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PubMed2000

Evidence from mutational specificity studies that yeast DNA polymerases delta and epsilon replicate different DNA strands at an intracellular replication fork.

Although polymerases delta and epsilon are required for DNA replication in eukaryotic cells, whether each polymerase functions on a separate template strand remains an open question. To begin examining the relative intracellular roles of the two polymerases, we used a plasmid-borne yeast tRNA gene and yeast strains that are mutators due to the elimination of proofreading by DNA polymerases delta or epsilon. Inversion of the tRNA gene to change the sequence of the leading and lagging strand templates altered the specificities of both mutator polymerases, but in opposite directions. That is, the specificity of the polymerase delta mutator with the tRNA gene in one orientation bore similarities to the specificity of the polymerase epsilon mutator with the tRNA gene in the other orientation, and vice versa. We also obtained results consistent with gene orientation having a minor influence on mismatch correction of replication errors occurring in a wild-type strain. However, the data suggest that neither this effect nor differential replication fidelity was responsible for the mutational specificity changes observed in the proofreading-deficient mutants upon gene inversion. Collectively, the data argue that polymerases delta and epsilon each encounter a different template sequence upon inversion of the tRNA gene, and so replicate opposite strands at the plasmid DNA replication fork.

Alleles↗
PubMed1996

Ligation of portal vein branch induces DNA polymerases alpha, delta, and epsilon in nonligated lobes.

Ligation of a portal vein branch supplying 70% of the rat liver causes compensatory hypertrophy of the nonligated hepatic lobes with concomitant atrophy of the ligated lobes. To elucidate the mechanism of this response, the induction of the replication enzymes DNA polymerases alpha, delta, epsilon, as well as proliferating cell nuclear antigen (PCNA), were investigated in nonligated lobes after portal branch ligation. The induction patterns were compared with the well studied liver regeneration after 70% partial hepatectomy. DNA polymerases alpha, delta, and epsilon in the liver were extracted with 5 mM KCl (low-salt extract), then with 600 mM KCl (high-salt extract). DNA polymerases alpha, delta, and epsilon in low-salt extract were partially separated on a hydroxyapatite column and quantified. All enzyme activities in the nonligated lobes started to increase within 24 hr and reached maximum levels by 48 hr after portal branch ligation. These patterns were quite similar to those obtained with the remnant liver after partial hepatectomy. In low-salt extract, DNA polymerase delta and epsilon were prominent, while, in high-salt extract, largely DNA polymerases alpha and some activity of epsilon were recovered. PCNA was also induced after both portal branch ligation and partial hepatectomy, reaching maximum levels at 48 hr. From the similar changes in DNA polymerases and PCNA, our data indicate that portal branch ligation induces hepatocyte proliferation in the nonligated lobes in a way similar to partial hepatectomy.

Animals↗
PubMed1990

Fidelity of animal cell DNA polymerases alpha and delta and of a human DNA replication complex.

We are investigating the mechanisms by which mutations are produced or avoided during DNA synthesis. Using in vitro fidelity assays, we have defined the error frequency and mutational specificity of the replicative animal cell DNA polymerases (alpha and delta). With DNA polymerase alpha or the four-subunit DNA polymerase alpha-DNA primase complex, neither of which contains detectable associated exonuclease activity, the fidelity of the polymerization step is low relative to spontaneous mutation rates in vivo. DNA polymerase delta is much more accurate, partly due to proofreading by the 3'----5' exonuclease activity associated with this polymerase. These fidelity studies have been extended to the replication apparatus present in extracts of human HeLa cells. The replication complex is highly accurate, suggesting that additional fidelity components are operating in the extract during bidirectional, semiconservative replication of double-stranded DNA. Nevertheless, in highly sensitive reversion assays, base substitution errors can be readily detected at frequencies greater than the estimated rate of spontaneous mutation in vivo. This suggests that fidelity components may be missing and/or that human cells depend heavily on postreplicative repair processes to correct replication errors.

Animals↗
PubMed1995

DNA polymerases alpha, delta, and epsilon of Novikoff hepatoma cells differ from those of normal rat liver in physicochemical and catalytic properties.

To investigate whether DNA replication in malignant cells deviates from that of normal cells we compared DNA polymerases alpha, delta, and epsilon from normal rat liver to the enzymes from fast-growing (malignant) Novikoff hepatoma cells. DNA polymerases were purified 300-fold by three chromatographic steps. Characterization included measurement of physicochemical constants (including sedimentation coefficients, diffusion coefficients, calculation of relative molecular masses), quantitation of catalytic activities using specific DNA primer templates (Km values) and inhibitors (Ki values), and identification of polypeptides which are strongly associated with DNA polymerases. Comparison of physicochemical and catalytic properties of DNA polymerases from both sources revealed similarities but also some important differences. DNA primase associated with DNA polymerase alpha, and 3'-5' exonuclease accompanying DNA polymerases delta and epsilon had similar activities. In contrast, the DNA-binding domain of DNA polymerases alpha and epsilon from hepatoma cells was altered since Km values, determined with the specific primer templates gapped calf thymus DNA and poly(dA.dT), were higher. Furthermore, sedimentation and diffusion coefficients, Stokes' radii, and frictional coefficient ratios of DNA polymerases alpha and epsilon from malignant cells significantly deviated. In addition, when the dNTP-binding sites were probed with specific inhibitors (aphidicolin, butylphenyl-dGTP, carbonyldiphosphonate, and dideoxy-TTP), significantly lower Ki values were obtained for the polymerases from Novikoff cells indicating lower affinity of the dNTP binding site to deoxyribonucleoside 5'-triphosphates. Altered catalytic and molecular properties are possibly a consequence of malignant transformation. It is to be expected that similar changes occur in DNA polymerases of other tumors. In particular, diminished affinity to primer templates and weakened nucleotide binding leads to lowered specificity of nucleotide selection in the base-pairing process and is therefore likely to cause an enhanced mutation rate during malignant progression.

Animals↗
PubMed1992

Structural and functional relationships of human DNA polymerases.

A continuing theme of our laboratory has been the understanding of human DNA polymerases at the structural level. We have purified DNA polymerases delta, epsilon and alpha from human placenta. Monoclonal antibodies to these polymerases were isolated and used as tools to study their immunochemical relationships. These studies have shown that while DNA polymerases delta, epsilon and alpha are discrete proteins, they must share common structural features by virtue of the ability of several of our monoclonal antibodies to exhibit cross-reactivity. A second approach we have taken is the molecular cloning of human DNA polymerase delta and epsilon. We have cloned the DNA polymerase delta cDNA, and this has allowed us to compare its primary structure to those of human polymerase alpha and other members of this polymerase family. Multiple sequence alignments have revealed that human DNA polymerase delta is also closely related to the herpes virus family of DNA polymerases. In situ hybridization has shown that the human DNA polymerase delta gene is localized to chromosome 19 q13.3-q13.4. In order to further determine the functional regions of the DNA polymerase delta structure we are currently expressing human pol delta in E. coli and baculovirus systems. Other work in our laboratory is directed toward examining the expression of DNA polymerase delta during the cell cycle.

Amino Acid Sequence↗
PubMed1992

Expression of the catalytic subunits of pol alpha and pol delta from fission yeast Schizosaccharomyces pombe.

This paper reports on expression and posttranslational modifications of the catalytic subunits of pol alpha and pol delta from fission yeast Schizosaccharomyces pombe. Okadaic acid treatment of S. pombe spheroplasts in amounts known to inhibit phosphatases 1 and 2A resulted in decreased proteolysis of both pol alpha and pol delta. Computer analysis of pol alpha and pol delta sequences confirmed the presence of consensus motifs for protein phosphorylation. Indirect immunofluorescence microscopy of S. pombe cells showed nuclear location of both proteins in wild type cells. However, whereas cells transformed with a vector expressing pol alpha produced a clear increase of the nuclear signal, no increase was detectable in cells transformed with pol delta. This observation suggests the existence of a mechanism limiting the cell concentration of pol delta in the cell. Constitutive expression of S. pombe pol delta in E. coli was possible only with vectors containing truncated forms of its gene, indicating a toxic effect of pol delta on E. coli growth.

Amino Acid Sequence↗
PubMed1992

DNA polymerase delta and epsilon holoenzymes from calf thymus.

Replication of singly-DNA primed M13 DNA by DNA polymerase (pol) delta completely relies on the simultaneous addition of proliferating cell nuclear antigen (PCNA), replication factor C (RF-C) and replication protein A (RP-A) (or E. coli single-strand DNA binding protein, SSB). Pol epsilon core alone is able to synthesize the products on singly-primed ssDNA. However, DNA synthesis by pol epsilon was stimulated up to 10-fold upon addition of the three auxiliary proteins PCNA, RF-C and SSB. This stimulation of pol epsilon by PCNA/RF-C/SSB appears to be the superposition of two events: pol epsilon holoenzyme (pol epsilon, PCNA, RF-C) synthesized longer products than its pol epsilon core counterpart, but elongated less primers. Furthermore, we analyzed the cooperative action of pol alpha/primase with pol delta or pol epsilon holoenzymes on unprimed M13 DNA. While pol delta displayed higher dNMP incorporation than pol epsilon, when a single primer was preannealed to DNA, pol epsilon was more efficient in the utilization of the primers synthesized by pol alpha/primase. Under these conditions both longer products and a higher amount of dNMP incorporation was found for pol epsilon holoenzyme, than for pol delta. Our data support the hypothesis of pol delta as the leading and pol epsilon as the second lagging strand replication enzyme.

Animals↗
PubMed1992

The 3'-->5' exonuclease associated with HeLa DNA polymerase epsilon.

The 3'-->5' exonuclease activity of highly purified large form of human DNA polymerase epsilon was studied. The activity removes mononucleotides from the 3' end of an oligonucleotide with a non-processive mechanism and leaves 5'-terminal trinucleotide non-hydrolyzed. This is the case both with single-stranded oligonucleotides and with oligonucleotides annealed to complementary regions of M13DNA. However, the reaction rates with single-stranded oligonucleotides are at least ten-fold when compared to those with completely base-paired oligonucleotides. Conceivably, mismatched 3' end of an oligonucleotide annealed to M13DNA is rapidly removed and the hydrolysis is slowed down when double-stranded region is reached. The preferential removal of a non-complementary 3' end and the nonprocessive mechanism are consistent with anticipated proofreading function. In addition to the 3'-->5' exonuclease activity, an 5'-->3' exonuclease activity is often present even in relatively highly purified DNA polymerase epsilon preparates suggesting that such an activity may be an essential component for the action of this enzyme in vivo. Contrary to the 3'-->5' exonuclease activity, the 5'-->3' exonuclease is separable from the polymerase activity.

Base Sequence↗
PubMed1997

Involvement of the RE V3 gene in the methylated base-excision repair system. Co-operation of two DNA polymerases, delta and Rev3p, in the repair of MMS-induced lesions in the DNA of Saccharomyces cerevisiae.

The ability of four yeast DNA polymerase mutant strains to carry out the repair of DNA treated with MMS was studied. Mutation in DNA polymerase Rev3, as well as the already known mutation in the catalytic subunit of DNA polymerase delta, were both found to lead to the accumulation of single-strand breaks, which indicates defective repair. A double-mutant strain carrying mutations in DNA polymerase delta and a deletion in the REV3 gene had a complete repair defect, both at permissive (23 degrees C) and restrictive (38 degrees C) temperatures, which was not observed in other pairwise combinations of tested polymerase mutants. Other polymerases are not involved in the repair of exogenous DNA methylation damage, since neither mutation in the DNA polymerase epsilon, nor deletion in the DNA polymerase IV (beta70) gene, caused defective repair. The data obtained suggest that DNA polymerases delta and Rev3p are both necessary to perform repair synthesis in the base-excision repair of methylation damage. The results are discussed in the light of current concepts on the role of DNA polymerase Rev3 in mutagenesis.

DNA Damage↗
PubMed2000

DNA polymerases and Ki-67 nuclear antigen are induced in correlation with the resected mass of rat liver up to 90%.

BACKGROUND AND AIMS: We studied the regeneration potential by measuring induction of DNA polymerases in the remnant rat liver after a partial hepatectomy (PHx) that is maximal but compatible with survival. METHODS: The regenerating rat liver was obtained after the 90% PHx. The induction of activities of DNA polymerase alpha, delta, and epsilon were measured after partial purification. The Ki-67 nuclear antigen was also detected histochemically. These parameters were compared with those after both 30% and 70% PHx. RESULTS: The 90% hepatectomy resulted in the strong inductions of DNA polymerase alpha, delta, and epsilon, at 48 h after operation, in association with increases in wet weight and total DNA in the remnant liver. The enzyme induction was much higher after 90% PHx than after 30% and 70% hepatectomy, in correlation with the resection volume. At 48 h after 90% hepatectomy, the Ki-67 positive cells increased up to 47.2% of hepatocytes in the remnant liver. CONCLUSION: The higher induction of replication enzymes by 90% hepatectomy reflects more cells entering mitogenic cell cycle, which supports the fast regeneration of the remnant liver. The number of proliferating hepatocytes is stringently controlled by an unknown mechanism sensing the mass of resected liver parenchyma.

Animals↗
PubMed2001

Stationary-phase mutations in proofreading exonuclease-deficient strains of the yeast Saccharomyces cerevisiae.

In order to understand the role of yeast polymerases in spontaneous mutagenesis in non-growing cells we have studied the effects of mutations that impair the 3'--> 5' exonuclease function of polymerases delta (pol3-01) and epsilon (pol2-4) on the spontaneous reversion frequency of the frameshift mutation his7-2 in cells starved for histidine. We showed that for each exonuclease-deficient mutant the rate of reversion per viable cell per day observed in stationary-phase cells remained constant up to the 9th day of starvation (while the number of viable cells dropped), and was very similar to that observed in the same mutants during the growth phase. These data suggest that both DNA polymerases are involved in the control of mutability in non-growing cells.

DNA Polymerase II↗
PubMed2006

Transgenic RNAi: Accelerating and expanding reverse genetics in mammals.

Reverse genetics in mammals has relied on gene targeting strategies and has mostly been limited to the mouse. Gene targeting through homologous recombination in mouse ES cells has drawbacks which include time, expense and complexity. Recently, a new approach has been developed based on RNA-interference (RNAi) which is simpler, faster, less expensive, and should be applicable to mammalian species other than mouse. The advent of RNAi is poised to accelerate the pace at which reverse genetics can be applied to study gene function in mammals.

Animals↗
PubMed1987

Biochemical characterization and development of DNA polymerases alpha and delta in the neonatal rat heart.

The ontogeny of DNA polymerase activity in the neonatal rat heart was studied. The DNA polymerase activities in rat heart extracts were identified as DNA polymerase alpha and DNA polymerase delta activities by their purification and characterization, by the use of a specific inhibitor (BuAdATP), and by a specific monoclonal antibody against DNA polymerase alpha. Using these inhibitors, it was shown that the two activities declined in parallel during the maturation and terminal differentiation of the heart.

Adenosine Triphosphate↗
PubMed1989

Immunochemical studies of DNA polymerase delta: relationships with DNA polymerase alpha.

A panel of murine hybridoma cell lines which produce antibodies against polypeptides present in human placental DNA polymerase delta preparations was developed. Eight of these antibodies were characterized by virtue of their ability to inhibit DNA polymerase delta activity and immunoblot the 170-kDa catalytic polypeptide. Six of these eight antibodies inhibit DNA polymerase delta but not DNA polymerase alpha, showing that the two proteins are distinct. However, the other two monoclonal antibodies inhibited both DNA polymerase delta and alpha activities, providing the first evidence that these two proteins have a structural relationship. In addition to antibodies against the catalytic polypeptide we also identified 11 antibodies which recognize 120-, 100-, 88-, 75-, 62-, 36-, and 22-kDa polypeptides in DNA polymerase delta preparations, suggesting that these proteins might be part of a replication complex. The antibody to the 36-kDa polypeptide was shown to be directed against proliferating cell nuclear antigen/cyclin. These antibodies should prove useful for studies aimed at distinguishing between DNA polymerases alpha and delta and for the investigation of the functional roles of DNA polymerase delta polypeptides.

Animals↗