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.     

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)     

Aphidicolin inhibits DNA polymerizing activity but not nucleolytic activity of Escherichia coli DNA polymerase II.

We have purified the DNA polymerase II of Escherichia coli from the recombinant strain carrying the plasmid which encodes the polB gene. We confirmed that the purified protein, of molecular weight 90,000, possesses a 3'----5' exonuclease activity in addition to DNA polymerizing activity in a single polypeptide. Its DNA polymerizing activity was sensitive to the drug aphidicoline, which is a specific and direct inhibitor of the alpha-like DNA polymerases including eukaryotic replicative DNA polymerases. Aphidicolin had no detectable effect on the 3'----5' exonuclease activity. The inhibition by aphidicolin on the polymerizing activity of polymerase II was competitive with respect to dNTP and uncompetitive with respect to template DNA. This mode of action is the same as that on eukaryotic DNA polymerase alpha. The apparent Ki value calculated from Lineweaver-Burk plots was 55.6 microM.     

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.     

Evidence favouring the hypothesis of a conserved 3'-5' exonuclease active site in DNA-dependent DNA polymerases.

The complete amino acid (aa) alignment of the N-terminal domain of 33 DNA-dependent DNA polymerases encompassing the putative segments Exo I, Exo II and Exo III, proposed by Bernad et al. [Cell 59 (1989) 219-228] to form a conserved 3'-5' exonuclease active site in prokaryotic and eukaryotic DNA polymerases, allowed us to identify and/or correct some of the most conserved segments (Exo I, II and III) in certain DNA polymerases. In particular, the aa region of T4 DNA polymerase and other eukaryotic (viral and cellular) DNA polymerases previously proposed as Exo I segment 1, did not align with the Exo I segment of Escherichia coli DNA polymerase I (PolI)-like and protein-primed DNA polymerases; instead, a new conserved region of aa similarity was identified in T4 DNA polymerase and eukaryotic (viral and cellular) DNA polymerases as their corresponding Exo I segment. Therefore, according to our alignment, the recently reported T4 DNA polymerase site-directed mutants, D189A and E191A [Reha-Krantz et al., Proc. Natl. Acad. Sci. USA 88 (1991) 2417-2421], do not correspond to what we now consider the critical Exo I motif of PolI. As discussed in this communication, the functional importance of conserved segments Exo I, Exo II and Exo III is supported by site-directed mutagenesis in PolI, and in phi 29, T7 and delta(Sc) DNA polymerases. Furthermore, genetically selected T4 DNA polymerase mutator mutants form two main clusters, centered in the conserved segment Exo III and in the newly identified Exo I segment.     

Primary structure of T4 DNA polymerase. Evolutionary relatedness to eucaryotic and other procaryotic DNA polymerases

Bacteriophage T4 gene 43 codes for the viral DNA polymerase. We report here the sequence of gene 43 and about 70 nucleotides of 5'- and 3'-flanking sequences, determined by both DNA and RNA sequencing. We have also purified T4 DNA polymerase from T4 infected Escherichia coli and from E. coli containing a gene 43 overexpression vector. A major portion of the deduced amino acid sequence has been verified by peptide mapping and sequencing of the purified DNA polymerase. All these results are consistent with T4 DNA polymerase having 898 amino acids with a calculated Mr = 103,572. Comparison of the primary structure of T4 DNA polymerase with the sequence of other procaryotic and eucaryotic DNA polymerases indicates that T4 DNA polymerase has regions of striking similarity with animal virus DNA polymerases and human DNA polymerase alpha. Surprisingly, T4 DNA polymerase shares only limited similarity with E. coli polymerase I and no detectable similarity with T7 DNA polymerase. Based on the location of specific mutations in T4 DNA polymerase and the conservation of particular sequences in T4 and eucaryotic DNA polymerases, we propose that the NH2-terminal half of T4 DNA polymerase forms a domain that carries out the 3'----5' exonuclease activity whereas the COOH-terminal half of the polypeptide contains the dNTP-binding site and is necessary for DNA synthesis.     

The DNA polymerase-encoding gene of Bacillus subtilis bacteriophage SPO1.

The bacteriophage SPO1 DNA polymerase-encoding gene, which contains a self-splicing intron, has been sequenced and its amino acid (aa) sequence has been deduced. The aa sequence of SPO1 DNA polymerase shows a high degree of similarity with that of DNA polymerase I from Escherichia coli (Po1I). Alignment with the sequences of Po1I, and the phi 29 and SPO1 DNA polymerases indicate that the aa residues that have been implicated in 3'----5' exonuclease activities are conserved.     

Mutations in the spacer region of Drosophila mitochondrial DNA polymerase affect DNA binding, processivity, and the balance between Pol and Exo function

The catalytic subunit (alpha) of mitochondrial DNA polymerase (pol gamma) shares conserved DNA polymerase and 3'-5' exonuclease active site motifs with Escherichia coli DNA polymerase I and bacteriophage T7 DNA polymerase. A major difference between the prokaryotic and mitochondrial proteins is the size and sequence of the region between the exonuclease and DNA polymerase domains, referred to as the spacer in pol gamma-alpha. Four gamma-specific conserved sequence elements are located within the spacer region of the catalytic subunit in eukaryotic species from yeast to humans. To elucidate the functional roles of the spacer region, we pursued deletion and site-directed mutagenesis of Drosophila pol gamma. Mutant proteins were expressed from baculovirus constructs in insect cells, purified to near homogeneity, and analyzed biochemically. We find that mutations in three of the four conserved sequence elements within the spacer alter enzyme activity, processivity, and/or DNA binding affinity. In addition, several mutations affect differentially DNA polymerase and exonuclease activity and/or functional interactions with mitochondrial single-stranded DNA-binding protein. Based on these results and crystallographic evidence showing that the template-primer binds in a cleft between the exonuclease and DNA polymerase domains in family A DNA polymerases, we propose that conserved sequences within the spacer of pol gamma may position the substrate with respect to the enzyme catalytic domains.     

On the fidelity of DNA replication: herpes DNA polymerase and its associated exonuclease.

Procaryotic DNA polymerases contain an associated 3'----5' exonuclease activity which provides a proofreading function and contributes substantially to replication fidelity. DNA polymerases of the eucaryotic herpes-type viruses contain similar associated exonuclease activities. We have investigated the fidelity of polymerases purified from wild type herpes simplex virus, as well as from mutator and antimutator strains. On synthetic templates, the herpes enzymes show greater relative exonuclease activities, and greater ability to excise a terminal mismatched base, than procaryotic DNA polymerases which proofread. On a phi X174 natural DNA template, the herpes enzymes are more accurate than purified eucaryotic DNA polymerases; the error rate is similar to E. coli polymerase I. However, conditions which abnegate proofreading by E. coli polymerase I have little effect on the herpes enzymes. We conclude that either these viral polymerases are accurate in the absence of proofreading, or the conditions examined have little effect on proofreading by the herpes DNA polymerases.     

Purification and properties of 2-aminoadipate: 2-oxoglutarate aminotransferase from bovine kidney.

Escherichia coli endonuclease IV hydrolyses the C(3')-O-P bond 5' to a 3'-terminal base-free deoxyribose. It also hydrolyses the C(3')-O-P bond 5' to a 3'-terminal base-free 2',3'-unsaturated sugar produced by nicking 3' to an AP (apurinic or apyrimidinic) site by beta-elimination; this explains why the unproductive end produced by beta-elimination is converted by the enzyme into a 3'-OH end able to prime DNA synthesis. The action of E. coli endonuclease IV on an internal AP site is more complex: in a first step the C(3')-O-P bond 5' to the AP site is hydrolysed, but in a second step the 5'-terminal base-free deoxyribose 5'-phosphate is lost. This loss is due to a spontaneous beta-elimination reaction in which the enzyme plays no role. The extreme lability of the C(3')-O-P bond 3' to a 5'-terminal AP site contrasts with the relative stability of the same bond 3' to an internal AP site; in the absence of beta-elimination catalysts, at 37 degrees C the half-life of the former is about 2 h and that of the latter 200 h. The extreme lability of a 5'-terminal AP site means that, after nicking 5' to an AP site with an AP endonuclease, in principle no 5'----3' exonuclease is needed to excise the AP site: it falls off spontaneously. We have repaired DNA containing AP sites with an AP endonuclease (E. coli endonuclease IV or the chromatin AP endonuclease from rat liver), a DNA polymerase devoid of 5'----3' exonuclease activity (Klenow polymerase or rat liver DNA polymerase beta) and a DNA ligase. Catalysts of beta-elimination, such as spermine, can drastically shorten the already brief half-life of a 5'-terminal AP site; it is what very probably happens in the chromatin of eukaryotic cells. E. coli endonuclease IV also probably participates in the repair of strand breaks produced by ionizing radiations: as E. coli endonuclease VI/exonuclease III, it is a 3'-phosphoglycollatase and also a 3'-phosphatase. The 3'-phosphatase activity of E. coli endonuclease VI/exonuclease III and E. coli endonuclease IV can also be useful when the AP site has been excised by a beta delta-elimination reaction.     

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.     

Rat DNA polymerase beta gene can join in excision repair of Escherichia coli.

Though DNA polymerase I (poll) of Escherichia (E.) coli is understood to play a role in repair synthesis of excision repair, it is still obscure whether DNA polymerase beta (pol beta) plays a similar role in eukaryotic cells. To estimate the role of pol beta in excision repair processes, we inserted the rat pol beta gene into several mutant E. coli defective in a diverse set of enzymatic activities of poll. UV resistance was seen only when the 5'----3' exonuclease (exo) activity of poll molecules remained. Therefore it is suggested that 5'----3' exo activity as well as pol beta activity are essential for repair synthesis of excision repair in eukaryotic cells.     

DNA polymerase epsilon: the latest member in the family of mammalian DNA polymerases

DNA polymerase epsilon is a mammalian polymerase that has a tightly associated 3'----5' exonuclease activity. Because of this readily detectable exonuclease activity, the enzyme has been regarded as a form of DNA polymerase delta, an enzyme which, together with DNA polymerase alpha, is in all probability required for the replication of chromosomal DNA. Recently, it was discovered that DNA polymerase epsilon is both catalytically and structurally distinct from DNA polymerase delta. The most striking difference between the two DNA polymerases is that processive DNA synthesis by DNA polymerase delta is dependent on proliferating cell nuclear antigen (PCNA), a replication factor, while DNA polymerase epsilon is inherently processive. DNA polymerase epsilon is required at least for the repair synthesis of UV-damaged DNA. DNA polymerases are highly conserved in eukaryotic cells. Mammalian DNA polymerases alpha, delta and epsilon are counterparts of yeast DNA polymerases I, III and II, respectively. Like DNA polymerases I and III, DNA polymerase II is also essential for the viability of cells, which suggests that DNA polymerase II (and epsilon) may play a role in DNA replication.     

Interaction of Escherichia coli DNA polymerase I with azidoDNA and fluorescent DNA probes: identification of protein-DNA contacts

The synthesis of an azidoDNA duplex and its use to photolabel DNA polymerases have been previously described (Gibson & Benkovic, 1987). We now present detailed experiments utilizing this azidoDNA photoprobe as a substrate for Escherichia coli DNA polymerase I (Klenow fragment) and the photoaffinity labeling of the protein. The azidoDNA duplex is an efficient substrate for both the polymerase and 3'----5' exonuclease activities of the enzyme. However, the hydrolytic degradation of the azido-bearing base is dramatically impaired. On the basis of the ability of these duplexes to photolabel the enzyme, we have determined that the protein contacts between five and seven bases of duplex DNA. Incubation of azidoDNA with the Klenow fragment in the presence of magnesium results in the in situ formation of a template-primer with the azido-bearing base bound at the polymerase catalytic site of the enzyme. Photolysis of this complex followed by proteolytic digestion and isolation of DNA-labeled peptides results in the identification of a single residue modified by the photoreactive DNA substrate. We identify Tyr766 as the modified amino acid and thus localize the catalytic site for polymerization in the protein. A mansyl-labeled DNA duplex has been prepared as a fluorescent probe of protein structure. This has been utilized to determine the location of the primer terminus when bound to the Klenow fragment. When the duplex contains five unpaired bases in the primer strand of the duplex, the primer terminus resides predominantly at the exonuclease catalytic site of the enzyme. Removal of the mismatched bases by the exonuclease activity of the enzyme yields a binary complex with the primer terminus now bound predominantly at the polymerase active site. Data are presented which suggest that the rate-limiting step in the exonuclease activity of the enzyme is translocation of the primer terminus from polymerase to exonuclease catalytic sites.     

Proofreading activity of DNA polymerase III responds like elongation activity to auxiliary subunits

A comparison of the 3'----5' proofreading properties between Escherichia coli DNA polymerase III holoenzyme and DNA polymerase III' was conducted. This study indicated that the influence of the holoenzyme auxiliary subunits on the proofreading exonuclease parallels their effect on the elongation reaction. At physiological ionic strengths the auxiliary subunits markedly stimulated the exonuclease rate in an ATP-dependent reaction, while the exonuclease rate of DNA polymerase III' was not affected by ATP. E. coli single-stranded DNA binding protein stimulated the 3'----5' exonuclease activity of holoenzyme and inhibited DNA polymerase III'. Similarly, the auxiliary subunits and ATP converted the proofreading activity to a highly processive exonuclease. Our observation, that the exonuclease activity of the DNA polymerase III holoenzyme responded to ATP, salt, and E. coli single-stranded DNA-binding protein like the elongation activity, is consistent with the polymerase and exonuclease subunits acting within the same complex in a coordinated reaction.     

Detection of an epitope, not required for polymerization, that is conserved between E.coli DNA polymerases I and III and bacteriophage T4 DNA polymerase.

Monoclonal antibodies directed against the alpha subunit of the DNA polymerase III holoenzyme (1) of E. coli were tested for cross-reactivity with a variety of polymerases. We found that one monoclonal antibody bound to E. coli DNA polymerase I as well as to DNA polymerase III. A weaker, but specific, interaction was also detected with T4 DNA polymerase. We exploited the proteolysis procedure developed by Setlow, Brutlag and Kornberg (2) to determine which domain of DNA polymerase I contained the conserved epitope. Contrary to expectations, it was not found in the polymerase domain, but in the 5'----3' exonuclease domain. This reveals a sequence or structure, sufficiently important to be conserved among these polymerases, that is not directly involved in the polymerization reaction.     

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.     

Hydrolysis of 3'-terminal mispairs in vitro by the 3'----5' exonuclease of DNA polymerase delta permits subsequent extension by DNA polymerase alpha

Purified DNA polymerase alpha, the major replicating enzyme found in mammalian cells, lacks an associated 3'----5' proofreading exonuclease that, in bacteria, contributes significantly to the accuracy of DNA replication. Calf thymus DNA polymerase alpha cannot remove mispaired 3'-termini, nor can it extend them efficiently. We designed a biochemical assay to search in cell extracts for a putative proofreading exonuclease that might function in concert with DNA polymerase alpha in vivo but dissociates from it during purification. Using this assay, we purified a 3'----5' exonuclease from calf thymus that preferentially hydrolyzes mispaired 3'-termini, permitting subsequent extension of the correctly paired 3'-terminus by DNA polymerase alpha. This exonuclease copurifies with a DNA polymerase activity that is biochemically distinct from DNA polymerase alpha and exhibits characteristics described for a second replicative DNA polymerase, DNA polymerase delta. In related studies, we showed that the 3'----5' exonuclease of authentic DNA polymerase delta, like the purified exonuclease, removes terminal mispairs, allowing extension by DNA polymerase alpha. These data suggest that a single proofreading exonuclease could be shared by DNA polymerases alpha and delta, functioning at the site of DNA replication in mammalian cells.     

Metal activation of synthetic and degradative activities of phi 29 DNA polymerase, a model enzyme for protein-primed DNA replication.

Analysis of metal activation on the synthetic and degradative activities of phi 29 DNA polymerase was carried out in comparison with T4 DNA polymerase and Escherichia coli DNA polymerase I (Klenow fragment). In the three DNA polymerases studied, both the polymerization and the 3'----5' exonuclease activity had clear differences in their metal ion requirements. The results obtained support the existence of independent metal binding sites for the synthetic and degradative activities of phi 29 DNA polymerase, according with the distant location of catalytic domains (N-terminal for the 3'----5' exonuclease and C-terminal for DNA polymerization) proposed for both Klenow fragment and phi 29 DNA polymerase. Furthermore, DNA competition experiments using phi 29 DNA polymerase suggested that the main differences observed in the metal usage to activate polymerization may be the consequence of metal-induced changes in the enzyme-DNA interactions, whose strength distinguishes processive and nonprocessive DNA polymerases. Interestingly, the initiation of DNA polymerization using a protein as a primer, a special synthetic activity carried out by phi 29 DNA polymerase, exhibited a strong preference for Mn2+ as metal activator. The molecular basis for this preference is mainly the result of a large increase in the affinity for dATP.     

Genetic Evidence for Two Protein Domains and a Potential New Activity in Bacteriophage T4 DNA Polymerase

Intragenic complementation was detected within the bacteriophage T4 DNA polymerase gene. Complementation was observed between specific amino (N)-terminal, temperature-sensitive (ts) mutator mutants and more carboxy (C)-terminal mutants lacking DNA polymerase polymerizing functions. Protein sequences surrounding N-terminal mutation sites are similar to sequences found in Escherichia coli ribonuclease H (RNase H) and in the 5'----3' exonuclease domain of E. coli DNA polymerase I. These observations suggest that T4 DNA polymerase, like E. coli DNA polymerase I, contains a discrete N-terminal domain.