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At least 127 records · Page 7Linked to original sources
PubMed1994

Elevated DNA polymerase alpha, DNA polymerase beta, and DNA topoisomerase II in a melphalan-resistant rhabdomyosarcoma xenograft that is cross-resistant to nitrosoureas and topotecan.

Previous investigations have revealed that the human TE-671 MR human rhabdomyosarcoma xenograft selected in vivo for melphalan resistance (M. C. Rosenberg, et al., Cancer Res., 49: 6917-6922, 1989) is cross-resistant to a wide variety of alkylating agents and to bleomycin, but is collaterally sensitive to etoposide. Although glutathione levels were noted to be elevated in TE-671 MR compared to the melphalan-sensitive parental TE-671 xenograft, treatment with buthionine sulfoximine to deplete glutathione levels did not fully restore melphalan sensitivity in the TE-671 MR xenograft. The present studies were undertaken to search for additional mechanisms of resistance in the TE-671 MR xenograft. Drug sensitivity testing performed at the dose of agents that was lethal to 10% of the animals revealed that the TE-671 MR xenograft maintained resistance to the bifunctional cross-linking agent 1,3-bis(2-chloroethyl)-1-nitrosourea and was cross-resistant to the topoisomerase I poison topotecan. Treatment with buthionine sulfoximine did not sensitize the TE-671 MR xenograft to 1,3-bis(2-chloroethyl)-1-nitrosourea. Further, even though O6-alkylguanine-DNA alkyltransferase levels were high in both the TE-671 and TE-671 MR xenografts, depletion of O6-alkylguanine-DNA alkyltransferase activity by treatment with O6-benzylguanine substantially sensitized the TE-671 xenografts but not the TE-671 MR xenografts, suggesting an additional mechanism of resistance. Measurement of additional enzyme activities that might be involved in DNA repair revealed significant elevations in DNA polymerase alpha (46 +/- 8 (SD) units/mg protein in TE-671, 69 +/- 6 units/mg protein in TE-671 MR, P < 0.05) and DNA polymerase beta (0.43 +/- 0.01 units/mg protein in TE-671, 0.78 +/- 0.12 units/mg protein in TE-671 MR, P < 0.05) but not DNA polymerase delta or total DNA ligase. Examination of topoisomerases by activity assays and Western blotting revealed a 2-fold increase in topoisomerase II and a 2-fold decrease in topoisomerase I in the TE-671 MR xenograft compared to the parental xenograft, apparently explaining the collateral sensitivity to etoposide and cross-resistance to topotecan. These results suggest that TE-671 MR xenografts contain multiple changes in activities of DNA repair-related proteins and other nuclear proteins that could contribute to alkylating agent resistance.

Alkyl and Aryl Transferases↗
PubMed1989

DNA polymerase I is required for premeiotic DNA replication and sporulation but not for X-ray repair in Saccharomyces cerevisiae.

We have used a set of seven temperature-sensitive mutants in the DNA polymerase I gene of Saccharomyces cerevisiae to investigate the role of DNA polymerase I in various aspects of DNA synthesis in vivo. Previously, we showed that DNA polymerase I is required for mitotic DNA replication. Here we extend our studies to several stages of meiosis and repair of X-ray-induced damage. We find that sporulation is blocked in all of the DNA polymerase temperature-sensitive mutants and that premeiotic DNA replication does not occur. Commitment to meiotic recombination is only 2% of wild-type levels. Thus, DNA polymerase I is essential for these steps. However, repair of X-ray-induced single-strand breaks is not defective in the DNA polymerase temperature-sensitive mutants, and DNA polymerase I is therefore not essential for repair of such lesions. These results suggest that DNA polymerase II or III or both, the two other nuclear yeast DNA polymerases for which roles have not yet been established, carry out repair in the absence of DNA polymerase I, but that DNA polymerase II and III cannot compensate for loss of DNA polymerase I in meiotic replication and recombination. These results do not, however, rule out essential roles for DNA polymerase II or III or both in addition to that for DNA polymerase I.

DNA Polymerase I↗
PubMed1989

DNA polymerase III, a second essential DNA polymerase, is encoded by the S. cerevisiae CDC2 gene.

Three nuclear DNA polymerases have been described in yeast: DNA polymerases I, II, and III. DNA polymerase I is encoded by the POL1 gene and is essential for DNA replication. Since the S. cerevisiae CDC2 gene has recently been shown to have DNA sequence similarity to the active site regions of other known DNA polymerases, but to nevertheless be different from DNA polymerase I, we examined cdc2 mutants for the presence of DNA polymerases II and III. DNA polymerase II was not affected by the cdc2 mutation. DNA polymerase III activity was significantly reduced in the cdc2-1 cell extracts. We conclude that the CDC2 gene encodes yeast DNA polymerase III and that DNA polymerase III, therefore, represents a second essential DNA polymerase in yeast.

Cell Cycle↗
PubMed1981

DNA polymerase, DNA ligase, and thymidine kinase activity in chicken lens, related to DNA X-ray lesion repair.

The activity of DNA polymerases alpha, beta, and gamma; DNA ligases I and II; and thymidine kinase in chicken lenses is determined. These enzymes are present in embryonic intact lenses freshly isolated at 6 days and 11 days of development and in lenses isolated at 11 days of development and cultured for three days. They are also found in both epithelium and fibers when separated at 10 days of embryonic development and in the epithelium of 141/2-month-old hen lenses. In the anucleate mature hen lens fibers, the only detected enzyme is thymidine kinase. Previous results showed that repair of X-ray irradiated DNA was total in 11-day-old embryonic lenses while repair was not detected at six days. On the other hand, repair was very much impaired when the 11-day-old lenses were cultured for three days. Enzymic analyses suggest that the defect in DNA repair and the spontaneous DNA degradation observed in chick lenses at certain embryonic stages, are not due to the absence of any of the above enzymic activities. Alternative explanations are discussed.

Animals↗
PubMed1979

Replication at restrictive temperatures in Escherichia coli containing a polCts mutation.

Escherichia coli cells with a polCts mutation contain a temperature-sensitive DNA polymerase II and fall to replicate DNA at the restrictive temperature (43 degreees C). Mutants deficient in polymerizing activity of the other two recognized DNA polymerases in E. coli can replicate DNA. We have isolated temperature-resistant revertants from a strain containing polA-, polB-, and polCts mutations. These revertants grow at 43 degrees C, but analysis of partially purified DNA polymerase III from several such revertants shows a temperature-sensitive DNA polymerase III activity. Genetic analysis by P1 transduction confirms that such revertants can contain a polCts mutation and also a polA- mutation. We find that such revertants behave phenotypically as PolI+ cells (DNA polymerase I-containing), and extracts of such cells show a DNA polymerase I-like activity. Revertants of polA-, dnaAts and polA-, dnaBts strains do not show such a DNA polymerase activity.

DNA Polymerase I↗
PubMed1976

Deoxyribonucleic acid polymerase II activity in an Escherichia coli mutator strain.

The polB gene encoding deoxyribonucleic acid (DNA) polymerase II has been located close to a mutator gene, mutT1, in Escherichia coli. We find the DNA polymerase II prepared from mutT1, strains to be normal in reaction requirements, heat stability, and ability to remove mismatched bases at termini. Recombinants formed from a mutant defective in DNA polymerase II (polB100) and mutT1 are deficient in polymerase II and have the same mutator phenotype as mutT1. Our linkage analysis indicates that mutT1 and polB100 are not isoallelic.

Cell-Free System↗
PubMed1992

Post-incision steps of nucleotide excision repair in Escherichia coli. Disassembly of the UvrBC-DNA complex by helicase II and DNA polymerase I.

UvrA, UvrB, and UvrC initiate nucleotide excision repair by incising a damaged DNA strand on each side of the damaged nucleotide. This incision reaction is substoichiometric with regard to UvrB and UvrC, suggesting that both proteins remain bound following incision and do not "turn over." The addition of only helicase II to such reaction mixtures turns over UvrC; UvrB turnover requires the addition of helicase II, DNA polymerase I, and deoxynucleoside triphosphates. Column chromatography and psoralen photocross-linking experiments show that following incision, the damaged oligomer remains associated with the undamaged strand, UvrB, and UvrC in a post-incision complex. Helicase II releases the damaged oligomer and UvrC from this complex, making repair synthesis possible; DNase I footprinting experiments show that UvrB remains bound to the resulting gapped DNA until displaced by DNA polymerase I. The specific binding of UvrB to a psoralen adduct in DNA inhibits psoralen-mediated DNA-DNA cross-linking, yet promotes the formation of UrvB-psoralen-DNA cross-links. The discovery of psoralen-UvrB photocross-linking offers the potential of active-site labeling.

Adenosine Triphosphatases↗
PubMed1994

The 3'-->5' exonucleases of both DNA polymerases delta and epsilon participate in correcting errors of DNA replication in Saccharomyces cerevisiae.

DNA polymerases II (epsilon) and III (delta) are the only nuclear DNA polymerases known to possess an intrinsic 3'-->5' exonuclease in Saccharomyces cerevisiae. We have investigated the spontaneous mutator phenotypes of DNA polymerase delta and epsilon 3'-->5' exonuclease-deficient mutants, pol3-01 and pol2-4, respectively. pol3-01 and pol2-4 increased spontaneous mutation rates by factors of the order of 10(2) and 10(1), respectively, measured as URA3 forward mutation and his7-2 reversion. Surprisingly, a double mutant pol2-4 pol3-01 haploid was inviable. This was probably due to accumulation of unedited errors, since a pol2-4/pol2-4 pol3-01/pol3-01 diploid was viable, with the spontaneous his7-2 reversion rate increased by about 2 x 10(3)-fold. Analysis of mutation rates of double mutants indicated that the 3'-->5' exonucleases of DNA polymerases delta and epsilon can act competitively and that, like the 3'-->5' exonuclease of DNA polymerase delta, the 3'-->5' exonuclease of DNA polymerase epsilon acts in series with the PMS1 mismatch correction system. Mutational spectra at a URA3 gene placed in both orientations near to a defined replication origin provided evidence that the 3'-->5' exonucleases of DNA polymerases delta and epsilon act on opposite DNA strands, but were in sufficient to distinguish conclusively between different models of DNA replication.

Amino Acid Sequence↗
PubMed1976

Changes in deoxyribonucleic acid polymerase activities in synthesis of deoxyribonucleic acid during sporulation of Bacillus subtilis.

The deoxyribonucleic acid (DNA) polymerase activities in Bacillus subtilis strains Marburg 168 (thy-trp2) and D22, a DNA polymerase I-deficient mutant, were measured at various stages of sporulation. The DNA polymerase I activity, which had decreased after the exponential growth, began to increase at the early stage of sporulation, reached a maximum and then again decreased. The activity of neither DNA polymerase II nor III was observed to change so drastically as that of DNA polymerase I during sporulation. The incorporation of [3H]deoxythymidine 5'-triphosphate ([3H]dTTP) into Brij 58-treated permeable cells increased during sporulation. The stimulation of [3H]dTTP incorporation into the cells by irradiation with ultraviolet light was also observed to coincide with DNA polymerase I activity. In strain D22 the activities of DNA polymerase II and III were almost constant with time. Neither change of [3H]dTTP incorporation into Brij 58-treated cells nor stimulation of incorporation by irradiation with ultraviolet light was observed.

Bacillus subtilis↗
PubMed1998

DNA polymerase epsilon encoded by cdc20+ is required for chromosomal DNA replication in the fission yeast Schizosaccharomyces pombe.

BACKGROUND: DNA polymerase II (PolII), the homologue of mammalian DNA polymerase epsilon, is essential for chromosomal DNA replication in the budding yeast Saccharomyces cerevisiae and also participates in S-phase checkpoint control. An important issue is whether chromosomal DNA replication in other eukaryotes, including the fission yeast Schizosaccharomyces pombe--in which the characteristics of replication origins are poorly defined--also requires DNA polymerase epsilon. It has been shown that DNA polymerase epsilon is not required for the in vitro replication of SV40 DNA by human cell extracts. RESULTS: We have cloned and sequenced S. pombe pol2+, which is identical to the cell-cycle gene cdc20+, encoding the catalytic polypeptide of DNA polymerase epsilon (Pol epsilon). The predicted amino acid sequence of Pol epsilon is highly homologous to that of S. cerevisiae PolII and human Pol epsilon. Consistent with this, the Pol epsilon polypeptide was recognized by polyclonal antibodies against S. cerevisiae PolII holoenzyme (PolII*). The terminal morphology of cells containing the disrupted pol2 gene was similar to that of DNA replication mutant cells and cdc20 mutant cells. Furthermore, the Pol epsilon activity from temperature-sensitive S. pombe cdc20 mutant cells was temperature-sensitive, and chromosomal DNA replication in the mutant cells was inhibited at the restrictive temperatures. CONCLUSION: These data strongly suggest that Pol epsilon is required for normal chromosomal DNA replication in S. pombe, as is PolII in S. cerevisiae. Thus, eukaryotic chromosomal DNA is replicated differently from that of viral SV40 DNA.

Amino Acid Sequence↗
PubMed1978

DNA-dependent RNA polymerase II from Acanthamoeba castellanii. Comparison of the catalytic properties and subunit architectures of the trophozoite and cyst enzymes.

The actively growing cells (trophozoites) of the amoeba Acanthamoeba castellanii were found to contain three or perhaps four different forms of class II DNA-dependent RNA polymerase (EC 2.7.7.6). The chromatographic and catalytic properties of all forms of the Acanthamoeba class II polymerases suggest them to be cognates of the class II polymerases previously reported. The predominant form was purified to near homogeneity and its subunit composition determined. Nine different polypeptides were found associated with the purified enzyme: 21 000; 185 000; 140 000; 70 000; 35 000; 21 000; 19 000; 18 500 and 16 200. These polypeptides were interpreted in terms of two class II RNA polymerases which differ in the molecular weight of their largest subunit. When A. castellanii is transferred to a medium lacking nutrients, the cells undergo cellular differentiation resulting in the formation of metabolically inactive cells (cyst formation). During this process there are significant changes in the RNA sequences transcribed. In contrast to this, we find that the chromatographic and catalytic properties of all of the class II RNA polymerases remain unchanged. Further, the subunit architecture of the predominant form(s) of polymerase II is unaltered. These findings suggest that although new RNA sequences are transcribed during encystment their appearance is not a consequence of extensive alterations in the subunit composition of the major class II RNA polymerase.

Amanitins↗
PubMed2001

Mutations in human DNA polymerase eta motif II alter bypass of DNA lesions.

Human DNA polymerase eta (hPol eta) is one of the newly identified Y-family of DNA polymerases. These polymerases synthesize past template lesions that are postulated to block replication fork progression. hPol eta accurately bypasses UV-associated cis-syn cyclobutane thymine dimers in vitro and contributes to normal resistance to sunlight-induced skin cancer. We describe here mutational analysis of motif II, a highly conserved sequence, recently reported to reside in the fingers domain and to form part of the active site in Y-family DNA polymerases. We used a yeast-based complementation system to isolate biologically active mutants created by random sequence mutagenesis, synthesized the mutant proteins in vitro and assessed their ability to bypass thymine dimers. The mutability of motif II in 210 active mutants has parallels with natural evolution and identifies Tyr52 and Ala54 as prime candidates for involvement in catalytic activity or bypass. We describe the ability of hPol eta S62G, a mutant polymerase with enhanced activity, to bypass five other site-specific lesions. Our results may serve as a prototype for studying other members of the Y-family DNA polymerases.

Amino Acid Sequence↗
PubMed1977

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.

Amanitins↗
PubMed1991

Eukaryotic DNA polymerase amino acid sequence required for 3'----5' exonuclease activity.

We have identified an amino-proximal sequence motif, Phe-Asp-Ile-Glu-Thr, in Saccharomyces cerevisiae DNA polymerase II that is almost identical to a sequence comprising part of the 3'----5' exonuclease active site of Escherichia coli DNA polymerase I. Similar motifs were identified by amino acid sequence alignment in related, aphidicolin-sensitive DNA polymerases possessing 3'----5' proofreading exonuclease activity. Substitution of Ala for the Asp and Glu residues in the motif reduced the exonuclease activity of partially purified DNA polymerase II at least 100-fold while preserving the polymerase activity. Yeast strains expressing the exonuclease-deficient DNA polymerase II had on average about a 22-fold increase in spontaneous mutation rate, consistent with a presumed proofreading role in vivo. In multiple amino acid sequence alignments of this and two other conserved motifs described previously, five residues of the 3'----5' exonuclease active site of E. coli DNA polymerase I appeared to be invariant in aphidicolin-sensitive DNA polymerases known to possess 3'----5' proofreading exonuclease activity. None of these residues, however, appeared to be identifiable in the catalytic subunits of human, yeast, or Drosophila alpha DNA polymerases.

Amino Acid Sequence↗
PubMed1997

Phosphorylation of the PCNA binding domain of the large subunit of replication factor C by Ca2+/calmodulin-dependent protein kinase II inhibits DNA synthesis.

Replication factor C (RF-C) is a heteropentameric protein essential for DNA replication and DNA repair. It is a molecular matchmaker required for loading of the proliferating cell nuclear antigen (PCNA) sliding clamp onto double-strand DNA and for PCNA-dependent DNA synthesis by DNA polymerases delta and epsilon. The DNA and PCNA binding domains of the large 140 kDa subunit of human RF-C have been recently cloned [Fotedar, R., Mossi, R., Fitzgerald, P., Rousselle, T., Maga, G., Brickner, H., Messier, H., Khastilba. S., Hübscher, U., & Fotedar, A. (1996) EMBO J. 15, 4423-4433]. Here we show that the PCNA binding domain is phosphorylated by the Ca2+/calmodulin-dependent protein kinase II (CaMKII), an enzyme required for cell cycle progression in eukaryotic cells. The DNA binding domain, on the other hand, is not phosphorylated. Phosphorylation by CaMKII reduces the binding of PCNA to RF-C and consequently inhibits RF-C-dependent DNA synthesis by DNA polymerases delta1 and epsilon. Once bound to PCNA and DNA, RF-C is protected from phosphorylation by CaMKII, suggesting a possible role of CaMKII in regulating the dynamics of interaction between PCNA and RF-C and thus interfering in the formation of an active sliding clamp by DNA polymerases delta and epsilon.

Animals↗
PubMed1997

DNA polymerase I in constitutive stable DNA replication in Escherichia coli.

We examined the effects of mutations in the polA (encoding DNA polymerase I) and polB (DNA polymerase II) genes on inducible and constitutive stable DNA replication (iSDR and cSDR, respectively), the two alternative DNA replication systems of Escherichia coli. The polA25::miniTn10spc mutation severely inactivated cSDR, whereas polA1 mutants exhibited a significant extent of cSDR. cSDR required both the polymerase and 5'-->3' exonuclease activities of DNA polymerase I. A similar requirement for both activities was found in replication of the pBR322 plasmid in vivo. DNA polymerase II was required neither for cSDR nor for iSDR. In addition, we found that the lethal combination of an rnhA (RNase HI) and a polA mutation could be suppressed by the lexA(Def) mutation.

Bacterial Proteins↗
PubMed1998

Modulation of DNA polymerases alpha, delta and epsilon by lactate dehydrogenase and 3-phosphoglycerate kinase.

Literature documents that glycolytic enzymes (among them lactate dehydrogenase and 3-phosphoglycerate kinase) can reside in nuclei of mammalian cells and exert functions in DNA replication, transcription and DNA repair, in addition to their role as catalysts in the cytoplasm. Transfer of glycolytic enzymes to cell nuclei requires modification, for example phosphorylation. We studied the effects of phosphorylated lactate dehydrogenase and 3-phosphoglycerate kinase on (i) UV-induced DNA repair, using permeabilized human fibroblasts, and (ii) in vitro DNA synthesis catalyzed by purified DNA polymerases alpha, delta, and epsilon from proliferating rat liver. (i) Phosphorylated lactate dehydrogenase stimulated UV-induced DNA repair synthesis in normal fibroblasts in a dose-dependent manner; the unphosphorylated enzyme slightly inhibited. In repair-deficient xeroderma pigmentosum fibroblasts reparative synthesis was not enhanced whether lactate dehydrogenase was phosphorylated or not, indicating that reparative DNA synthesis must be possible in order to be stimulated. (ii) Activity of purified DNA polymerases alpha, delta, and epsilon was differentially stimulated or inhibited, according to the phosphorylation status of lactate dehydrogenase. DNA polymerases were also modulated by 3-phosphoglycerate kinase, depending on the primer-templates used which were gapped DNA (mimicking a repair mode of DNA synthesis) or single-stranded M13 DNA (representing the replicative mode of DNA synthesis). Since glycolytic enzymes in cell nuclei retain binding ability for their cofactors, cytoplasmic substrates and inhibitors, a regulatory linkage might exist between the energy state of a cell and its replicative and reparative functions.

Animals↗