DNA polymerases β and λ and their roles in cell

Among the set of mammalian DNA polymerases, DNA polymerases belonging to the X and Y families have a special place. The majority of these enzymes are involved in repair, including base excision repair and non-homologous end joining. Some of them play a crucial role during the specific process which is referred to as translesion synthesis (TLS). TLS intends for the cell surviving during the replication of damaged DNA templates. Additionally, specific activities of TLS-polymerases have to be useful for repair of double-stranded clustered lesions: if the synthesis is proceeded via base excision repair process, the role of DNA polymerases β or λ will be important. In this review we discussed the biochemical properties and functional relevance of X family DNA polymerases β and λ.     

The peculiarities of repair DNA-polymerases in the loach embryogenesis

The current concept of eukaryotic DNA polymerases is considered, which are involved in nuclear DNA repair. The data are given on a new group of DNA polymerases that maintain the integrity of DNA structures without preliminary excision of damaged regions. A special attention is paid to specific features of the functioning of repair DNA polymerases in embryogenesis of the loach. A possible existence is discussed of the previously unknown pathway of DNA repair with participation of DNA polymerase delta as independent from the nuclear antigen of proliferating cells.     

DNA polymerases β and λ and their roles in DNA replication and repair

DNA-dependent DNA polymerases are the main enzymes that catalyze DNA replication. Higher eukaryotic cells have 19 DNA polymerases with strikingly different properties [1]. Mitochondrial DNA polymerase γ of the A family and most of the nuclear enzymes of the B family are high-fidelity DNA polymerases that are involved not only in genomic DNA replication but also in DNA repair. Among the other 15 proteins, DNA polymerases belonging to the X and Y families have a special place. The majority of these enzymes are also involved in repair, including base excision repair and nonhomologous end joining. Some of them play a specific role in replication of damaged DNA templates. This process is referred to as translesion synthesis (TLS). DNA polymerases β and λ, which belong to the X structural family, are polyfunctional enzymes; their properties and functions are discussed.     

Directed evolution of novel polymerases

DNA and RNA polymerases evolved to function in specific environments with specific substrates to propagate genetic information in all living organisms. The commercial availability of these polymerases has revolutionized the biotechnology industry, but for many applications native polymerases are limited by their stability or substrate recognition. Thus, there is great interest in the directed evolution of DNA and RNA polymerases to generate enzymes with novel, desired properties, such as thermal stability, resistance to inhibitors, and altered substrate specificity. Several screening and selection approaches have been developed, both in vivo and in vitro, and have been used to evolve polymerases with a variety of important activities. Both the techniques and the evolved polymerases are reviewed here, along with a comparison of the in vivo and in vitro approaches.     

Structural and functional relationships between prokaryotic and eukaryotic DNA polymerases.

The Bacillus subtilis phage luminal diameter 29 DNA polymerase, involved in protein-primed viral DNA replication, was inhibited by phosphonoacetic acid (PAA), a known inhibitor of alpha-like DNA polymerases, by decreasing the rate of elongation. Three highly conserved regions of amino acid homology, found in several viral alpha-like DNA polymerases and in the luminal diameter 29 DNA polymerase, one of them proposed to be the PAA binding site, were also found in the T4 DNA polymerase. This prokaryotic enzyme was highly sensitive to the drugs aphidicolin and the nucleotide analogues butylanilino dATP (BuAdATP) and butylphenyl dGTP (BuPdGTP), known to be specific inhibitors of eukaryotic alpha-like DNA polymerases. Two potential DNA polymerases from the linear plasmid pGKL1 from yeast and the S1 mitochondrial DNA from maize have been identified, based on the fact that they contain the three conserved regions of amino acid homology. Comparison of DNA polymerases from prokaryotic and eukaryotic origin showed extensive amino acid homology in addition to highly conserved domains. These findings reflect evolutionary relationships between hypothetically unrelated DNA polymerases.     

In vitro production and screening of DNA polymerase eta mutants for catalytic diversity

Mutant DNA polymerases have become an increasingly important tool in biotechnology. The ability to examine the activity and specific properties of enzymes has a crucial role in the characterization of the enzyme. We have developed several systems for characterizing DNA polymerases that combine random mutagenesis with in vivo selection systems. However in vivo screening systems for specific properties are sometimes unavailable. The ability to quickly screen for polymerase activity has many applications, including the identification of compounds that can inhibit polymerase activity, identifying the properties of newly discovered polymerases, and engineering new biological properties into existing polymerases. These applications can both expand the knowledge of the basic science of polymerases and can further industrial efforts to identify new drugs that specifically target polymerase activity. Here we present a high-throughput in vitro assay to select for active polymerases. We show the applicability of this assay by measuring the level of activity for a set of in vitro synthesized polymerase mutants and by screening for the incorporation of a fluorescent nucleotide analog by DNA polymerases.     

Mammalian proliferating cell nuclear antigen stimulates the processivity of two wheat embryo DNA polymerases.

Multiple DNA polymerases have been described in all organisms studied to date. Their specific functions are not easy to determine, except when powerful genetic and/or biochemical tools are available. However, the processivity of a DNA polymerase could reflect the physiological role of the enzyme. In this study, analogies between plant and animal DNA polymerases have been investigated by analyzing the size of the products synthesized by wheat DNA polymerases A, B, CI, and CII as a measure of their processivity. Thus, incubations have been carried out with poly(dA)-oligo(dT) as a template-primer under varying assay conditions. In the presence of MgCl2, DNA polymerase A was highly processive, whereas DNA polymerases B, CI, and CII synthesized much shorter products. With MnCl2 instead of MgCl2, DNA polymerase A was highly processive, DNA polymerases B and CII were moderately processive, and DNA polymerase CI remained strictly distributive. The effect of calf thymus proliferating cell nuclear antigen (PCNA) on wheat polymerases was studied as described for animal DNA polymerases. The high processivity of DNA polymerase A was PCNA independent, whereas both enzyme activity and processivity of wheat DNA polymerases B and CII were significantly stimulated by PCNA. On the other hand, DNA polymerase CI was not stimulated by PCNA and, like animal DNA polymerase beta, was distributive in all cases. From these results, we propose that wheat DNA polymerase A could correspond to a DNA polymerase alpha, DNA polymerases B and CII could correspond to the delta-like enzyme, and DNA polymerase CI could correspond to DNA polymerase beta.     

DNA polymerases beta and lambda, and their roles in the DNA replication and repair

One of the key stages of life of a cell is genome duplication. The main enzymes which lead this process are DNA-dependent DNA polymerases. At the moment, 19 DNA polymerases with striking properties are listed in the eukaryotic cells. Mitochondrial DNA polymerase gamma from A family and most of the nuclear enzymes from B family are high fidelity DNA polymerases which are participate in genome DNA replication process as well as in DNA repair. Among the other 1 5 proteins, the D N A polymerases belonging to the X and Y families have a special place. They participate in a different repair processes such as base excision repair and non-homologous end joining. Moreover, some of them play a specific role in the replication of the damaged DNA templates. This process is referred as translesion synthesis or TLS. The DNA polymerases beta and lambda members of X family are enclosed in polyfunctional enzymes, and their properties and functions will be discussed in this review.     

Butylanilinouracil: a selective inhibitor of HeLa cell DNA synthesis and HeLa cell DNA polymerase alpha.

A series of 6-anilinouracils, dGTP analogues which selectively inhibit specific bacterial DNA polymerases, were examined for their capacity to inhibit purified DNA polymerases from HeLa cells. The p-n-butyl derivative (BuAU) was found to inhibit DNA polymerase alpha with a Ki of approximately 60 microM. The inhibitory effect of BuAU was reversed specifically by dGTP and was observed only for DNA polymerase alpha; polymerases beta and lambda were not inhibited by drug at concentrations as high as 1 mM. BuAU also was inhibitory in vivo in HeLa cell culture; at 100 microM it reversibly inhibited cell division and selectively depressed DNA synthesis. The results of these studies indicate that BuAU is an inhibitor with considerable potential as a specific probe with which to dissect the structure of mammalian polymerase alpha and its putative role in cellular DNA replication.     

The evolution of DNA polymerases with novel activities

DNA and RNA polymerases have evolved in nature to function in specific environments with specific substrates. Thus, although the commercial availability of these enzymes has revolutionized the biotechnology industry, their applications are limited. The availability of polymerases that have unnatural properties would be of even greater utility. Towards this goal, several activity-based screening and selection approaches have been developed. Using these techniques, polymerases that synthesize a variety of different polymers, including those containing 2'-O-methyl-modified nucleotides or unnatural base pairs, have been evolved. These results suggest that polymerases tailored for any specific application could soon be available.     

Inhibitors acting on Nucleic Acid Synthesis in an Oncogenic RNA Virus

IN infection with an oncogenic RNA virus, synthesis of viral RNA seems to be catalysed by an RNA dependent DNA polymerase in the host cell^1–4. Several specific inhibitors of viral DNA polymerases have been found^5–7 and Spiegelman⁸ has shown that the activity of viral enzymes depends strongly on the chemical composition of the template. We report here first a new highly specific poison of the Rauscher murine leukaemia virus (RMLV) DNA polymerases; second, several inactivators of the RNA and DNA template involved in the RMLV enzyme systems; and third, the action of actinomycin D on viral DNA polymerases and on host DNA/RNA polymerase. The results are discussed with respect to the influence of actinomycin D on virus multiplication.     

Analysis of Rev1p and Pol zeta in mitochondrial mutagenesis suggests an alternative pathway of damage tolerance

Ultraviolet light is a potent DNA damaging agent that induces bulky lesions in DNA which block the replicative polymerases. In order to ensure continued DNA replication and cell viability, specialized translesion polymerases bypass these lesions at the expense of introducing mutations in the nascent DNA strand. A recent study has shown that the N-terminal sequences of the nuclear translesion polymerases Rev1p and Pol zeta can direct GFP to the mitochondrial compartment of Saccharomyces cerevisiae. We have investigated the role of these polymerases in mitochondrial mutagenesis. Our analysis of mitochondrial DNA point mutations, microsatellite instability, and the spectra of mitochondrial mutations indicate that these translesion polymerases function in a less mutagenic pathway in the mitochondrial compartment than they do in the nucleus. Mitochondrial phenotypes resulting from the loss of Rev1p and Pol zeta suggest that although these polymerases are responsible for the majority of mitochondrial frameshift mutations, they do not greatly contribute to mitochondrial DNA point mutations. Analysis of spontaneous mitochondrial DNA point mutations suggests that Pol zeta may play a role in general mitochondrial DNA maintenance. In addition, we observe a 20-fold increase in UV-induced mitochondrial DNA point mutations in rev deficient strains. Our data provides evidence for an alternative damage tolerance pathway that is specific to the mitochondrial compartment.     

Specific Features of Repair DNA-Polymerases in Embryogenesis of the Loach

The current concept of eukaryotic DNA polymerases is considered, which are involved in nuclear DNA repair. The data are given on a new group of DNA polymerases that maintain the integrity of DNA structures without preliminary excision of damaged regions. A special attention is paid to specific features of the functioning of repair DNA polymerases in embryogenesis of the loach. A possible existence is discussed of the previously unknown pathway of DNA repair with participation of DNA polymerase as independent from the nuclear antigen of proliferating cells.     

Cell cycle dependent activities of DNA polymerases alpha and delta in Chinese hamster ovary cells

The activities of DNA polymerases alpha and delta, in extracts from Chinese hamster ovary (CHO) cells, were assayed in order to determine whether these polymerases are regulated during the cell cycle. An exponential population of CHO cells was separated into enriched populations of G-1, S, and G-2/M phases of cell cycle by centrifugal elutriation. Total cell homogenates from each population were assayed for DNA polymerase activity by measuring labeled nucleotide incorporation into the exogenous templates oligo(dT).poly(dA) and DNase I activated calf thymus DNA. In these experiments, specific DNA polymerase inhibitors were added to assays of the cellular extracts to allow for the independent measurement of activities of DNA polymerases alpha and delta. Comparisons of total DNA polymerase activity from cellular extracts, sampled from each portion of the cell cycle, demonstrated no significant change with respect to the concentration of total protein. However, results indicate that the activity of DNA polymerase delta increases with respect to that of DNA polymerase alpha in the G-2/M portion of the cell cycle. This difference in relative activities of DNA polymerases alpha and delta suggests a coordinate regulation of a specific species of DNA polymerase during the cell cycle.     

On/off switch mediated by Exo+ polymerases: experimental analysis for its physiological and technological implications

The potential physiological role and technological application of the premature termination of DNA polymerization through the off-switch of exo+ polymerases were studied using 3' phosphorothioate-modified or unmodified primers with single base mismatch distal to the 3' terminus. With exonuclease-digestible unmodified primers, a gradient premature termination of DNA polymerization was observed when amplified with exo+ polymerases. With 3' allele specific phosphorothioate-modified primers, an efficient off-switch effect occurred in the discrimination of a single nucleotide polymorphism when directly using genomic DNA. Clearly, the off-switch of exo+ polymerases is useful in biomedical research.     

Computational Investigation of Locked Nucleic Acid (LNA) Nucleotides in the Active Sites of DNA Polymerases by Molecular Docking Simulations

Aptamers constitute a potential class of therapeutic molecules typically selected from a large pool of oligonucleotides against a specific target. With a scope of developing unique shorter aptamers with very high biostability and affinity, locked nucleic acid (LNA) nucleotides have been investigated as a substrate for various polymerases. Various reports showed that some thermophilic B-family DNA polymerases, particularly KOD and Phusion DNA polymerases, accepted LNA-nucleoside 5′-triphosphates as substrates. In this study, we investigated the docking of LNA nucleotides in the active sites of RB69 and KOD DNA polymerases by molecular docking simulations. The study revealed that the incoming LNA-TTP is bound in the active site of the RB69 and KOD DNA polymerases in a manner similar to that seen in the case of dTTP, and with LNA structure, there is no other option than the locked C3′-endo conformation which in fact helps better orienting within the active site.     

Phi29 DNA polymerase residues Tyr59, His61 and Phe69 of the highly conserved ExoII motif are essential for interaction with the terminal protein

Phage Φ29 encodes a DNA-dependent DNA polymerase belonging to the eukaryotic-type (family B) subgroup of DNA polymerases that use a protein as the primer for initiation of DNA synthesis. In one of the most important motifs present in the 3′→5′ exonucleolytic domain of proofreading DNA polymerases, the ExoII motif, Φ29 DNA polymerase contains three amino acid residues, Y59, H61 and F69, which are highly conserved among most proofreading DNA polymerases. These residues have recently been shown to be involved in proper stabilization of the primer terminus at the 3′→5′ exonuclease active site. Here we investigate by means of site-directed mutagenesis the role of these three residues in reactions that are specific for DNA polymerases utilizing a protein-primed DNA replication mechanism. Mutations introduced at residues Y59, H61 and F69 severely affected the protein-primed replication capacity of Φ29 DNA polymerase. For four of the mutants, namely Y59L, H61L, H61R and F69S, interaction with the terminal protein was affected, leading to few initiation and transition products. These findings, together with the specific conservation of Y59, H61 and F69 among DNA polymerases belonging to the protein-primed subgroup, strongly suggest a functional role of these amino acid residues in the DNA polymerase–terminal protein interaction.     

DNA polymerases alpha beta and gamma in inherited diseases affecting DNA repair.

Fibroblasts derived from patients with diseases affecting DNA repair processes, such as Xeroderma Pigmentosum (classical and variant), Fanconi's anemia, Bloom's syndrome, Ataxia Telangiectasica, Progeria and Werner's syndrome, were assayed for the three DNA polymerases. The specific activities of these enzymes were found within the limits observed in normal human fibroblasts. Also the sedimentation properties of the three polymerases were unaltered.     

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 α 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 δ 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 ζ, for mutagenesis. The function of DNA polymerase ɛ in DNA replication may be restricted to that of Okazaki fragment maturation. In contrast, either polymerase δ or ɛ suffices for the repair of UV-induced damage. The role of DNA polymerase β in base-excision repair is well established for mammalian systems, but in yeast, DNA polymerase δ appears to fullfill that function. Received: 20 April 1998 / Accepted: 8 May 1998