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.     

Characterization of a 3''----5'' exonuclease activity in the phage phi 29-encoded DNA polymerase.

Purified protein p2 of phage phi 29, characterized as a specific DNA polymerase involved in the initiation and elongation of phi 29 DNA replication, contains a 3'----5' exonuclease active on single-stranded DNA, but not on double-stranded DNA. No 5'----3' exonuclease activity was found. The 3'----5' exonuclease activity was shown to be associated with the DNA polymerase since 1) the two activities were heat-inactivated with identical kinetics and 2) both activities, present in purified protein p2, cosedimented in a glycerol gradient.     

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.     

High expression of a 3''----5'' exonuclease activity is specific to B lymphocytes.

V(D)J joining, the immunoglobulin heavy-chain (IgH) class switch, and somatic hypermutation directed at variable regions are unique genetic recombination or mutation events which occur during B-cell differentiation. The enzymatic process directing and controlling these events remains obscure. An assay for exonucleolytic activity has been devised, and an exonuclease activity expressed at high levels in normal B lymphocytes has been detected. The high expression of this enzyme is specific to B lymphocytes and may be developmentally regulated. We have partially purified a B-cell-associated nuclease by column chromatography. Using this preparation, we have begun a rigorous analysis of its activity. This activity is a nonprocessive, 3'----5' exonuclease with a requirement for divalent cations. Our studies demonstrate that EDTA, poly(dI-dC), and glycerol are all inhibitory to B-cell-associated exonucleolytic activity. The exonuclease displays sequence preference but no sequence specificity when tested on a variety of native DNA substrates. This nuclease is distinct from other exonuclease activities previously described.     

The requirement for both DNA polymerase and 5'' to 3'' exonuclease activities of DNA polymerase I during Tn5 transposition

Summary By assaying transposition of Tn5 from b221 cI857 rex::Tn5 (Berg 1977) in PolA-proficient and deficient cells, both the polymerase activity and 5 to 3 exonuclease acivity of DNA polymerase I have been shown to be required for transposition. This requirement could not be observed in three other systems in which the transposon donor replicon had existed in the PolA-proficient and deficient cells before the transposition event to be assayed occurred. By analogy to Tn3, this may indicate that the repressor encoded by Tn5 has already been expressed and hence become rate-limiting in the overall transposition process, even in PolA-deficient cells still possessing a residual activity. One polA mutant was found among more than 50 transposition-deficient (tnp) mutants isolated by the use of b221 cI857 rex::Tn5.     

Deoxyribonucleic acid repair in Escherichia coli mutants deficient in the 5''----3'' exonuclease activity of deoxyribonucleic acid polymerase I and exonuclease VII.

A series of Escherichia coli strains deficient in the 5'----3' exonuclease activity associated with deoxyribonucleic acid (DNA) polymerase I (exonuclease VI) and exonuclease VII has been constructed. Both of these enzymes are capable of pyrimidine dimer excision in vitro. These strains were examined for conditional lethality, sensitivity to ultraviolet (UV) and X-irradiation, postirradiation DNA degradation, and ability to excise pyrimidine dimers. It was found that strains deficient in both exonuclease VI (polAex-) and exonuclease VII (xseA-) are significantly reduced in their ability to survive incubation at elevated temperature (43 degrees C) beyond the reduction previously observed for the polAex single mutants. The UV and X-ray sensitivity of the exonuclease VI-deficient strains was not increased by the addition of the xseA7 mutation. Mutants deficient in both enzymes are about as efficient as wild-type strains at excising dimers produced by up to 40 J/m2 UV. At higher doses strains containing only polAex- mutations show reduced ability to excise dimers; however, the interpretation of dimer excision data at these doses is complicated by extreme postirradiation DNA degradation in these strains. The additional deficiency in the polAex xseA7 double-mutant strains has no significant effect on either postirradiation DNA degradation or the apparent deficiency in dimer excision at high UV doses observed in polAex single mutants.     

The 5'' to 3'' exonuclease activity of DNA polymerase I is essential for Streptococcus pneumoniae

Three different mutations were introduced in the polA gene of Streptococcus pneumoniae by chromosomal transformation. One mutant gene encodes a truncated protein that possesses 5' to 3' exonuclease but has lost polymerase activity. This mutation does not affect cell viability. Other mutated forms of polA that encode proteins with only polymerase activity or with no enzymatic activity could not substitute for the wild-type polA gene in the chromosome unless the 5' to 3' exonuclease domain was encoded elsewhere in the chromosome. Thus, it appears that the 5' to 3' exonuclease activity of the DNA polymerase I is essential for cell viability in S. pneumoniae. Absence of the polymerase domain of DNA polymerase I slightly diminished the ability of S. pneumoniae to repair DNA lesions after ultraviolet irradiation. However, the polymerase domain of the pneumococcal DNA polymerase I gave almost complete complementation of the polA5 mutation in Escherichia coli with respect to resistance to ultraviolet irradiation.     

Streptococcus pneumoniae DNA polymerase I lacks 3''-to-5'' exonuclease activity: localization of the 5''-to-3'' exonucleolytic domain.

The Streptococcus pneumoniae polA gene was altered at various positions by deletions and insertions. The polypeptides encoded by these mutant polA genes were identified in S. pneumoniae. Three of them were enzymatically active. One was a fused protein containing the first 11 amino acid residues of gene 10 from coliphage T7 and the carboxyl-terminal two-thirds of pneumococcal DNA polymerase I; it possessed only polymerase activity. The other two enzymatically active proteins, which contained 620 and 351 amino acid residues from the amino terminus, respectively, lacked polymerase activity and showed only exonuclease activity. These two polymerase-deficient proteins and the wild-type protein were hyperproduced in Escherichia coli and purified. In contrast to the DNA polymerase I of Escherichia coli but similar to the corresponding enzyme of Thermus aquaticus, the pneumococcal enzyme appeared to lack 3'-to-5' exonuclease activity. The 5'-to-3' exonuclease domain was located in the amino-terminal region of the wild-type pneumococcal protein. This exonuclease activity excised deoxyribonucleoside 5'-monophosphate from both double- and single-stranded DNAs. It degraded oligonucleotide substrates to a decameric final product.     

Selective inactivation of the 3'' to 5'' exonuclease activity of Escherichia coli DNA polymerase I by heat

Heat selectively inactivates the 3' to 5' exonuclease activity of E. coli DNA polymerase I, resulting in reduced dNTP turnover and lower fidelity of replication of homopolymer and natural DNA templates.     

Polymerization activity of an alpha-like DNA polymerase requires a conserved 3''-5'' exonuclease active site.

Most DNA polymerases are multifunctional proteins that possess both polymerizing and exonucleolytic activities. For Escherichia coli DNA polymerase I and its relatives, polymerase and exonuclease activities reside on distinct, separable domains of the same polypeptide. The catalytic subunits of the alpha-like DNA polymerase family share regions of sequence homology with the 3'-5' exonuclease active site of DNA polymerase I; in certain alpha-like DNA polymerases, these regions of homology have been shown to be important for exonuclease activity. This finding has led to the hypothesis that alpha-like DNA polymerases also contain a distinct 3'-5' exonuclease domain. We have introduced conservative substitutions into a 3'-5' exonuclease active site homology in the gene encoding herpes simplex virus DNA polymerase, an alpha-like polymerase. Two mutants were severely impaired for viral DNA replication and polymerase activity. The mutants were not detectably affected in the ability of the polymerase to interact with its accessory protein, UL42, or to colocalize in infected cell nuclei with the major viral DNA-binding protein, ICP8, suggesting that the mutation did not exert global effects on protein folding. The results raise the possibility that there is a fundamental difference between alpha-like DNA polymerases and E. coli DNA polymerase I, with less distinction between 3'-5' exonuclease and polymerase functions in alpha-like DNA polymerases.     

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.     

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.     

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 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.     

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.     

Characterization of mutational specificity within the lacI gene for a mutD5 mutator strain of Escherichia coli defective in 3''----5'' exonuclease (proofreading) activity.

The mutD (dnaQ) gene of Escherichia coli codes for the epsilon subunit of the DNA polymerase III holoenzyme which is involved in 3'----5' exonuclease proofreading activity. We determined the mutational specificity of the mutator allele, mutD5, in the lacI gene of E. coli. The mutD5 mutation preferentially produces single base substitutions as judged from the enhanced fraction of lacI nonsense mutations and the spectrum of sequenced dominant lacI (lacId) and constitutive lacO (lacOc) mutations which were predominantly (69/71) single nucleotide substitutions. The distribution of amber lacI and sequenced lacId mutations revealed that transitions occur more frequently than transversions. A . T----G . C and G . C----A . T transitions were equally frequent and, with one major exception, evenly distributed among numerous sites. Among the transversions, A . T----T . A events were the most common, A . T----C . G substitutions were rare, and G . C----C . G changes were not detected. Transversions were unequally distributed among a limited number of sites with obvious hotspots. All 11 sequenced transversions had a consensus neighboring sequence of 5'-C-C-(mutated G or A)-C-3'. Although no large deletions or complex mutational events were recovered, sequencing revealed that mutD5 induced single nucleotide deletions within consecutive G X C sequences. An extraordinary A . T----G . C transition hotspot occurred at nucleotide position +6 in the lac operator region; the mutD5 mutation frequency of this single base pair was calculated to be 1.2 X 10(-3).     

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.