A novel DNA polymerase family found in Archaea

One of the most puzzling results from the complete genome sequence of the methanogenic archaeon Methanococcus jannaschii was that the organism may have only one DNA polymerase gene. This is because no other DNA polymerase-like open reading frames (ORFs) were found besides one ORF having the typical alpha-like DNA polymerase (family B). Recently, we identified the genes of DNA polymerase II (the second DNA polymerase) from the hyperthermophilic archaeon Pyrococcus furiosus, which has also at least one alpha-like DNA polymerase (T. Uemori, Y. Sato, I. Kato, H. Doi, and Y. Ishino, Genes Cells 2:499-512, 1997). The genes in M. jannaschii encoding the proteins that are homologous to the DNA polymerase II of P. furiosus have been located and cloned. The gene products of M. jannaschii expressed in Escherichia coli had both DNA polymerizing and 3'-->5' exonuclease activities. We propose here a novel DNA polymerase family which is entirely different from other hitherto-described DNA polymerases.     

Purification and characterization of a new DNA polymerase from budding yeast Saccharomyces cerevisiae. A probable homolog of mammalian DNA polymerase beta

A new DNA polymerase activity was identified and purified to near homogeneity from extracts of mitotic and meiotic cells of the yeast Saccharomyces cerevisiae. This activity increased at least 5-fold during meiosis, and it was shown to be associated with a 68-kDa polypeptide as determined by SDS-polyacrylamide gel electrophoresis. This new DNA polymerase did not have any detectable 3'-->5' exonuclease activity and preferred small gapped DNA as a template-primer. The activity was inhibited by dideoxyribonucleoside 5'-triphosphates and N-ethylmaleimide but not by concentrations of aphidicolin which completely inhibit either DNA polymerases I (alpha), II (epsilon), or III (delta). Since no polypeptide(s) in the extensively purified DNA polymerase fractions cross-reacted with antibodies raised against yeast DNA polymerases I, II, and III, we called this enzyme DNA polymerase IV. The DNA polymerase IV activity increased at least 10-fold in a yeast strain overexpressing the gene product predicted from the YCR14C open-reading frame (identified on S. cerevisiae chromosome III and provisionally called POLX), while no activity was detected in a strain where POLX was deleted. These results strongly suggest that DNA polymerase IV is encoded by the POLX gene and is a probable homolog of mammalian DNA polymerase beta.     

Anti-mutagenic activity of DNA damage-binding proteins mediated by direct inhibition of translesion replication

DNA lesions that block replication can be bypassed in Escherichia coli by a special DNA synthesis process termed translesion replication. This process is mutagenic due to the miscoding nature of the DNA lesions. We report that the repair enzyme formamido-pyrimidine DNA glycosylase and the general DNA damage recognition protein UvrA each inhibit specifically translesion replication through an abasic site analog by purified DNA polymerases I and II, and DNA polymerase III (alpha subunit) from E. coli. In vivo experiments suggest that a similar inhibitory mechanism prevents at least 70% of the mutations caused by ultraviolet light DNA lesions in E. coli. These results suggest that DNA damage-binding proteins regulate mutagenesis by a novel mechanism that involves direct inhibition of translesion replication. This mechanism provides anti-mutagenic defense against DNA lesions that have escaped DNA repair.     

Replication of O6-methylguanine-containing DNA by repair and replicative DNA polymerases

The biological consequences of O6-methylguanine (m6G) in DNA are well recognized. When template m6G is encountered by DNA polymerases, replication is hindered and trans-lesion replication results in the preferential incorporation of dTMP opposite template m6G. Thus, unrepaired m6G in DNA is both cytotoxic and mutagenic. Yet, cell lines tolerant to m6G in DNA have been isolated, which indicates that some cellular DNA polymerases may replicate m6G-containing DNA with reasonable efficiency. Previous reports suggested that mammalian pol beta could not replicate m6G-containing DNA, but we find that pol beta can catalyze trans-lesion replication; however, the lesion must reside in the optimal context for pol beta activity, single- or short nucleotide gapped substrates. Primed single-stranded DNA templates, with or without template m6G, were poor substrates for pol beta as reported in earlier studies. In contrast, trans-lesion replication by bacteriophage T4 DNA polymerase was observed for primed single-stranded DNA templates. Replication of m6G-containing DNA by T4 DNA polymerase required the gp45 accessory protein that clamps the polymerase to the DNA template. The rate-limiting step in replicating m6G-containing DNAs by both DNA polymerases tested was incorporation of dTMP across from the lesion.     

Comparative sequence analysis of ribonucleases HII, III, II PH and D

Escherichia coli ribonucleases (RNases) HII, III, II, PH and D have been used to characterise new and known viral, bacterial, archaeal and eucaryotic sequences similar to these endo- (HII and III) and exoribonucleases (II, PH and D). Statistical models, hidden Markov models (HMMs), were created for the RNase HII, III, II and PH and D families as well as a double-stranded RNA binding domain present in RNase III. Results suggest that the RNase D family, which includes Werner syndrome protein and the 100 kDa antigenic component of the human polymyositis scleroderma (PMSCL) autoantigen, is a 3'-->5' exoribonuclease structurally and functionally related to the 3'-->5' exodeoxyribonuclease domain of DNA polymerases. Polynucleotide phosphorylases and the RNase PH family, which includes the 75 kDa PMSCL autoantigen, possess a common domain suggesting similar structures and mechanisms of action for these 3'-->5' phosphorolytic enzymes. Examination of HMM-generated multiple sequences alignments for each family suggest amino acids that may be important for their structure, substrate binding and/or catalysis.     

Residues at the carboxy terminus of T4 DNA polymerase are important determinants for interaction with the polymerase accessory proteins

Three T4 DNA polymerase accessory proteins (44P/62P and 45P) stimulate the polymerase (pol) activity and the 3'-5' exonuclease (exo) activity of T4 DNA polymerase (43P) on long, double-stranded DNA substrates. The 44P/62P "clamp loader" facilitates the binding of 45P, the "sliding clamp", to DNA that is primed for replication. Using a series of truncated 43P mutants, we identified a region at the extreme carboxy terminus of the DNA polymerase that is required for its interaction with accessory proteins. Truncation mutants of 43P lacking the carboxy-terminal 3, 6, or 11 residues retained full pol and exo activity on short synthetic primer-templates. However, the ability of the accessory proteins to enhance these activities on long double-stranded DNA templates was drastically reduced, and the extent of the reduction in activity was greater as more residues were deleted. One of the truncation mutants (N881), which had 17 residues removed from the carboxy terminus, showed reduced binding affinity and diminished pol activity but enhanced exo activity upon incubation with a small primer-template. The exo activity of the N881 mutant, on short, single-stranded DNA was unchanged, however, compared to the wild-type enzyme. These results are consistent with inferences drawn from the crystal structure of a DNA polymerase from a related T-even phage, RB69, where the carboxy-terminal 12 residues (equivalent to the 11 residues of 43P from phage T4) protrude from the thumb domain and are free to interact with complementary surfaces of the accessory proteins. The structural integrity of the thumb region in the N881 mutant is probably perturbed and could account for its reduced binding affinity and pol activity when incubated with short, double-stranded DNA substrates.     

Mutational analysis of the 3'-->5' proofreading exonuclease of Escherichia coli DNA polymerase III

The epsilon subunit of Escherichia coli DNA polymerase III holoenzyme, the enzyme primarily responsible for the duplication of the bacterial chromosome, is a 3'-->5' exonuclease that functions as a proofreader for polymerase errors. In addition, it plays an important structural role within the pol III core. To gain further insight into how epsilon performs these joint structural and catalytic functions, we have investigated a set of 20 newly isolated dnaQ mutator mutants. The mutator effects ranged from strong (700-8000-fold enhancement) to moderate (6-20-fold enhancement), reflecting the range of proofreading deficiencies. Complementation assays revealed most mutators to be partially or fully dominant, suggesting that they carried an exonucleolytic defect but retained binding to the pol III core subunits. One allele, containing a stop codon 3 amino acids from the C-terminal end of the protein, was fully recessive. Sequence analysis of the mutants revealed mutations in the Exo I, Exo II and recently proposed Exo IIIepsilon motifs, as well as in the intervening regions. Together, the data support the functional significance of the proposed motifs, presumably in catalysis, and suggest that the C-terminus of straightepsilon may be specifically involved in binding to the alpha (polymerase) subunit.     

The hyperthermophilic bacterium Thermotoga maritima has two different classes of family C DNA polymerases: evolutionary implications

Bacterial DNA polymerase III (family C DNA polymerase), the principal chromosomal replicative enzyme, is known to occur in at least three distinct forms which have provisionally been classified as class I ( Escherichia coli DNA pol C-type), class II ( Bacillus subtilis DNA pol C-type) and class III (cyanobacteria DNA pol C-type). We have identified two family C DNA polymerase sequences in the hyperthermophilic bacterium Thermotoga maritima. One DNA polymerase consisting of 842 amino acid residues and having a molecular weight of 97 213 belongs to class I. The other one, consisting of 1367 amino acid residues and having a molecular weight of 155 361, is a member of class II. Comparative sequence analyses suggest that the class II DNA polymerase is the principal DNA replicative enzyme of the microbe and that the class I DNA polymerase may be functionally inactive. A phylogenetic analysis using the class II enzyme indicates that T.maritima is closely related to the low G+C Gram-positive bacteria, in particular to Clostridium acetobutylicum, and mycoplasmas. These results are in conflict with 16S rRNA-based phylogenies, which placed T.maritima as one of the deepest branches of the bacterial tree.     

Eukaryotic DNA polymerases in DNA replication and DNA repair

DNA polymerases carry out a large variety of synthetic transactions during DNA replication, DNA recombination and DNA repair. Substrates for DNA polymerases vary from single nucleotide gaps to kilobase size gaps and from relatively simple gapped structures to complex replication forks in which two strands need to be replicated simultaneously. Consequently, one would expect the cell to have developed a well-defined set of DNA polymerases with each one uniquely adapted for a specific pathway. And to some degree this turns out to be the case. However, in addition we seem to find a large degree of cross-functionality of DNA polymerases in these different pathways. DNA polymerase alpha is almost exclusively required for the initiation of DNA replication and the priming of Okazaki fragments during elongation. In most organisms no specific repair role beyond that of checkpoint control has been assigned to this enzyme. DNA polymerase delta functions as a dimer and, therefore, may be responsible for both leading and lagging strand DNA replication. In addition, this enzyme is required for mismatch repair and, together with DNA polymerase zeta, for mutagenesis. The function of DNA polymerase epsilon in DNA replication may be restricted to that of Okazaki fragment maturation. In contrast, either polymerase delta or epsilon suffices for the repair of UV-induced damage. The role of DNA polymerase beta in base-excision repair is well established for mammalian systems, but in yeast, DNA polymerase delta appears to fulfill that function.     

Effect of immunization on the RNA polymerase activity of guinea-pig macrophage nuclei

Macrophages from peritoneal exudate contain three types of DNA dependent RNA polymerase. The activity of RNA polymerases in macrophages derived from normal animals is very low. Guinea-pigs were immunized by sheep red blood cells. The immunization enhanced the activity of the RNA polymerase of macrophages derived from peritoneal exudate. The RNA polymerase activity was tested after the solubilization and chromatographic resolution of the three types of polymerases with exogenous template. The results obtained indicated that the immunization enhances the levels of polymerase I and III 10 fold while the level of polymerase II increased 5 fold.     

Schizosaccharomyces pombe cdc20+ encodes DNA polymerase epsilon and is required for chromosomal replication but not for the S phase checkpoint

In fission yeast both DNA polymerase alpha (pol alpha) and delta (pol delta) are required for DNA chromosomal replication. Here we demonstrate that Schizosaccharomyces pombe cdc20+ encodes the catalytic subunit of DNA polymerase epsilon (pol epsilon) and that this enzyme is also required for DNA replication. Following a shift to the restrictive temperature, cdc20 temperature-sensitive mutant cells block at the onset of DNA replication, suggesting that cdc20+ is required early in S phase very near to the initiation step. In the budding yeast Saccharomyces cerevisiae, it has been reported that in addition to its proposed role in chromosomal replication, DNA pol epsilon (encoded by POL2) also functions directly as an S phase checkpoint sensor [Navas, T. A., Zhou, Z. & Elledge, S. J. (1995) Cell 80, 29-39]. We have investigated whether cdc20+ is required for the checkpoint control operating in fission yeast, and our data indicate that pol epsilon does not have a role as a checkpoint sensor coordinating S phase with mitosis. In contrast, germinating spores disrupted for the gene encoding pol alpha rapidly enter mitosis in the absence of DNA synthesis, suggesting that in the absence of pol alpha, normal coordination between S phase and mitosis is lost. We propose that the checkpoint signal operating in S phase depends on assembly of the replication initiation complex, and that this signal is generated prior to the elongation stage of DNA synthesis.     

phi29 DNA polymerase residue Ser122, a single-stranded DNA ligand for 3'-5' exonucleolysis, is required to interact with the terminal protein

Three amino acid residues highly conserved in most proofreading DNA polymerases, a phenylalanine contained in the Exo II motif and a serine and a leucine belonging to the S/TLx2h motif, were recently shown to be critical for 3'-5' exonucleolysis by acting as single-stranded DNA ligands (de Vega, M., Lázaro, J.M., Salas, M. and Blanco, L. (1998) J. Mol. Biol. 279, 807-822). In this paper, site-directed mutants at these three residues were used to analyze their functional importance for the synthetic activities of phi29 DNA polymerase, an enzyme able to start linear phi29 DNA replication using a terminal protein (TP) as primer. Mutations introduced at Phe65, Ser122, and Leu123 residues of phi29 DNA polymerase severely affected the replication capacity of the enzyme. Three mutants, F65S, S122T, and S122N, were strongly affected in their capacity to interact with a DNA primer/template structure, suggesting a dual role during both polymerization and proofreading. Interestingly, mutant S122N was not able to maintain a stable interaction with the TP primer, thus impeding the firsts steps (initiation and transition) of phi29 DNA replication. The involvement of Ser122 in the consecutive binding of TP and DNA is compatible with the finding that the TP/DNA polymerase heterodimer was not able to use a DNA primer/template structure. Assuming a structural conservation among the eukaryotic-type DNA polymerases, a model for the interactions of phi29 DNA polymerase with both TP and DNA primers is presented.     

A comparison of DNA polymerase alpha from untransformed and SV40-transformed human fibroblasts

1. DNA polymerase alpha (pol alpha) isolated from Simian virus 40 (SV40)-transformed cells showed more than 3-fold higher specific activity than pol alpha from normal cells. The enzymes from untransformed and transformed cells also differed in molecular size, thermolability, sensitivity to inhibitors and specificity of template-primer utilization. 2. Western analysis using anti-Tag to probe both a crude cell homogenate and partially purified pol alpha from SV40 transformed cells showed multiple immunoreactive bands with different molecular sizes. 3. While alpha polymerases from both normal and transformed cells exhibited tightly associated primase activity, they showed different DNA binding affinities. 4. These data suggest that T antigen binding to pol alpha alters the initiation of DNA replication and/or the function of pol alpha in SV40-transformed cells, and that pol alpha from SV40-transformed human fibroblasts have different catalytic subunit characteristics than pol alpha from untransformed cells.     

The plastid genome of the cryptophyte alga, Guillardia theta: complete sequence and conserved synteny groups confirm its common ancestry with red algae

Previously, we have purified and characterized DNA helicase III from the yeast Saccharomyces cerevisiae [Shimizu, K. and Sugino, A. (1993) J. Biol. Chem. 268, 9578-9584]. Here, we have further characterized DNA helicase III activity. It was found that the combined action of the helicase III, yeast DNA topoisomerase I (yTop I), and yeast RPA protein on a covalently closed, circular DNA generates a highly underwound DNA species that has been called form I* or form U. Furthermore, these underwound structures can be accessed by yeast DNA polymerase I (alpha)-primase to initiate DNA synthesis. These reactions mimic in vivo initiation of chromosomal DNA replication. In order to clone the gene encoding DNA helicase III, a partial amino acid sequence of the purified DNA helicase III polypeptide was determined. Using a mix oligonucleotides synthesized based on the amino acid sequence of the helicase, we cloned the gene encoding the helicase III and found it to be identical to YER176W (HEL1) on chromosome V. The amino acid sequence predicted from the nucleotide sequence of the gene has conserved DNA helicase domains that are highly homologous to those of DNA helicases required for DNA replication. However, complete deletion of the gene from the chromosome did not result in any growth defect, suggesting that the gene product is not required for DNA synthesis or that it is functionally substituted by other helicase(s). Furthermore, the deletion strain does not exhibit sensitivity to any DNA-damaging reagents, although it is hypersensitive to calcofluor white, hygromycin, and papulacandin.