3'-->5'-exonucleases of the rat liver and the correction of replication errors]

Mammalian nuclear DNA polymerases alpha and beta are lack of the proofreading 3'-->5' exonucleolytic activity. 40 and 50 kDa 3'-->5' exonucleases were isolated from rat liver. The exonucleases were shown to excise mismatched nucleotides from poly[d(A--T)] template 10 and 2 fold faster than matched ones. The addition of either exonuclease to DNA polymerase alpha from rat liver or calf thymus 5-10 times increased the accuracy of reproduction of primed DNA from bacteriophage phi X174 amber 3, values of exonuclease and DNA polymerase activities being approximately equal. The exonuclease activity surpasses the DNA polymerase one by an order of magnitude in chromatin and nuclear membrane. These data, taken together, are indicative of potent proofreading into hepatocytes.     

Functional interdependence of DNA polymerizing and 3'-->5' exonucleolytic activities in Pyrococcus furiosus DNA polymerase I

Pyrococcus furiosus DNA polymerase I (Pol BI) belongs to the family B (alpha-like) DNA polymerases and has a strong 3'-->5' exonucleolytic activity, in addition to its DNA polymerizing activity. To understand the relationship between the structure and function of this DNA polymerase, three deletion mutants, Delta1 (DeltaLeu746-Ser775), Delta2 (DeltaLeu717-Ser775) and Delta3 (DeltaHis672-Ser775), and two substituted mutants of Asp405, D405A and D405E, were constructed. These substitutions affected both the DNA polymerizing and the 3'-->5' exonucleolytic activities. The Delta1 mutant protein had DNA polymerizing activity with higher specific activity than that of the wild-type Pol BI, but retained only 10% of the exonucleolytic activity of the wild-type. The other two deletion mutants lost most of both activities. These results suggest that the DNA polymerizing and exonucleolytic activities are closely related to each other in the folded structure of this DNA polymerase, as proposed in the family B DNA polymerases.     

A 70-kDa chloroplast DNA polymerase from pea (<Emphasis Type="Italic">Pisum sativum</Emphasis>) that shows high processivity and displays moderate fidelity

A 70-kDa chloroplast (ct) DNA polymerase from pea has been purified to apparent homogeneity. The ct DNA polymerase was insensitive to dideoxynucleotides (d(2) NTP) but showed high sensitivity to phosphonoacetic acid. The enzyme lacked any detectable 5'-->3' exonuclease activity but showed 3'-->5' exonuclease activity. The polymerase displayed high processivity (3 kb) and moderate fidelity, which may be sufficient for the faithful replication of the 140-kb pea ct genome. A 43-kDa accessory protein increased the polymerization rate but did not affect the rate of mis-incorporation in vitro, thus indicating that the domains for polymerisation and proof reading may be spatially separate.     

The fidelity of DNA synthesis by the catalytic subunit of yeast DNA polymerase alpha alone and with accessory proteins

The fidelity of DNA synthesis catalyzed by the 180-kDa catalytic subunit (p180) of DNA polymerase alpha from Saccharomyces cerevisiae has been determined. Despite the presence of a 3'----5' exonuclease activity (Brooke et al., 1991, J. Biol. Chem., 266, 3005-3015), its accuracy is similar to several exonuclease-deficient DNA polymerases and much lower than other DNA polymerases that have associated exonucleolytic proofreading activity. Average error rates are 1/9900 and 1/12,000, respectively, for single base-substitution and minus-one nucleotide frameshift errors; the polymerase generates deletions as well. Similar error rates are observed with reactions containing the 180-kDa subunit plus an 86-kDa subunit (p86), or with these two polypeptides plus two additional subunits (p58 and p49) comprising the DNA primase activity required for DNA replication. Finally, addition of yeast replication factor-A (RF-A), a protein preparation that stimulates DNA synthesis and has single-stranded DNA-binding activity, yields a polymerization reaction with 7 polypeptides required for replication, yet fidelity remains low relative to error rates for semiconservative replication. The data suggest that neither exonucleolytic proofreading activity, the beta subunit, the DNA primase subunits nor RF-A contributes substantially to base substitution or frameshift error discrimination by the DNA polymerase alpha catalytic subunit.     

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.     

The correcting role of autonomous 3'-->5' exonucleases in mammalian multienzyme DNA polymerase complexes

A study was made of the correcting role of autonomous 3'-->5' exonucleases (AE) contained in multienzyme DNA polymerase complexes of rat hepatocytes or calf thymocytes. DNA was synthesized on phage psi X174 amber3 or M13mp2 primer-templates, and used to transfect Escherichia coli spheroplasts. Frequencies were estimated for direct and reverse mutations resulting from mistakes made in the course of in vitro DNA synthesis. The mistake rate of the hepatocytic complex was estimated at 3 x 10(-6) with equimolar dNTP, and increased tenfold when proteins accounting for 70% of the total 3'-->5' exonuclease activity of the complex were removed. The fidelity of DNA synthesis was completely restored in the presence of exogenous AE (epsilon subunit of E. coli DNA polymerase III). Nuclear (Pol delta n) and cytosolic (Pol delta c) forms of DNA polymerase delta were isolated from calf thymocytes. The former was shown to contain an AE (TREX2) absent from the latter. As compared with Pol delta c, Pol delta n had a 20-fold higher exo/pol ratio and allowed 4-5 times higher fidelity of DNA synthesis. The mistake rate of DNA polymerase complexes changed when dNTP were used in nonequimolar amounts.     

The multiple biological roles of the 3'-->5' exonuclease of Saccharomyces cerevisiae DNA polymerase delta require switching between the polymerase and exonuclease domains

Until recently, the only biological function attributed to the 3'-->5' exonuclease activity of DNA polymerases was proofreading of replication errors. Based on genetic and biochemical analysis of the 3'-->5' exonuclease of yeast DNA polymerase delta (Pol delta) we have discerned additional biological roles for this exonuclease in Okazaki fragment maturation and mismatch repair. We asked whether Pol delta exonuclease performs all these biological functions in association with the replicative complex or as an exonuclease separate from the replicating holoenzyme. We have identified yeast Pol delta mutants at Leu523 that are defective in processive DNA synthesis when the rate of misincorporation is high because of a deoxynucleoside triphosphate (dNTP) imbalance. Yet the mutants retain robust 3'-->5' exonuclease activity. Based on biochemical studies, the mutant enzymes appear to be impaired in switching of the nascent 3' end between the polymerase and the exonuclease sites, resulting in severely impaired biological functions. Mutation rates and spectra and synergistic interactions of the pol3-L523X mutations with msh2, exo1, and rad27/fen1 defects were indistinguishable from those observed with previously studied exonuclease-defective mutants of the Pol delta. We conclude that the three biological functions of the 3'-->5' exonuclease addressed in this study are performed intramolecularly within the replicating holoenzyme.     

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.     

The 3'-->5' exonucleases of DNA polymerases delta and epsilon and the 5'-->3' exonuclease Exo1 have major roles in postreplication mutation avoidance in Saccharomyces cerevisiae

Replication fidelity is controlled by DNA polymerase proofreading and postreplication mismatch repair. We have genetically characterized the roles of the 5'-->3' Exo1 and the 3'-->5' DNA polymerase exonucleases in mismatch repair in the yeast Saccharomyces cerevisiae by using various genetic backgrounds and highly sensitive mutation detection systems that are based on long and short homonucleotide runs. Genetic interactions were examined among DNA polymerase epsilon (pol2-4) and delta (pol3-01) mutants defective in 3'-->5' proofreading exonuclease, mutants defective in the 5'-->3' exonuclease Exo1, and mismatch repair mutants (msh2, msh3, or msh6). These three exonucleases play an important role in mutation avoidance. Surprisingly, the mutation rate in an exo1 pol3-01 mutant was comparable to that in an msh2 pol3-01 mutant, suggesting that they participate directly in postreplication mismatch repair as well as in other DNA metabolic processes.     

Single-base discrimination mediated by proofreading 3' phosphorothioate-modified primers

It has been well known for decades that deoxyribonucleic acid (DNA) polymerases with proofreading function have a higher fidelity in primer extension as compared to those without 3' exonuclease activities. However, polymerases with proofreading function have not been used in single nucleotide polymorphism (SNP) assays. Here, we describe a new method for single-base discrimination by proofreading the 3' phosphorothioate-modified primers using a polymerase with proofreading function. Our data show that the combination of a polymerase with 3' exonuclease activity and the 3' phosphorothioate-modified primers work efficiently as a single-base mismatch-operated on/off switch. DNA polymerization only occurred from matched primers, whereas mismatched primers were not extended at the broad range of annealing temperature tested in our study. This novel single-base discrimination method has potential in SNP assays.     

Eukaryotic error prone DNA polymerases: suggested roles in replication, repair and mutagenesis

A number of error-prone DNA polymerases is found among eukaryotes from yeasts up to mammalia including humans. According to the partial homology of a primary structure, they are united in families B, X, Y and display high infidelity on uninjured DNA-template, whereas they are rather accurate on DNA injuries. These DNA polymerases are characterized by the probability of base substitutions or frame shifts of 10(-3) to 7.5 x 10(-1) on DNA injuries, whereas the probability of spontaneous mutagenesis per replicated nucleotide accounts 10(-10) - 10(-12). Inaccurate DNA polymerases are terminal deoxynucleotidyl transferase (TdT), DNA polymerases beta, zeta, kappa, eta, iota, lamda, mu, and Rev1. Their principal properties are described in this review. All of the polymerases under study are deprived of the corrective 3'-->5' exonucleolytic activity. The specialization of these polymerases is contained in the capability to synthesize opposite DNA lesions (not eliminated by multiple repair systems) that is explained by the flexibility of their active sites or by the limited capability to exhibit the TdT activity. Classic DNA polymerases alpha, delta, epsilon, and gamma cannot elongate the primers with mismatched nucleotides on their 3'-ends (that leads to the replication block), whereas some of the specialized polymerases can do it. It is accompanied by the overcoming of a replication block, often with the expense of an elevated mutagenesis. How can a cell live under the conditions of such a huge infidelity of many DNA polymerases? Error-prone DNA polymerases are not found in all tissues though some of them are essential for an organism survival. Furthermore, cells must not allow for these polymerases to work effectively on uninjured DNA. After bypass of a lesion on DNA-template, it is necessary, as soon as possible, to switch catalysis of the DNA synthesis from the specialized polymerases on the relatively accurate DNA polymerases delta and epsilon (fidelity of 10(-5) - 10(-6)). It is made by the formation of the complexes of polymerases delta or epsilon with PCNA and replicative factors RP-A and RF-C. Such highly processive complexes manifest the bigger affinity to the correct primers than the specialized DNA polymerases do. The switching is stimulated by distributivity or weak processivity of the specialized DNA polymerases. The accuracy of these polymerases are augmented by the action of the corrective 3'-exonucleolytic function of DNA polymerases delta and epsilon as well as by the autonomous 3'-->5' exonucleases which are widespread among the representatives of the whole phylogenetic tree. Exonucleolytic correction slows down the replication in the presence of lesions in DNA-template but makes the replication more accurate that decreases the probability of mutagenesis and carcinogenesis.     

Phosphorothioate primers improve the amplification of DNA sequences by DNA polymerases with proofreading activity.

Two thermostable DNA polymerases with proofreading activity--Vent DNA polymerase and Pfu DNA polymerase--have attracted recent attention, mainly because of their enhanced fidelities during amplification of DNA sequences by the polymerase chain reaction. A severe disadvantage for their practical application, however, results from the observation that due to their 3' to 5' exonuclease activities these enzymes degrade the oligodeoxynucleotides serving as primers for the DNA synthesis. It is demonstrated that this exonucleolytic attack on the primer molecules can be efficiently prevented by the introduction of single phosphorothioate bonds at their 3' termini. This strategy, which can be easily accomplished using routine DNA synthesis methodology, may open the way to a widespread use of these novel enzymes in the polymerase chain reaction.     

Antimutagenic role of autonomous 3'-->5'-exonucleases

Our own and literary data about antimutagenic role of autonomous 3'-->5'-exonucleases (AE) are analyzed. AE are not bound covalently to DNA polymerases but often involved in replicative complexes. Intracellular overproduction of AE in bacteria is accompanied with the sharp suppression of mutagenesis, whereas the inactivation of AE in bacteria and higher fungi results in the increase of mutation rates by 2-3 orders of magnitude. The addition of AE in biologically meaningful concentrations to DNA polymerases elevates substantially the accuracy of their work in vitro. In these cases, the reverse mutation rates were measured in the DNA from phage (X174 amber 3, whereas the direct mutation rates--in the DNA from phage M13mp2, both being used as primer-templates for DNA synthesis and then transfected into spheroplasts of Escherichia coli. The accuracy of action of nuclease-free DNA polymerases alpha and beta are shown to raise in the presence of AE by 2-3 orders, the accuracy of moderately processive DNA polymerase I--by 2 orders, the accuracy of highly processive DNA polymerase delta--by 5-10 times, though the latter 2 polymerases display and their own 3'-->5'-exonucleolytic activity. AE, involved in the multienzyme DNA polymerase complexes, augment the accuracy of complexes action by 5-10 times. The model of "external" corrective role of AE in DNA biosynthesis is proposed. Study of 30 objects from all 3 kingdoms of live beings (from archae- and eubacteria to mammalia including human) has shown that AE account, as minimum, from 30 to 90% of the total cellular 3'-->5'-exonucleolytic activity. So AE increase essentially the intracellular ratio of values of 3'-->5'-exonuclease to DNA polymerase activities in the very various representatives from a phylogenetic tree that results always in the augmentation of the accuracy of DNA biosynthesis.     

The proofreading 3'-->5' exonuclease activity of DNA polymerases: a kinetic barrier to translesion DNA synthesis

The 3'-->5' exonuclease activity intrinsic to several DNA polymerases plays a primary role in genetic stability; it acts as a first line of defense in correcting DNA polymerase errors. A mismatched basepair at the primer terminus is the preferred substrate for the exonuclease activity over a correct basepair. The efficiency of the exonuclease as a proofreading activity for mispairs containing a DNA lesion varies, however, being dependent upon both the DNA polymerase/exonuclease and the type of DNA lesion. The exonuclease activities intrinsic to the T4 polymerase (family B) and DNA polymerase gamma (family A) proofread DNA mispairs opposite endogenous DNA lesions, including alkylation, oxidation, and abasic adducts. However, the exonuclease of the Klenow polymerase cannot discriminate between correct and incorrect bases opposite alkylation and oxidative lesions. DNA damage alters the dynamics of the intramolecular partitioning of DNA substrates between the 3'-->5' exonuclease and polymerase activities. Enzymatic idling at lesions occurs when an exonuclease activity efficiently removes the same base that is preferentially incorporated by the DNA polymerase activity. Thus, the exonuclease activity can also act as a kinetic barrier to translesion synthesis (TLS) by preventing the stable incorporation of bases opposite DNA lesions. Understanding the downstream consequences of exonuclease activity at DNA lesions is necessary for elucidating the mechanisms of translesion synthesis and damage-induced cytotoxicity.     

Use of 2-aminopurine fluorescence to study the role of the beta hairpin in the proofreading pathway catalyzed by the phage T4 and RB69 DNA polymerases

For DNA polymerases to proofread a misincorporated nucleotide, the terminal 3-4 nucleotides of the primer strand must be separated from the template strand before being bound in the exonuclease active center. Genetic and biochemical studies of the bacteriophage T4 DNA polymerase revealed that a prominent beta-hairpin structure in the exonuclease domain is needed to efficiently form the strand-separated exonuclease complexes. We present here further mutational analysis of the loop region of the T4 DNA polymerase beta-hairpin structure, which provides additional evidence that residues in the loop, namely, Y254 and G255, are important for DNA replication fidelity. The mechanism of strand separation was probed in in vitro reactions using the fluorescence of the base analogue 2-aminopurine (2AP) and mutant RB69 DNA polymerases that have modifications to the beta hairpin, to the exonuclease active site, or to both. We propose from these studies that the beta hairpin in the exonuclease domain of the T4 and RB69 DNA polymerases functions to facilitate strand separation, but residues in the exonuclease active center are required to capture the 3' end of the primer strand following strand separation.     

Complexes of nuclear DNA-polymerases with 3'----5'-exonucleases from the rat liver

"Editing" 3'----5' exonuclease activity of DNA polymerases corrects replication errors. This activity associated with procaryotic DNA polymerases is not intrinsic to purified mammalian DNA polymerases. By means of extraction and subsequent gel filtration, several subspecies of complexes of 3'----5' exonuclease (E.C. 3.1.4.26) with DNA polymerases alpha, beta (E.C. 2.7.7.7) and some other proteins were isolated from chromatin, nucleoplasm, nuclear membrane, and cytosol. Complexes containing 3'----5' exonuclease manifest from 40 to 70% of total DNA polymerase activity revealed in different compartments of a hepatocyte. Molecular masses of the complexes amount from 250 to 1500 kDa They dissociate as a result of solution hydrophobization. DNA polymerase alpha activity enhances 5--8 folds during cell transition from G0 to S-period. The value of the ratio of 3'----5' exonuclease activity of different complexes to their DNA polymerase activity varies from 0.5 to 12. Other cases of discovery of the complexes of DNA polymerases with 3'----5' exonucleases are discussed. It is suggested that the absence of 3'----5' exonuclease active site in the DNA polymerase polypeptide is compensated by the complex formation of the corresponding enzymes.     

Eukaryotic DNA polymerases

Knowledge about eukaryotic DNA polymerases has increased considerably during recent years. Much have been learnt about both the structures and the functions of "classical" DNA polymerases alpha, beta, delta, epsilon and gamma. New DNA polymerases that possess very unusual functions have been identified. They are able to perform translesional synthesis, take part in somatic hypermutation and prevent some cancers. Much attention has also been devoted to the role of 3'-->5' exonuclease activity in the accuracy of DNA synthesis. On the other hand, it have been shown that there are also negative aspects of the activity of DNA polymerases. Lack of some DNA polymerases or even their altered functions may lead to carcinogenesis and accelerate the process of ageing.     

Characterization of two DNA polymerases from the hyperthermophilic euryarchaeon Pyrococcus abyssi.

The complete genome sequence of the hyperthermophilic archaeon Pyrococcus abyssi revealed the presence of a family B DNA polymerase (Pol I) and a family D DNA polymerase (Pol II). To extend our knowledge about euryarchaeal DNA polymerases, we cloned the genes encoding these two enzymes and expressed them in Escherichia coli. The DNA polymerases (Pol I and Pol II) were purified to homogeneity and characterized. Pol I had a molecular mass of approximately 90 kDa, as estimated by SDS/PAGE. The optimum pH and Mg(2+) concentration of Pol I were 8.5-9.0 and 3 mm, respectively. Pol II is composed of two subunits that are encoded by two genes arranged in tandem on the P. abyssi genome. We cloned these genes and purified the Pol II DNA polymerase from an E. coli strain coexpressing the cloned genes. The optimum pH and Mg(2+) concentration of Pol II were 6.5 and 15-20 mm, respectively. Both P. abyssi Pol I and Pol II have associated 3'-->5' exonuclease activity although the exonuclease motifs usually found in DNA polymerases are absent in the archaeal family D DNA polymerase sequences. Sequence analysis has revealed that the small subunit of family D DNA polymerase and the Mre11 nucleases belong to the calcineurin-like phosphoesterase superfamily and that residues involved in catalysis and metal coordination in the Mre11 nuclease three-dimensional structure are strictly conserved in both families. One hypothesis is that the phosphoesterase domain of the small subunit is responsible for the 3'-->5' exonuclease activity of family D DNA polymerase. These results increase our understanding of euryarchaeal DNA polymerases and are of importance to push forward the complete understanding of the DNA replication in P. abyssi.