-->5' exonuclease hydrolyzes 3'-terminal mispairs approximately 15-fold more efficiently than 3'-terminal base pairs on primed single-stranded DNA substrates, whereas it does not discriminate between any of three specific mispairs (dAMP:dAMP;dGMP:dGMP; dGMP:dAMP). Under polymerization conditions, gamma polymerase does not extend a 3'-terminal mispair from the "stationary" state, even in the presence of a large excess of the next correct nucleotide. Instead, 3'-terminal mispairs are hydrolyzed quantitatively by the 3'-->5' exonuclease over the reaction time course. During DNA synthesis by gamma polymerase in the "polymerization" mode, limited misincorporation and subsequent mispair extension do occur. Here, it appears that misincorporation and not mispair extension is rate-limiting. Template-primer challenge experiments suggest that the mechanism of template-primer transfer from the 3'-->5' exonuclease active site to the DNA polymerase active site is intermolecular; transfer from the exonuclease to polymerase mode appears to require dissociation and reassociation of mitochondrial DNA polymerase.     

Contribution of 3' leads to 5' exonuclease activity of DNA polymerase III holoenzyme from Escherichia coli to specificity

The effects of deoxynucleoside monophosphates on the 3' leads to 5' exonuclease activity of DNA polymerase III holoenzyme have been correlated with their effects on the fidelity of DNA replication. In particular, dGMP inhibits the proofreading activity of the enzyme and decreases the fidelity in those cases where a "following nucleotide effect" is also noted. This is strong evidence for proofreading. However, the absence of the effects of proofreading inhibitors or following nucleotides need not be evidence against the occurrence of proofreading: a theoretical analysis shows that these effects may not be observed even though there is active proofreading. This is suggested to be the case with the phage T4 enzyme system. The proofreading activity of Pol III appears to be directed primarily towards removing purine x pyrimidine-mediated rather than purine x purine-mediated misincorporations. recA protein inhibits the proofreading activity of Pol III on synthetic templates containing mismatched 3' termini. This is paralleled by a decrease in the fidelity of DNA replication in vitro. The inhibition is increased in the presence of dGMP or dAMP but there is no further increase in the infidelity of replication. The presence of both dNMPs and recA protein does not enable Pol III to copy past pyrimidine photodimers.     

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.     

Mechanisms of selective inhibition of 3' to 5' exonuclease activity of Escherichia coli DNA polymerase I by nucleoside 5'-monophosphates.

The 3' to 5' exonuclease activity of Escherichia coli DNA polymerase I can be selectively inhibited by nucleoside 5'-monophosphates, wherease the DNA polymerase activity is not inhibited. The results of kinetic studies show that nucleotides containing a free 3'-hydroxy group and a 5'-phosphoryl group are competitive inhibitors of the 3' to 5' exonuclease. Previous studies by Huberman and Kornberg [Huberman, J., and Kornberg, A. (1970), J. Biol. Chem. 245, 5326] have demonstrated a binding site for nucleoside 5'-monophosphates on DNA polymerase I. The Kdissoc values for nucleoside 5'-monophosphates determined in that study are comparable to the Ki values determined in the present study, suggesting that the specific binding site for nucleoside 5'-monophosphates represents the inhibitor site of the 3' to 5' exonuclease activity. We propose that (1) the binding site for nucleoside 5'-monophosphates on DNA polymerase I may represent the product site of the 3' to 5' exonuclease activity. (2) the primer terminus site for the 3' to 5' exonuclease activity is distinct from the primer terminus site for the polymerase activity, and (3) nucleoside 5'-monophosphates bind at the primer terminus site for the 3' to 5' exonuclease activity.     

Selective affinity chromatography of DNA polymerases with associated 3' to 5' exonuclease activities

The use of 5'-AMP as a ligand for the affinity chromatography of DNA polymerases with intrinsic 3' to 5' exonuclease activities was investigated. The basis for this is that 5'-AMP would be expected to act as a ligand for the associated 3' to 5' exonuclease. The requirements for binding of Escherichia coli DNA polymerase I, T4 DNA polymerase, and calf thymus DNA polymerase delta, all of which have associated 3' to 5' exonuclease activities, to several commercially available 5'-AMP supports with different linkages of 5'-AMP to either agarose or cellulose were examined. The DNA polymerases which possessed 3' to 5' exonuclease activities were bound to agarose types in which the 5'-phosphoryl group and the 3'-hydroxyl group of the AMP were unsubstituted. Bound enzyme could be eluted by either an increase in ionic strength or competitive binding of nucleoside 5'-monophosphates. Magnesium was found to reinforce the binding of the enzyme to these affinity supports. DNA polymerase alpha, which does not have an associated 3' to 5' exonuclease activity, did not bind to any of these columns. These differences can be used to advantage for the purification of DNA polymerases that have associated 3' to 5' exonuclease activities, as well as a means for establishing the association of 3' to 5' exonuclease activities with DNA polymerases.     

Mechanism of 3'' to 5'' exonuclease associated with phage T5-induced DNA polymerase: processiveness and template specificity.

T5-induced DNA polymerase has an associated 3' to 5' exonuclease activity. Both single-stranded and duplex DNA are hydrolyzed by this enzyme in a quasi-processive manner. This is indicated by the results of polymer-challenge experiments utilizing product analysis techniques. Due to the quasi-processive mode of hydrolysis, the kinetics of label release from the 3'-terminally labeled oligonucleotide substrates, annealed to complementary homopolymers, show an initial high rate of hydrolysis. In the case of both single-stranded and duplex DNA substrates, hydrolysis seems to continue, at best, up to the point where the enzyme is five or six nucleotides away from the 5-end. The enzyme carries out mismatch repair, as evidenced by experiments with primer molecules containing improper base residues at the 3'-OH terminus. Control experiments with complementary base residues at the 3'-end indicate that extensive removal of terminal residue takes place in the presence of dNTP's only when such residues are "improper" in the Watson-Crick sense.     

Characterization of the 5'' to 3'' exonuclease associated with Thermus aquaticus DNA polymerase.

Thermus aquaticus DNA polymerase was shown to contain an associated 5' to 3' exonuclease activity. Both polymerase and exonuclease activities cosedimented with a molecular weight of 72,000 during sucrose gradient centrifugation. Using a novel in situ activity gel procedure to simultaneously detect these two activities, we observed both DNA polymerase and exonuclease in a single band following either nondenaturing or denaturing polyacrylamide gel electrophoresis: therefore, DNA polymerase and exonuclease activities reside in the same polypeptide. As determined by SDS-polyacrylamide gel electrophoresis this enzyme has an apparent molecular weight of 92,000. The exonuclease requires a divalent cation (MgCl2 or MnCl2), has a pH optimum of 9.0 and excises primarily deoxyribonucleoside 5'-monophosphate from double-stranded DNA. Neither heat denatured DNA nor the free oligonucleotide (24-mer) were efficient substrates for exonuclease activity. The rate of hydrolysis of a 5'-phosphorylated oligonucleotide (24-mer) annealed to M13mp2 DNA was about twofold faster than the same substrate containing a 5'-hydroxylated residue. Hydrolysis of a 5'-terminal residue from a nick was preferred threefold over the same 5'-end of duplex DNA. The 5' to 3' exonuclease activity appeared to function coordinately with the DNA polymerase to facilitate a nick translational DNA synthesis reaction.     

Evidence that the nuclease activities associated with the herpes simplex type 1 DNA polymerase are due to the 3'-5' exonuclease.

We investigated nuclease activities associated with the catalytic subunit of herpes simplex virus type 1 DNA polymerase. We confirm that a 3'-5' exonuclease copurifies with this enzyme. Previous reports suggested that a 5' DNase was intrinsic to the polymerase. Our preparation lacks such activity.     

Excision of gamma-ray damaged thymine by E. coli extracts is due to the 5'leads to 3' exonuclease associated with DNA polymerase I

Extracts of $\text{E. coli pol}$Aexl which contains a temperature sensitive 5′→3′ exonuclease function of polymerase I accomplish the selective excision of products of the 5,6-dihydroxy-dihydrothymine type from γ-irradiated DNA and OsO₄-oxidized polyd(A-T) at the permissive temperature (30°) but not at the nonpermissive temperature (42°). The 5′→3′ exonuclease activity of polymerase I, therefore, acts as a repair exonuclease in γ-ray excision repair.     

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.     

Characterization of a 3'-5' exonuclease associated with VDJP.

VDJP (V(D)J RSS Dependent DNA Joining Protein) was cloned based on binding to the nonamer portion of the V(D)J recombinational signal sequence (RSS), and genetic analysis revealed that VDJP is encoded by the same gene as the large subunit of Replication Factor C (RF-C). Recombinant VDJP has a site directed DNA joining activity and is capable of forming a covalent bond between DNA fragments containing an RSS element near their ends and exhibits 3' to 5' exonuclease activity. In this report, we examine the biochemical properties of the VDJP exonuclease activity such as directionality of nuclease action (3' to 5' or 5' to 3'), single-strand substrate preference, cleavage products, dependence on cofactors and metal cations, and optimal reaction conditions. From this analysis, we conclude that VDJP has an intrinsic 3'-5' exonuclease activity that produces mononucleotide products.