Bacteriophage PRD1 DNA polymerase: evolution of DNA polymerases

A small lipid-containing bacteriophage PRD1 specifies its own DNA polymerase that utilizes terminal protein as a primer for DNA synthesis. The PRD1 DNA polymerase gene has been sequenced, and its amino acid sequence has been deduced. This protein-primed DNA polymerase consists of 553 amino acid residues with a calculated molecular weight of 63,300. Thus, it appears to be the smallest DNA polymerase ever isolated from prokaryotic cells. Comparison of the PRD1 DNA polymerase sequence with other DNA polymerase sequences that have been published yielded segmental but significant homologies. These results strongly suggest that many prokaryotic and eukaryotic DNA polymerase genes, regardless of size, have evolved from a common ancestral gene. The results further indicate that those DNA polymerases that use either an RNA or protein primer are related. We propose to classify DNA polymerases on the basis of their evolutionary relatedness.     

DNA polymerase I gene of Saccharomyces cerevisiae: nucleotide sequence, mapping of a temperature-sensitive mutation, and protein homology with other DNA polymerases.

A 5600-base pair segment spanning the coding region of the Saccharomyces cerevisiae DNA polymerase I gene was sequenced and found to contain an open reading frame of 1468 codons, corresponding to a polypeptide of Mr 166,794. A pol1 temperature-sensitive mutation, encoding a DNA-polymerase-primase complex with altered stability, has a single base-pair substitution that changes the glycine at position 493 to a positively charged arginine. Protein sequence comparison with other prokaryotic and eukaryotic DNA polymerases reveals three major regions of homology. This observation suggests that certain DNA polymerases might require the conservation of critical amino acid residues for activity.     

Cloning and expression of T4 DNA polymerase.

The structural gene coding for bacteriophage T4 DNA polymerase (gene 43) has been cloned into inducible plasmid vectors, which provide a source for obtaining large amounts of this enzyme after induction. The T4 DNA polymerase produced in this fashion was purified by an innovative three-step procedure and was fully active.     

DNA sequence analysis with a modified bacteriophage T7 DNA polymerase.

A chemically modified phage T7 DNA polymerase has three properties that make it ideal for DNA sequencing by the chain-termination method. The enzyme is highly processive, catalyzing the polymerization of thousands of nucleotides without dissociating. By virtue of the modification the 3' to 5' exonuclease activity is eliminated. The modified polymerase efficiently uses nucleotide analogs that increase the electrophoretic resolution of bands in gels. Consequently, dideoxynucleotide-terminated fragments have highly uniform radioactive intensity throughout the range of a few to thousands of nucleotides in length. There is virtually no background due to terminations at pause sites or secondary-structure impediments. Processive synthesis with dITP in place of dGTP eliminates band compressions, making possible the unambiguous determination of sequences from a single orientation.     

Fidelity of DNA polymerases in DNA amplification.

Denaturing gradient gel electrophoresis (DGGE) was used to separate and isolate the products of DNA amplification by polymerase chain reaction (PCR). The strategy permitted direct enumeration and identification of point mutations created by T4, modified T7, Klenow fragment of polymerase I, and Thermus aquaticus (Taq) DNA polymerases. Incorrectly synthesized sequences were separated from the wild type by DGGE as mutant/wild-type heteroduplexes and the heteroduplex fraction was used to calculate the average error rate (mutations per base duplication). The error rate induced in the 104-base-pair low-temperature melting domain of exon 3 of the human hypoxanthine/guanine phosphoribosyltransferase (HPRT) gene was approximately 3.4 x 10(-5) for modified T7, 1.3 x 10(-4) for Klenow fragment, and 2.1 x 10(-4) for Taq polymerases after a 10(6)-fold amplification. The error rate for T4 DNA polymerase was not more than 3 x 10(-6) error per base duplication. The predominant mutations were sequenced and found to be transitions of G.C to A.T for T4 and modified T7 DNA polymerases, and A.T to G.C for Taq polymerase. Klenow fragment induced both possible transitions and deletions of 2 and 4 base pairs.     

Homology between DNA polymerases of poxviruses, herpesviruses, and adenoviruses: nucleotide sequence of the vaccinia virus DNA polymerase gene.

A 5400-base-pair segment of the vaccinia virus genome was sequenced and an open reading frame of 938 codons was found precisely where the DNA polymerase had been mapped by transfer of a phosphonoacetate-resistance marker. A single nucleotide substitution changing glycine at position 347 to aspartic acid accounts for the drug resistance of the mutant vaccinia virus. The 5' end of the DNA polymerase mRNA was located 80 base pairs before the methionine codon initiating the open reading frame. Correspondence between the predicted Mr 108,577 polypeptide and the 110,000 purified enzyme indicates that little or no proteolytic processing occurs. Extensive homology, extending over 435 amino acids, was found upon comparing the DNA polymerase of vaccinia virus and DNA polymerase of Epstein-Barr virus. A highly conserved sequence of 14 amino acids in the carboxyl-terminal regions of the above DNA polymerases is also present at a similar location in adenovirus DNA polymerase. This structure, which is predicted to form a turn flanked by beta-pleated sheets, may form part of an essential binding or catalytic site that accounts for its presence in DNA polymerases of poxviruses, herpesviruses, and adenoviruses.     

Escherichia coli thioredoxin: a subunit of bacteriophage T7 DNA polymerase.

T7 DNA polymerase (DNA nucleotidyltransferase; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) is composed of an 84,000 dalton protein specified by the gene 5 of the phage and a 12,000 dalton protein (TsnC protein) specified by the tsnC gene of E. coli [Modrich, P. & Richardson, C. C. (1975) J. Biol. Chem. 250 5515-5522]. Both proteins are necessary for T7 DNA polymerase activity and for the replication of T7 DNA. The TsnC protein is identical to thioredoxin of E. coli by the following criteria: (1) Homogeneous preparations of both proteins have TsnC and thioredoxin activity. (2) Both proteins show similar stability to heat. (3) They have identical mobilities, corresponding to a molecular weight of 12,000, on polyacrylamide gels containing sodium dodecyl sulfate. (4) Their amino-acid compositions are indistinguishabe. (5) Antibody prepared against thioredoxin inhibits TsnC activity. (6) TsnC protein isolated from purified T7 DNA polymerase has thioredoxin activity. In addition, preparations of T7 DNA polymerase itself exhibit thioredoxin activity and are partially inhibited by antibody to thioredoxin.     

A ganciclovir-resistant clinical isolate of human cytomegalovirus exhibiting cross-resistance to other DNA polymerase inhibitors.

Clinical isolates of human cytomegalovirus (HCMV) were screened for susceptibility to ganciclovir by plaque-reduction assay and in situ ELISA. A pretreatment isolate of HCMV obtained from the bronchial brushing of a heart transplant recipient contained both ganciclovir-susceptible and -resistant virus. Ganciclovir-susceptible (P8) and -resistant (D16) strains were further isolated by plaque purification. Both strains phosphorylated ganciclovir at levels similar to the ganciclovir-susceptible strain AD169. D16 was also resistant to phosphonoformic acid and to (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)adenine and cytosine. These data suggest that the resistance of D16 to these drugs results from a mutation in the viral DNA polymerase gene.     

DNA nick processing by exonuclease and polymerase activities of bacteriophage T4 DNA polymerase accounts for acridine-induced mutation specificities in T4.

Acridine-induced frameshift mutagenesis in bacteriophage T4 has been shown to be dependent on T4 topoisomerase. In the absence of a functional T4 topoisomerase, in vivo acridine-induced mutagenesis is reduced to background levels. Further, the in vivo sites of acridine-induced deletions and duplications correlate precisely with in vitro sites of acridine-induced T4 topoisomerase cleavage. These correlations suggest that acridine-induced discontinuities introduced by topoisomerase could be processed into frameshift mutations. The induced mutations at these sites have a specific arrangement about the cleavage site. Deletions occur adjacent to the 3' end and duplications occur adjacent to the 5' end of the cleaved bond. It was proposed that at the nick, deletions could be produced by the 3'-->5' removal of bases by DNA polymerase-associated exonuclease and duplications could be produced by the 5'-->3' templated addition of bases. We have tested in vivo for T4 DNA polymerase involvement in nick processing, using T4 phage having DNA polymerases with altered ratios of exonuclease to polymerase activities. We predicted that the ratios of the deletion to duplication mutations induced by acridines in these polymerase mutant strains would reflect the altered exonuclease/polymerase ratios of the mutant T4 DNA polymerases. The results support this prediction, confirming that the two activities of the T4 DNA polymerase contribute to mutagenesis. The experiments show that the influence of T4 DNA polymerase in acridine-induced mutation specificities is due to its processing of acridine-induced 3'-hydroxyl ends to generate deletions and duplications by a mechanism that does not involve DNA slippage.     

The enzymological basis for resistance of herpesvirus DNA polymerase mutants to acyclovir: Relationship to the structure of α-like DNA polymerases

Acyclovir (ACV), like many antiviral drugs, is a nucleoside analog. In vitro, ACV triphosphate inhibits herpesvirus DNA polymerase by means of binding, incorporation into primer/template, and dead-end complex formation in the presence of the next deoxynucleoside triphosphate. However, it is not known whether this mechanism operates in vivo. To address this and other questions, we analyzed eight mutant polymerases encoded by drug-resistant viruses, each altered in a region conserved among α-like DNA polymerases. We measured K_m and k_cat values for dGTP and ACV triphosphate incorporation and K_i values of ACV triphosphate for dGTP incorporation for each mutant. Certain mutants showed increased K_m values for ACV triphosphate incorporation, suggesting a defect in inhibitor binding. Other mutants showed reduced k_cat values for ACV triphosphate incorporation, suggesting a defect in incorporation of inhibitor into DNA, while the rest of the mutants exhibited both altered k_m and k_cat values. In most cases, the fold increase in K_i of ACV triphosphate for dGTP incorporation relative to wild-type polymerase was similar to fold resistance conferred by the mutation in vivo; however, one mutation conferred a much greater increase in resistance than in K_i. The effects of mutations on enzyme kinetics could be explained by using a model of an α-like DNA polymerase active site bound to primer/template and inhibitor. The results have implications for mechanisms of action and resistance of antiviral nucleoside analogs in vivo, in particular for the importance of incorporation into DNA and for the functional roles of conserved regions of polymerases.     

Enzymatic initiation of DNA synthesis by yeast DNA polymerases.

Partially purified yeast RNA polymerases (RNA nucleotidyltransferases) initiate DNA synthesis by yeast DNA polymerase (DNA nucleotidyltransferase) I and to a lesser extent yeast DNA polymerase II in the replication of single-stranded DNA. The enzymatic initiation of DNA synthesis on phage fd DNA template occurs with dNTPs alone and is further stimulated by the presence of rNTPs in DNA polymerase I reactions. The presence of rNTPs has no effect on the RNA polymerase initiation of the DNA polymerase II reaction. RNA polymerases I and III are more efficient in initiation of DNA synthesis than RNA polymerase II. Analyses of the products of fd DNA replication show noncovalent linkage between the newly synthesized DNA and the template DNA, and covalent linkage between the newly synthesized RNA and DNA.     

Elongation of primed DNA templates by eukaryotic DNA polymerases.

The combined action of DNA polymerase alpha and DNA polymerase beta leads to the synthesis of full-length linear DNA strands with phi X174 DNA templates containing an RNA primer. The reaction can be carried out in two stages. In the first stage, DNA polymerase alpha catalyzes the synthesis of a chain that averaged 230 deoxynucleotides long and was covalently linked to the RNA primer. In the second stage, DNA polymerase beta elongates the DNA strand covalently attached to the RNA primer to full length. With DNA primers, DNA polymerase alpha catalyzes only limited deoxynucleotide addition whereas DNA polymerase beta alone elongates DNA primed templates to full length. DNA polymerase beta can also stimulate the synthesis of adenovirus DNA in vitro in the presence of a cytosol extract from adenovirus-infected cells. In all of these systems, dNMP incorporation catalyzed by DNA polymerase beta was sensitive to N-ethylmaleimide; however, this polymerase activity was resistant to N-ethylmaleimide with poly(rA) x (dT) as the primer template.     

Exchange of DNA polymerases at the replication fork of bacteriophage T7

T7 gene 5 DNA polymerase (gp5) and its processivity factor, Escherichia coli thioredoxin, together with the T7 gene 4 DNA helicase, catalyze strand displacement synthesis on duplex DNA processively (>17,000 nucleotides per binding event). The processive DNA synthesis is resistant to the addition of a DNA trap. However, when the polymerase-thioredoxin complex actively synthesizing DNA is challenged with excess DNA polymerase-thioredoxin exchange occurs readily. The exchange can be monitored by the use of a genetically altered T7 DNA polymerase (gp5-Y526F) in which tyrosine-526 is replaced with phenylalanine. DNA synthesis catalyzed by gp5-Y526F is resistant to inhibition by chain-terminating dideoxynucleotides because gp5-Y526F is deficient in the incorporation of these analogs relative to the wild-type enzyme. The exchange also occurs during coordinated DNA synthesis in which leading- and lagging-strand synthesis occur at the same rate. On ssDNA templates with the T7 DNA polymerase alone, such exchange is not evident, suggesting that free polymerase is first recruited to the replisome by means of T7 gene 4 helicase. The ability to exchange DNA polymerases within the replisome without affecting processivity provides advantages for fidelity as well as the cycling of the polymerase from a completed Okazaki fragment to a new primer on the lagging strand.     

DNA polymerase of bacteriophage T4 is an autogenous translational repressor.

In bacteriophage T4 the protein product of gene 43 (gp43) is a multifunctional DNA polymerase that is essential for replication of the phage genome. The protein harbors DNA-binding, deoxyribonucleotide-binding, DNA-synthesizing (polymerase) and 3'-exonucleolytic (editing) activities as well as a capacity to interact with several other T4-induced replication enzymes. In addition, the T4 gp43 is a repressor of its own synthesis in vivo. We show here that this protein is an autogenous repressor of translation, and we have localized its RNA-binding sequence (translational operator) to the translation initiation domain of gene 43 mRNA. This mechanism for regulation of T4 DNA polymerase expression underscores the ubiquity of translational repression in the control of T4 DNA replication. Many T4 DNA polymerase accessory proteins and nucleotide biosynthesis enzymes are regulated by the phage-induced translational repressor regA, while the T4 single-stranded DNA-binding protein (T4 gp32) is, like gp43, autogenously regulated at the translational level.     

A 5'' to 3'' exonuclease functionally interacts with calf DNA polymerase epsilon.

Analysis of fractions containing purified DNA polymerase epsilon from calf thymus has revealed the presence of a 5' to 3' exonuclease activity that is specific for a single strand of duplex DNA. This activity is capable of degrading a 3'-labeled oligonucleotide hybridized to M13mp18 DNA. When a second oligonucleotide primer is annealed 3 bases upstream, degradation of the downstream primer is strictly dependent on DNA synthesis from the upstream primer. Replacement of the downstream primer by an oligoribonucleotide of identical sequence results in a similar pattern of exonucleolytic activity. The activity has been highly purified and found to cosediment in glycerol gradients with a peptide of 56 kDa as judged by SDS/PAGE analysis. Effects of calf DNA polymerase alpha and delta on exonuclease activity are also observed but with differences in the pattern of products.     

Macromolecular crowding increases binding of DNA polymerase to DNA: an adaptive effect.

Macromolecular crowding extends the range of ionic conditions supporting high DNA polymerase reaction rates. Reactions tested were nick-translation and gap-filling by DNA polymerase I of Escherichia coli, nuclease and polymerase activities of the large fragment of that polymerase, and polymerization by the T4 DNA polymerase. For all of these reactions, high concentrations of nonspecific polymers increased enzymatic activity under otherwise inhibitory conditions resulting from relatively high ionic strength. The primary mechanism of the polymer effect seems to be to increase the binding of polymerase to DNA. We suggest that this effect on protein-DNA complexes is only one example of a general "metabolic buffering" action of crowded solutions on a variety of macromolecular interactions.