DNA polymerases in midgestation mouse embryo, trophoblast, and decidua

The DNA polymerases of midgestation mouse embryo, trophoblast, and decidua have been examined. A low molecular weight, nuclear. DNA-dependent polymerase (D-DNA polymerase) and a higher molecular weight cytoplasmic enzyme were found in all three cell types. A DNA polymerase which utilized the poly(A) strand of oligo(dT) · poly(A) as template (R-DNA polymerase) was also found in the three cell types. This enzyme was present both in the nucleus and the cytoplasm. All enzyme levels were highest in the rapidly dividing embryonic cells, substantially lower in the DNA replicating but nondividing trophoblast cells, and lowest in the nonreplicating, nondividing decidual cells. Our observations are consistent with the idea that the nuclear and cytoplasmic D-DNA polymerases are under coordinate control. The relationship of these enzymes to DNA synthesis in vivo is discussed.     

Inhibition of eucaryotic DNA polymerases by phosphonoacetate and phosphonoformate

Phosphonoacetate was found to be an inhibitor of the DNA polymerase α from three human cells, HeLa, Wi-38, and phytohemagglutinin-stimulated lymphocytes. The inhibition patterns were determined. The apparent inhibition constants (K_ii) were about 30 μm. Thus the DNA polymerase α is 15 to 30 times less sensitive to Phosphonoacetate than the herpesvirus-induced DNA polymerase. The DNA polymerase α from Chinese hamster ovary cells and calf thymus was also inhibited. The DNA polymerases β and γ from the eucaryotic cells were relatively insensitive to phosphonoacetate. The sensitivity of the DNA polymerase α and the relative insensitivity of the DNA polymerase β and γ appeared to be general characteristics of the vertebrate polymerases, DNA polymerases from two other eucaryotic cells, yeast DNA polymerase A and B and tobacco cell DNA polymerase, were inhibited by phosphonoacetate, and to about the same extent as the α-polymerases. Fourteen phosphonate analogs were examined for inhibition of the HeLa DNA polymerase α. Only one, phosphonoformate, was an inhibitor. The mechanism of inhibition for phosphonoformate was analogous to that for phosphonoacetate.     

The relationship between two murine DNA-dependent DNA polymerases from the cytosol and the low molecular weight DNA polymerase.

After aqueous subcellular fractionation and partial purification by phosphocellulose chromatography, murine cells are found to contain a low molecular weight DNA-dependent DNA polymerase (beta) in the nuclear fraction and two distinguishable DNA-dependent DNA polymerases (C-I and C-II) in the cytosol. Both C-I and C-II are found in testis, liver, and regenerating liver; the amount of C-I being several fold increased in the regenerating liver and in immature testis. C-I and C-II are distinguishable by the criteria of salt sensitivity, inhibition by single-stranded DNA, elution from phosphocellulose, inhibition by 0.3 mM N-ethylmaleimide, template preference, and sedimentation coefficient. C-II is dissociated by 0.25 M KC1 to an active form of DNA polymerase of sedimentation coefficient 3.5 S while C-I is not dissociated, maintaining its sedimentation coefficient of 7.2 S. Many similar chemical and physical properties of C-II and the low molecular weight nuclear DNA polymerase (beta) suggest that C-II may represent an aggregate state of beta monomers, The size, reaction properties and the increase in enzyme activity under conditions of rapid cellular proliferation suggest C-I is analogous to the alpha DNA polymerase.     

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.     

DNA polymerases of rat liver. Partial characterization and effect of various inhibitors.

Three distinct DNA-dependent DNA polymerase activities have been partially purified from normal rat liver. Soluble activities are separable into two distinct fractions (P1 and P2) by phosphocellulose chromatography. A low-molecular-weight DNA polymerase was isolated from purified nuclei. The enzymes were characterized according to chromatographic and sedimentation behavior, enzymological properties, and response to various inhibitors. The results indicate that fraction P1 corresponds to the high-molecular-weight enzyme and suggest that polymerase P2 may be derived from partial dissociation of the high-molecular-weight enzyme. The molecular weight of polymerase P1 was estimated to be about 250 000 by Sephadex column chromatography. Both fraction P2 and nuclear DNA polymerase appeared to be low-molecular-weight enzymes. However, the molecular size of these activities was apparently different. The estimated molecular weights of nuclear and P2 enzyme are about 40 000 and 25 000, respectively. As with the nuclear enzyme, polymerase P2 (but not P1) appeared to be free of detectable exonuclease activity. All of these polymerases showed a marked preference for initiated polydeoxyribonucleotide templates. The rat liver polymerases differed in their ability to use poly[d(A-T)-A1 primer-template, as is shown by the ratios of their activity with this synthetic polymer to that with activated DNA: 0.5, 2.75, and 1.34 for P1, P2, and nuclear polymerase, respectively. Denatured DNA was a poor template for both enzymes P1 and P2, but it was inert as template for the nuclear enzyme. Although each of these polymerases required all four deoxynucleoside triphosphates for maximal activity, they catalyzed a high rate of synthesis in the absence of one or more deoxynucleoside triphosphates. Such a 'limited' synthesis was much more extensive for polymerase P2 and nuclear enzyme than for P1 was the most sensitive of the three to sulphydryl reagents, ehtidium bromide, heparin, and single-stranded DNA. The responses of P2 and nuclear enzymes to various inhibitors were very similar. However, these two enzymes respond differently to heat and high ionic strength.     

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.     

Nuclear DNA polymerases and the HeLa cell cycle.

Purified nuclei of HeLa S3 cells contain two DNA-dependent DNA polymerases that have distinct physical and enzymatic properties. We have investigated the variations in their activity during the cell cycle of a synchronized culture. Cells were synchronized by a double thymidine block, harvested at various phases of the cycle, and the two DNA polymerases were purified partially by DEAE-cellulose and phosphocellulose chromatography. The activity of DNA polymerase I (low molecular weight, N-ethylmaleimide-insensitive) remains essentially constant throughout the cycle. The activity of DNA polymerase II (high molecular weight, N-ethylmaleimide-sensitive), however, increases during G1 to mid-S and declines, 7- to 10-fold between late-S and G2. Addition of cycloheximide (60 mug/ml) to cultures 12 hours after the release from thymidine block abolishes the rise in the activity of DNA polymerase II. Cycloheximide also reduced the activity of DNA polymerase I by 60%. Addition of hydroxyurea (1mM) at 1 hour after release has no effect on the activity of either enzyme. We conclude that in HeLa cells, DNA polymerase I and II are distinct enzymes, that DNA polymerase II probably functions in DNA replication and is probably induced in response to stimuli for DNA biosynthesis.     

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.     

The DNA polymerases of Leishmania mexicana

Two previously isolated DNA polymerases from the parasitic protozoan Leishmania mexicana were further characterized by exposure to inhibitors of mammalian DNA polymerases. DNA polymerase A, a high molecular mass enzyme, and DNA polymerase B, a beta-like DNA polymerase were compared to each other and to their mammalian counterparts regarding pH optimum, utilization of templates, and response to various inhibitors and ionic strengths. The results suggest that the DNA polymerases from L. mexicana differ from the host enzymes and may offer a target for chemotherapeutic intervention.     

Multiple functions of DNA polymerases

The primary role of DNA polymerases is to accurately and efficiently replicate the genome in order to ensure the maintenance of the genetic information and its faithful transmission through generations. This is not a simple task considering the size of the genome and its constant exposure to endogenous and environmental DNA damaging agents. Thus, a number of DNA repair pathways operate in cells to protect the integrity of the genome. In addition to their role in replication, DNA polymerases play a central role in most of these pathways. Given the multitude and the complexity of DNA transactions that depend on DNA polymerase activity, it is not surprising that cells in all organisms contain multiple highly specialized DNA polymerases, the majority of which have only recently been discovered. Five DNA polymerases are now recognized in Escherichia coli, 8 in Saccharomyces cerevisiae, and at least 15 in humans. While polymerases in bacteria, yeast and mammalian cells have been extensively studied much less is known about their counterparts in plants. For example, the plant model organism Arabidopsis thaliana is thought to contain 12 DNA polymerases, whose functions are mostly unknown. Here we review the properties and functions of DNA polymerases focusing on yeast and mammalian cells but paying special attention to the plant enzymes and the special circumstances of replication and repair in plant cells.     

Fidelity of mammalian DNA replication and replicative DNA polymerases.

Current models suggest that two or more DNA polymerases may be required for high-fidelity semiconservative DNA replication in eukaryotic cells. In the present study, we directly compare the fidelity of SV40 origin-dependent DNA replication in human cell extracts to the fidelity of mammalian DNA polymerases alpha, delta, and epsilon using lacZ alpha of M13mp2 as a reporter gene. Their fidelity, in decreasing order, is replication greater than or equal to pol epsilon greater than pol delta greater than pol alpha. DNA sequence analysis of mutants derived from extract reactions suggests that replication is accurate when considering single-base substitutions, single-base frameshifts, and larger deletions. The exonuclease-containing calf thymus DNA polymerase epsilon is also highly accurate. When high concentrations of deoxynucleoside triphosphates and deoxyguanosine monophosphate are included in the pol epsilon reaction, both base substitution and frameshift error rates increase. This response suggests that exonucleolytic proofreading contributes to the high base substitution and frameshift fidelity. Exonuclease-containing calf thymus DNA polymerase delta, which requires proliferating cell nuclear antigen for efficient synthesis, is significantly less accurate than pol epsilon. In contrast to pol epsilon, pol delta generates errors during synthesis at a relatively modest concentration of deoxynucleoside triphosphates (100 microM), and the error rate did not increase upon addition of adenosine monophosphate. Thus, we are as yet unable to demonstrate that exonucleolytic proofreading contributes to accuracy during synthesis by DNA polymerase delta. The four-subunit DNA polymerase alpha-primase complex from both HeLa cells and calf thymus is the least accurate replicative polymerase. Fidelity is similar whether the enzyme is assayed immediately after purification or after being stored frozen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activities of DNA polymerases and RNA polymerases detected in situ in growing and differentiating cells of root cortex.

Activities of DNA polymerases and RNA polymerases were studied by autoradiographic methods in growing and differentiating root cortex cells of Zea mays - a species in which endomitosis occurs - and Tulipa kaufmanniana - in which this process does not occur. In Tulipa kaufmanniana, the highest activity of DNA polymerase appears in the nuclei of meristematic zone during the S phase of the cell cycle. In Zea mays, endomitotic replication of DNA occurs in all growth and differentiation zones and the activity of DNA polymerase in the nuclei is similar to that in the meristematic zone. In both species, nuclear RNA synthesis, measured with 3H uridine incorporation, is highest in the meristematic zone and declines steadily with development. Activity of nuclear RNA polymerase is present in all developmental zones in both species and is similar to that in the meristematic zone. 3H uridine incorporation into nucleoli decreases markedly in both species, whereas the activity of nucleolar RNA polymerase remains at a high level in all root segments in Zea mays and decreases slightly in Tulipa kaufmanniana. It is argued that the differences between the incorporation of 3H uridine and that or 3H UMP may be caused by a reduction of the pool of endogenous ribonucleoside triphosphates. Marked activities of DNA polymerase and RNA polymerase in cytoplasm are possibly related to the growth and division of plastids and mitochondria.     

DNA Polymerases from Human Cells

Nuclei of HeLa cells contain two separable DNA polymerases which have different properties and cytoplasm contains a single DNA polymerase activity similar to one of the nuclear DNA polymerases. The same distribution of DNA polymerases has been found in the nucleus and cytoplasm of WI-38 cells, a normal diploid human cell line.     

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.     

Gene duplications in evolution of archaeal family B DNA polymerases.

All archaeal DNA-dependent DNA polymerases sequenced to date are homologous to family B DNA polymerases from eukaryotes and eubacteria. Presently, representatives of the euryarchaeote division of archaea appear to have a single family B DNA polymerase, whereas two crenarchaeotes, Pyrodictium occultum and Sulfolobus solfataricus, each possess two family B DNA polymerases. We have found the gene for yet a third family B DNA polymerase, designated B3, in the crenarchaeote S. solfataricus P2. The encoded protein is highly divergent at the amino acid level from the previously characterized family B polymerases in S. solfataricus P2 and contains a number of nonconserved amino acid substitutions in catalytic domains. We have cloned and sequenced the ortholog of this gene from the closely related Sulfolobus shibatae. It is also highly divergent from other archaeal family B DNA polymerases and, surprisingly, from the S. solfataricus B3 ortholog. Phylogenetic analysis using all available archaeal family B DNA polymerases suggests that the S. solfataricus P2 B3 and S. shibatae B3 paralogs are related to one of the two DNA polymerases of P. occultum. These sequences are members of a group which includes all euryarchaeote family B homologs, while the remaining crenarchaeote sequences form another distinct group. Archaeal family B DNA polymerases together constitute a monophyletic subfamily whose evolution has been characterized by a number of gene duplication events.     

Sequential assembly of translesion DNA polymerases at UV-induced DNA damage sites

In response to DNA damage such as from UV irradiation, mammalian Y-family translesion synthesis (TLS) polymerases Polη and Rev1 colocalize with proliferating cell nuclear antigen at nuclear foci, presumably representing stalled replication sites. However, it is unclear whether the localization of one polymerase is dependent on another. Furthermore, there is no report on the in vivo characterization of the Rev3 catalytic subunit of the B-family TLS polymerase Polζ. Here we describe the detection of endogenous human Polη, Rev1, and Rev3 by immunocytochemistry using existing or newly created antibodies, as well as various means of inhibiting their expression, which allows us to examine the dynamics of endogenous TLS polymerases in response to UV irradiation. It is found that Rev1 and Polη are independently recruited to the nuclear foci, whereas the Rev3 nuclear focus formation requires Rev1 but not Polη. In contrast, neither Rev1 nor Polη recruitment requires Rev3. To further support these conclusions, we find that simultaneous suppression of Polη and Rev3 results in an additive cellular sensitivity to UV irradiation. These observations suggest a cooperative and sequential assembly of TLS polymerases in response to DNA damage. They also support and extend the current polymerase switch model.     

Three DNA polymerases of rat ascites hepatoma cells: properties of the enzymes and effect of RNA synthesis on the reactions.

Three different DNA polymerases have been isolated from rat ascites hepatoma cells [1--3]. The molecular weight of a DNA polymerase (polymerase C) purified from the soluble fraction of the cells was estimated to be 142 000 by sedimentation on a sucrose gradient, while the molecular weights of two DNA polymerases (polymerase P-1 and P-2) purified from nuclear membrane-chromatin fraction were estimated to be 117 000 and 44 000, respectively, by the same method. Under certain conditions, the poly (dT) strand of poly[(dA)-(dT)] was copied well by the polymerases, especially by the nuclear polymerases. Poly (dC) was a good template for the high molecular weight DNA polymerases C and P-1, but poly(dT) and poly(dA) were not effective templates. By addition of complementary oligoribonucleotides, the single-stranded deoxypolymers were copied by the high molecular weight polymerases C and P-1. When single-stranded fd phage DNA was used as template, the polymerization reactions by the high molecular weight polymerases were stimulated by the concomitant synthesis of RNA. This indicates that the oligoribonucleotide acts as a primer in these reactions.     

DNA polymerases alpha and delta are immunologically and structurally distinct

The relationship between DNA polymerases alpha and delta are evaluated immunologically by monoclonal antibody specifically against DNA polymerase alpha and murine polyclonal antiserum against calf thymus DNA polymerase delta. DNA polymerases alpha and delta are found to be immunologically distinct. The structural relationship between the proliferating cell nuclear antigen (PCNA)-dependent calf DNA polymerase delta and DNA polymerase alpha from human and calf was analyzed by two-dimensional tryptic peptide mapping of the catalytic polypeptides. The results demonstrate that the catalytic polypeptides of the PCNA-dependent calf polymerase delta and DNA polymerase alpha are distinct, unrelated, and do not share any common structural determinants. The immunological and structural relationship between a recently identified PCNA-independent form of DNA polymerase delta from HeLa cells was also assessed. This PCNA-independent human polymerase delta was found to be immunologically unrelated to human polymerase alpha but to share some immunological and structural determinants with the PCNA-dependent calf thymus polymerase delta.