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.     

Replication slippage of different DNA polymerases is inversely related to their strand displacement efficiency.

Replication slippage is a particular type of error caused by DNA polymerases believed to occur both in bacterial and eukaryotic cells. Previous studies have shown that deletion events can occur in Escherichia coli by replication slippage between short duplications and that the main E. coli polymerase, DNA polymerase III holoenzyme is prone to such slippage. In this work, we present evidence that the two other DNA polymerases of E. coli, DNA polymerase I and DNA polymerase II, as well as polymerases of two phages, T4 (T4 pol) and T7 (T7 pol), undergo slippage in vitro, whereas DNA polymerase from another phage, Phi29, does not. Furthermore, we have measured the strand displacement activity of the different polymerases tested for slippage in the absence and in the presence of the E. coli single-stranded DNA-binding protein (SSB), and we show that: (i) polymerases having a strong strand displacement activity cannot slip (DNA polymerase from Phi29); (ii) polymerases devoid of any strand displacement activity slip very efficiently (DNA polymerase II and T4 pol); and (iii) stimulation of the strand displacement activity by E. coli SSB (DNA polymerase I and T7 pol), by phagic SSB (T4 pol), or by a mutation that affects the 3' --> 5' exonuclease domain (DNA polymerase II exo(-) and T7 pol exo(-)) is correlated with the inhibition of slippage. We propose that these observations can be interpreted in terms of a model, for which we have shown that high strand displacement activity of a polymerase diminishes its propensity to slip.     

Highly tolerated amino acid substitutions increase the fidelity of Escherichia coli DNA polymerase I

Fidelity of DNA synthesis, catalyzed by DNA polymerases, is critical for the maintenance of the integrity of the genome. Mutant polymerases with elevated accuracy (antimutators) have been observed, but these mainly involve increased exonuclease proofreading or large decreases in polymerase activity. We have determined the tolerance of DNA polymerase for amino acid substitutions in the active site and in different segments of E. coli DNA polymerase I and have determined the effects of these substitutions on the fidelity of DNA synthesis. We established a DNA polymerase I mutant library, with random substitutions throughout the polymerase domain. This random library was first selected for activity. The essentiality of DNA polymerases and their sequence and structural conservation suggests that few amino acid substitutions would be tolerated. However, we report that two-thirds of single base substitutions were tolerated without loss of activity, and plasticity often occurs at evolutionarily conserved regions. We screened 408 members of the active library for alterations in fidelity of DNA synthesis in Escherichia coli expressing the mutant polymerases and carrying a second plasmid containing a beta-lactamase reporter. Mutation frequencies varied from 1/1000- to 1000-fold greater compared with wild type. Mutations that produced an antimutator phenotype were distributed throughout the polymerase domain, with 12% clustered in the M-helix. We confirmed that a single mutation in this segment results in increased base discrimination. Thus, this work identifies the M-helix as a determinant of fidelity and suggests that polymerases can tolerate many substitutions that alter fidelity without incurring major changes in activity.     

The gamma subfamily of DNA polymerases: cloning of a developmentally regulated cDNA encoding Xenopus laevis mitochondrial DNA polymerase gamma.

We used the known sequence of the Saccharomyces cerevisiae DNA polymerase gamma to clone the genes or cDNAs encoding this enzyme in two other yeasts, Pychia pastoris and Schizosaccharomyces pombe, and one higher eukaryote, Xenopus laevis. To confirm the identity of the final X.laevis clone, two antisera raised against peptide sequences were shown to react with DNA polymerase gamma purified from X.laevis oocyte mitochondria. A developmentally regulated 4.6 kb mRNA is recognized on Northern blots of oocyte RNA using the X.laevis cDNA. Comparison of the four DNA polymerase gamma gene sequences revealed several highly conserved sequence blocks, comprising an N-terminal 3'-->5'exonuclease domain and a C-terminal polymerase active center interspersed with gamma-specific gene sequences. The consensus sequences for the DNA polymerase gamma exonuclease and polymerase domains show extensive sequence similarity to DNA polymerase I from Escherichia coli. Sequence conservation is greatest for residues located near the active centers of the exo and pol domains of the E.coli DNA polymerase I structure. The domain separating the exonuclease and polymerase active sites is larger in DNA polymerase gamma than in other members of family A (DNA polymerase I-like) polymerases. The S.cerevisiae DNA polymerase gamma is atypical in that it includes a 240 residue C-terminal extension that is not found in the other members of the DNA polymerase gamma family, or in other family A DNA polymerases.     

The hyperthermophilic archaeon Pyrodictium occultum has two alpha-like DNA polymerases.

We cloned two genes encoding DNA polymerases from the hyperthermophilic archaeon Pyrodictium occultum. The deduced primary structures of the two gene products have several amino acid sequences which are conserved in the alpha-like (family B) DNA polymerases. Both genes were expressed in Escherichia coli, and highly purified gene products, DNA polymerases I and II (pol I and pol II), were biochemically characterized. Both DNA polymerase activities were heat stable, but only pol II was sensitive to aphidicolin. Both pol I and pol II have associated 5'-->3' and 3'-->5' exonuclease activities. In addition, these DNA polymerases have higher affinity to single-primed single-stranded DNA than to activated DNA; even their primer extension abilities by themselves were very weak. A comparison of the complete amino acid sequences of pol I and pol II with two alpha-like DNA polymerases from yeast cells showed that both pol I and pol II were more similar to yeast DNA polymerase III (ypol III) than to yeast DNA polymerase II (ypol II), in particular in the regions from exo II to exo III and from motif A to motif C. However, comparisons region by region of each polymerase showed that pol I was similar to ypol II and pol II was similar to ypol III from motif C to the C terminus. In contrast, pol I and pol II were similar to ypol III and ypol II, respectively, in the region from exo III to motif A. These findings suggest that both enzymes from P. occultum play a role in the replication of the genomic DNA of this organism and, furthermore, that the study of DNA replication in this thermophilic archaeon may lead to an understanding of the prototypical mechanism of eukaryotic DNA replication.     

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.     

DNA polymerase III, a second essential DNA polymerase, is encoded by the S. cerevisiae CDC2 gene

Three nuclear DNA polymerases have been described in yeast: DNA polymerases I, II, and III. DNA polymerase I is encoded by the POL1 gene and is essential for DNA replication. Since the S. cerevisiae CDC2 gene has recently been shown to have DNA sequence similarity to the active site regions of other known DNA polymerases, but to nevertheless be different from DNA polymerase I, we examined cdc2 mutants for the presence of DNA polymerases II and III. DNA polymerase II was not affected by the cdc2 mutation. DNA polymerase III activity was significantly reduced in the cdc2-1 cell extracts. We conclude that the CDC2 gene encodes yeast DNA polymerase III and that DNA polymerase III, therefore, represents a second essential DNA polymerase in yeast.     

DNA polymerase III from Saccharomyces cerevisiae. II. Inhibitor studies and comparison with DNA polymerases I and II

The newly identified yeast DNA polymerase III was compared to DNA polymerases I and II and the mitochondrial DNA polymerase. Inhibition by aphidicolin (I50) of DNA polymerases I, II, and III was 4, 6, and 0.6 micrograms/ml, respectively. The mitochondrial enzyme was insensitive to the drug. N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate strongly inhibited DNA polymerase I (I50 = 0.3 microM), whereas DNA polymerase III was less sensitive (I50 = 80 microM). Conditions that allowed proteolysis to proceed during the preparation of extracts converted DNA polymerase II from a sensitive form (I50 = 2.4 microM) to a resistant form (I50 = 2 mM). The mitochondrial DNA polymerase is insensitive (I50 greater than 5 mM). With most other inhibitors tested (N-ethylmaleimide, heparin, salt) only small differences were observed between the three nuclear DNA polymerases. Polyclonal antibodies to DNA polymerase III did not inhibit DNA polymerases I and II, nor were those polymerases recognized by Western blotting. Monoclonal antibodies to DNA polymerase I did not crossreact with DNA polymerases II and III. The results show that DNA polymerase III is distinct from DNA polymerase I and II.     

Competitive processivity-clamp usage by DNA polymerases during DNA replication and repair

Protein clamps are ubiquitous and essential components of DNA metabolic machineries, where they serve as mobile platforms that interact with a large variety of proteins. In this report we identify residues that are required for binding of the beta-clamp to DNA polymerase III of Escherichia coli, a polymerase of the Pol C family. We show that the alpha polymerase subunit of DNA polymerase III interacts with the beta-clamp via its extreme seven C-terminal residues, some of which are conserved. Moreover, interaction of Pol III with the clamp takes place at the same site as that of the delta-subunit of the clamp loader, providing the basis for a switch between the clamp loading machinery and the polymerase itself. Escherichia coli DNA polymerases I, II, IV and V (UmuC) interact with beta at the same site. Given the limited amounts of clamps in the cell, these results suggest that clamp binding may be competitive and regulated, and that the different polymerases may use the same clamp sequentially during replication and repair.     

Specificity of replicative and SOS-inducible DNA polymerases in frameshift mutagenesis: mutability of Salmonella typhimurium strains overexpressing SOS-inducible DNA polymerases to 30 chemical mutagens

DNA replication is frequently hindered because of the presence of DNA lesions induced by endogenous and exogenous genotoxic agents. To circumvent the replication block, cells are endowed with multiple specialized DNA polymerases that can bypass a variety of DNA damage. To better understand the specificity of specialized DNA polymerases to bypass lesions, we have constructed a set of derivatives of Salmonella typhimurium TA1538 harboring plasmids carrying the polB, dinB or mucAB genes encoding Escherichia coli DNA polymerase II, DNA polymerase IV or DNA polymerase RI, respectively, and examined the mutability to 30 chemicals. The parent strain TA1538 possesses CGCGCGCG hotspot sequence for -2 frameshift. Interestingly, the chemicals could be classified into four groups based on the mutagenicity to the derivatives: group I whose mutagenicity was highest in strain YG5161 harboring plasmid carrying dinB; group II whose mutagenicity was almost equally high in strain YG5161 and strain TA98 harboring plasmid carrying mucAB; group III whose mutagenicity was highest in strain TA98; group IV whose mutagenicity was not affected by the introduction of any of the plasmids. Introduction of plasmid carrying polB did not enhance the mutagenicity except for benz[a]anthracene. We also introduced a plasmid carrying polA encoding E. coli DNA polymerase I to strain TA1538. Strikingly, the introduction of the plasmid reduced the mutagenicity of chemicals belonging to groups I, II and III, but not the chemicals of group IV, to the levels observed in the derivative whose SOS-inducible DNA polymerases were all deleted. These results suggest that (i) DNA polymerase IV and DNA polymerase RI possess distinct but partly overlapping specificity to bypass lesions leading to -2 frameshift, (ii) the replicative DNA polymerase, i.e., DNA polymerase III, participates in the mutagenesis and (iii) the enhanced expression of E. coli polA may suppress the access of Y-family DNA polymerases to the replication complex.     

Sites of termination of in vitro DNA synthesis on cis-diamminedichloroplatinum(II) treated single-stranded DNA: a comparison between E. coli DNA polymerase I and eucaryotic DNA polymerases alpha.

We have compared the capacity of the large fragment of E. coli DNA polymerase I and highly purified DNA polymerases alpha from either Drosophila melanogaster embryos or calf thymus to replicate single-stranded M13 mp10 DNA treated with the antitumoral drug cis-diamminedichloroplatinum(II) (cis-DDP). We report that: a) although prokaryotic and eukaryotic enzymes have different structural complexity and dissimilar in vivo functions, their synthesis was blocked in vitro at similar sites on cis-DDP treated DNA; b) this inhibition occurred not only at d(G)n sequences, as previously reported for E. coli DNA polymerase I, (Pinto & Lippard (1985) Proc. Natl. Acad. Sci. USA, 82, 4616-4619) but also at other sequences which may represent putative cis-DDP-DNA adducts.     

A conserved 3'----5' exonuclease active site in prokaryotic and eukaryotic DNA polymerases

The 3'----5' exonuclease active site of E. coli DNA polymerase I is predicted to be conserved for both prokaryotic and eukaryotic DNA polymerases based on amino acid sequence homology. Three amino acid regions containing the critical residues in the E. coli DNA polymerase I involved in metal binding, single-stranded DNA binding, and catalysis of the exonuclease reaction are located in the amino-terminal half and in the same linear arrangement in several prokaryotic and eukaryotic DNA polymerases. Site-directed mutagenesis at the predicted exonuclease active site of the phi 29 DNA polymerase, a model enzyme for prokaryotic and eukaryotic alpha-like DNA polymerases, specifically inactivated the 3'----5' exonuclease activity of the enzyme. These results reflect a high evolutionary conservation of this catalytic domain. Based on structural and functional data, a modular organization of enzymatic activities in prokaryotic and eukaryotic DNA polymerases is also proposed.     

SOS-inducible DNA polymerase II of E coli is homologous to replicative DNA polymerase of eukaryotes

The polB gene of Escherichia coli encodes DNA polymerase II whose role in vivo is not defined. The polB gene has been cloned and shown to be identical to a DNA damage-inducible gene dinA which is regulated by the LexA repressor. Nucleotide sequencing of polB reveals that E coli DNA polymerase II is highly homologous to replicative DNA polymerases of eukaryotes which include human DNA polymerase alpha and Saccharomyces cerevisiae DNA polymerases I, II and III. The polB gene is not required for growth, UV-repair and UV-mutagenesis.     

Family A and family B DNA polymerases are structurally related: evolutionary implications.

In order to establish the evolutionary relationship between the family A and B DNA polymerases, we have closely compared the 3'-->5' exonuclease domains between the Klenow fragment of E.coli DNA polymerase I (a family A DNA polymerase) and the bacteriophage PRD1 DNA polymerase, the smallest member of the DNA polymerase family B. Although the PRD1 DNA polymerase has a smaller 3'-->5' exonuclease domain, its active sites appear to be very similar to those of the Klenow fragment. Site-directed mutagenesis studies revealed that the residues important for the 3'-->5' exonuclease activity, particularly metal binding ligands for the Klenow fragment, are all conserved in the PRD1 DNA polymerase as well. The metal binding ligands are also essential for the strand-displacement activity of the PRD1 DNA polymerase. Based on these results and the studies by others in various systems, we conclude that family A and B DNA polymerases, at least in the 3'-->5' exonuclease domain, are structurally as well as evolutionarily related.     

Domain exchange: chimeras of Thermus aquaticus DNA polymerase, Escherichia coli DNA polymerase I and Thermotoga neapolitana DNA polymerase

The intervening domain of the thermostable Thermus aquaticus DNA polymerase (TAQ: polymerase), which has no catalytic activity, has been exchanged for the 3'-5' exonuclease domain of the homologous mesophile Escherichia coli DNA polymerase I (E.coli pol I) and the homologous thermostable Thermotoga neapolitana DNA polymerase (TNE: polymerase). Three chimeric DNA polymerases have been constructed using the three-dimensional (3D) structure of the Klenow fragment of the E.coli pol I and 3D models of the intervening and polymerase domains of the TAQ: polymerase and the TNE: polymerase: chimera TaqEc1 (exchange of residues 292-423 from TAQ: polymerase for residues 327-519 of E.coli pol I), chimera TaqTne1 (exchange of residues 292-423 of TAQ: polymerase for residues 295-485 of TNE: polymerase) and chimera TaqTne2 (exchange of residues 292-448 of TAQ: polymerase for residues 295-510 of TNE: polymerase). The chimera TaqEc1 showed characteristics from both parental polymerases at an intermediate temperature of 50 degrees C: high polymerase activity, processivity, 3'-5' exonuclease activity and proof-reading function. In comparison, the chimeras TaqTne1 and TaqTne2 showed no significant 3'-5' exonuclease activity and no proof-reading function. The chimera TaqTne1 showed an optimum temperature at 60 degrees C, decreased polymerase activity compared with the TAQ: polymerase and reduced processivity. The chimera TaqTne2 showed high polymerase activity at 72 degrees C, processivity and less reduced thermostability compared with the chimera TaqTne1.     

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.     

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.     

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.