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M Goulian

Publications and source records attributed to M Goulian.

At least 37 records · Page 2Linked to original sources
PubMed1990

The mechanism of action of an accessory protein for DNA polymerase alpha/primase.

A previous paper reported the purification (from mouse cell extracts) and some of the properties of a protein, alpha accessory factor (AAF), that specifically stimulates DNA polymerase alpha/primase (1). We describe here studies on the mechanism of action of AAF. In the presence of AAF and a large excess of single-stranded circular DNA template, a molecule of DNA polymerase alpha/primase interacts with a single template DNA molecule priming and synthesizing multiple short DNA fragments covering thousands of nucleotides without detaching from the template, and, by many-fold repetition of the process, accomplishes serial replication of the population of DNA molecules. In contrast, without AAF the reaction involves the whole population of DNA molecules in parallel and with a very large number of binding events between DNA polymerase alpha/primase and DNA [corrected] template. The profound [corrected] increase in affinity of DNA polymerase alpha/primase for the DNA template that characterizes the mechanism suggests a functional identification of AAF as a template affinity protein. The resulting greater efficiency accounts for the ability of AAF to stimulate both the primase and polymerase activities of DNA polymerase alpha/primase. AAF also increases the processivity of DNA polymerase alpha/primase from approximately 15 to approximately 115 nucleotides, a size similar to that of mammalian Okazaki fragments, and it appears to allow DNA polymerase alpha/primase to traverse double-stranded regions of a DNA template. These features of the mechanism of AAF suggest that it may have a role in assisting DNA polymerase alpha/primase in synthesis of the lagging strand of a replication fork.

Animals↗
PubMed1990

Three cytoplasmic DNA polymerases that utilize poly(rA).oligo(dT).

Three DNA polymerases that use poly(rA).oligo(dT) were partially purified from cytoplasmic extracts of cultured mouse cells (after removal of mitochondria), and characterized. One is similar to, and may be the same as, the mitochondrial DNA polymerase gamma. The other two enzymes, one 7.5 S and the other 3.6 S, share some properties with DNA polymerases beta and gamma, e.g. their responses to certain inhibitors; however, they are not clearly identified with any previously well-characterized mammalian DNA polymerases. It is also demonstrated that the response of DNA polymerase gamma to N-ethylmaleimide is template dependent, and that DNA polymerase alpha has an authentic (albeit small) activity with poly(rA).oligo(dT).

Animals↗
PubMed1989

Intact DNA polymerase alpha/primase from mouse cells. Purification and structure.

A procedure is described for purification of DNA polymerase alpha/primase from cultured mouse lymphoblasts. Approximately 0.5 mg of enzyme, free of detectable contaminants, was obtained from 40 g of cells using seven conventional purification steps. The mouse enzyme contains subunits of 180, 70, 56, and 47 kDa, almost completely intact and similar to the subunit sizes reported for DNA polymerase alpha/primase from Drosophila embryos (Kaguni, L. S., Rossignol, J-M., Conaway, R. C., and Lehman, I. R. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2221-2225) and Saccharomyces (Plevani, P., Foiani, M., Valsasnini, P., Badaracco, G., Cheriathundam, E., and Chang, L.M.S. (1985) J. Biol. Chem. 260, 7102-7107). A similar structure has also been inferred for mammalian DNA polymerase alpha/primase; however, previous descriptions of the highly purified mammalian enzyme complex have included substantial evidence of proteolysis and/or partial loss of subunits. In particular, the intact 180-kDa subunit has ordinarily been a minor component, and the molecular mass of the complex and total number of subunits have not been established. Results reported here indicate that the native DNA polymerase alpha/primase consists of one each of the four subunit sizes for a total of 353 kDa, based on estimates from denaturing gels, or 344 kDa when sizes deduced from available nucleic acid sequence data are substituted for three of the four subunits. A figure of 313 kDa was calculated from the sedimentation coefficient (8.9 S) and Stokes radius (81.1 A), the values for which also indicate a frictional ratio of 1.80, corresponding to an axial ratio of approximately 16 and suggesting a highly extended structure.

Animals↗
PubMed1986

DNA fragmentation and cytotoxicity from increased cellular deoxyuridylate.

Previous results from this laboratory have shown that thymidylate deprivation results in dramatic elevation of intracellular dUTP and incorporation of dUMP into DNA. The goal of the present studies was to determine whether the latter changes may play a part in the associated cytotoxicity ("thymineless death"), which is ordinarily assumed to be a direct result of reduced intracellular dTTP. The approach used here was to increase intracellular dUTP without allowing dTTP to diminish and observe the effects on cell viability. dUMP pools were expanded by exposure of cells to deoxyuridine [in cell growth medium containing hypoxanthine, methotrexate, and thymidine (HAT medium)], resulting in accumulation of dUTP to levels that approached those of dTTP, which were at, or higher than, the levels in untreated cells. In conjunction with this the cells became nonviable, and newly synthesized DNA was fragmented, both of which occur with thymidylate depletion and, we assume, result from the active process of excision repair at the many uracil-containing sites in DNA. The results indicate that, although the relative importance of low dTTP remains unknown, elevated dUTP can account for the cytotoxicity caused by thymidine starvation. Most of the "dTTP" measured by the DNA polymerase assay in cells treated with methotrexate (MTX) (plus purine supplement) was, in fact, dUTP, which may explain some previous observations of only modest depression of dTTP in cells treated with MTX or similarly acting drugs.

Animals↗
PubMed1983

2'-deoxyribosyl analogues of UDP-N-acetylglucosamine in cells treated with methotrexate or 5-fluorodeoxyuridine.

dUDP-GlcNAc, the 2'-deoxyribosyl analogue of UDP-GlcNAc, has been identified in human lymphoid cells treated with the dihydrofolate reductase inhibitor, methotrexate. It was shown previously that elevation of dUTP accompanies the gross expansion in intracellular deoxyuridylate pools that results from the methotrexate-induced block in thymidylate synthetase activity (1). dUDP-GlcNAc presumably is formed from dUTP acting in place of UTP in the normal pathway for formation of UDP-GlcNAc. Neither dUTP nor dUDP-GlcNAc has been detected in untreated cells. Inhibition of thymidylate synthetase by treatment of cells with 5-fluorodeoxyuridine (5-FdUrd) also causes the appearance of dUDP-GlcNAc, and, in addition, 5-FdUDP-GlcNAc, synthesized from 5-FdUTP. The metabolic effects, if any, of these analogues are not known. Synthesis of the analogues may help to limit accumulation of dUTP and 5-FdUTP under circumstances in which the deoxyuridine triphosphatase mechanism is insufficient.

Cell Line↗
PubMed1982

DNA polymerase requirements for parvovirus H-1 DNA replication in vitro.

An in vitro system using nuclei from parvovirus H-1-infected cells was used to characterize the influence of inhibitors of mammalian DNA polymerases on viral DNA synthesis. The experiments tested the effects of aphidicolin, which is highly specific for DNA polymerase alpha, and 2',3'-dideoxythymidine-5'-triphosphate (ddTTP), which inhibits cellular DNA polymerases in the order gamma greater than beta greater than alpha. Both aphidicolin and ddTTP were inhibitory, indicating that both polymerase alpha and a ddttp-sensitive enzyme are required for viral DNA synthesis. This was seen more clearly in kinetic measurements, which indicated an initial period of rapid DNA synthesis with the participation of polymerase alpha, followed by a period of less rapid, but more sustained, rate of DNA synthesis carried out by a ddTTP-sensitive enzyme, probably polymerase gamma. One interpretation of the results is that polymerase alpha functions in a strand displacement stage of the viral DNA replication mechanism, whereas polymerase gamma serves to convert the displaced single strands back to double-strand replicative form.

Aphidicolin↗
PubMed1982

Nucleotide levels and incorporation of 5-fluorouracil and uracil into DNA of cells treated with 5-fluorodeoxyuridine.

Intracellular pools of 5-fluoro-2'-deoxyuridine (FdUrd) and dUrd nucleotides were measured in cultured human lymphoblasts treated with FdUrd. At 1 microM FdUrd, intracellular 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) was approximately 400 pmoles/10(6) cells, and FdUTP was approximately 0.1 pmole/10(6) cells. Intracellular dUMP and dUTP were elevated to values of approximately 1000 pmoles/10(6) cells and approximately 0.1 pmol/10(6) cells, respectively. With decrease in dTTP levels, utilization of FdUTP and dUTP as substrates for DNA synthesis became significant. FdUMP and dUMP, approximately 5 pmoles of each per micromole of DNA nucleotide, were found in DNA of cells treated with FdUrd (1 microM). The active removal of FUra and Ura from DNA of FdUrd-treated cells by the normal repair mechanism may lead to fragmentation of DNA and contribute to the cytotoxic effect of FdUrd.

DNA↗
PubMed1981

Synthesis of parvovirus H-1 replicative form from viral DNA by DNA polymerase gamma.

The initial event in the replication cycle of parvovirus H-1 is conversion of the single-stranded linear viral DNA to the double-stranded linear replicative form. We describe here detection of an activity in uninfected cell extracts that carries out this reaction. The activity was purified and identified as DNA polymerase gamma.

Cell Line↗
PubMed1980

The effect of methotrexate on levels of dUTP in animal cells.

The amounts of intracellular nucleotides of dThd and dUrd were measured in cultured human lymphoblasts with and without treatment with methotrexate (plus hypoxanthine) to inhibit thymidylate synthetase. dTTP fell from approximately 40 pmol/10(6) cells (untreated) to approximately 1 pmol/10(6) cells with drug treatment. dUMP, increased approximately 10(3)-fold with methotrexate. dUTP was not detected in the cell without drug treatment (less than or equal to 0.3 fmol/10(6) cells), but was easily measured at approximately 0.2 pmol/10(6) cells in drug-inhibited cells. Thus the ratio dUTP/dTTP incrased from less than or equal to 10(-5) in untreated cells to approximately 0.2 in cells treated with methotrexate, indicating that dUMP may be incorporated in substantial amounts into the DNA of the drug-treated cells. The magnitude of these effects on nucleotide pools suggests the possibility that the normal nucleolytic mechanism for removal of dUMP from DNA may play a part in the toxic effects on the cell that result from depression of thymidylate synthetase activity.

Cells, Cultured↗
PubMed1980

Initiator RNA synthesis upon ribonucleotide depletion. Evidence for base substitutions.

Nearest neighbor frequencies in initiator RNA (iRNA) of a human lymphocyte cell line have been measured in isolated nuclei under conditions where ribonucleotides have been omitted from the incubation. Deoxynucleotides are incorporated in iRNA to a limited extent for the omitted cognate ribonucleotide. In addition, upon omission of a ribonucleotide, changes in the nearest neighbor frequencies also occur indicating base substitution of another ribonucleotide. The base substitutions that have been observed are the incorporation of rA for rG or rU. Substitution for rA has not been observed. When rC is omitted, a change in all nearest neighbor frequencies occurs, suggesting possibly an unusual role for rC. The substitutions demonstrate the unique features of iRNA synthesis and point out possible alterations in iRNA that may occur in vivo when nucleotide pools are altered.

Base Sequence↗
PubMed1980

Methotrexate-induced misincorporation of uracil into DNA.

A line of human lymphoid cells was tested for the presence of dUMP in DNA with or without treatment with the dihydrofolate reductase inhibitor, methotrexate. Cells treated with methotrexate and labeled with [(3)H]dUrd contained dUMP in DNA in readily detectable amounts ( approximately 0.8 pmol of dUMP per mumol of total DNA nucleotide), and this was increased approximately 3-fold if the cells were also treated with Ura at the same time. No dUMP (<1 fmol/mumol of DNA) could be detected by these methods in DNA from cells not treated with methotrexate, regardless of whether Ura was present or absent. The presence of dUMP in DNA from cells treated with methotrexate is a result of the great increase in intracellular concentration of dUTP and the fall in dTTP that accompany inhibition of thymidylate synthetase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase; EC 2.1.1.45) by the drug. These changes are apparently sufficient to overcome the normal mechanisms that exclude dUMP from DNA, and the enhancement by Ura reflects suppression of one of the mechanisms, Ura removal from DNA by the enzyme Ura-DNA glycosylase. The results suggest an active lesion of DNA in cells in which thymidylate synthetase is inhibited. Under these conditions there appears to be a cyclic incorporation and removal of dUMP resulting from reinsertion of dUMP during gap repair at sites of Ura removal. This consequence of the normal excision-repair process, which occurs when intracellular levels of dUTP approach those of dTTP, may have effects related to the cytotoxicity of drug inhibitors of thymidylate synthetase, clinical deficiencies of folate and vitamin B-12, and thymineless death, in general.

Cell Line↗
PubMed1980

Mechanism for exclusion of 5-fluorouracil from DNA.

5-Fluorodeoxyuridine 5'-triphosphate is hydrolyzed by the enzyme deoxyuridine triphosphate diphosphohydrolase (EC 3.6.1.23). Uracil-DNA glycosylase removes 5-fluorouracil (FUra) from FUra-containing DNA similar to its removal of uracil from uracil-containing DNA. The absence of FUra in DNA following exposure of cells to FUra can be explained by the activities of these two enzymes.

DNA↗
PubMed1979

Covalent association of protein with replicative form DNA of parvovirus H-1.

The double-stranded replicative form (RF) DNA of the autonomous parvovirus H-1 can be isolated from infected cells in a covalent complex with protein. The protein is present on most or all of the RF DNA, including actively replicating molecules, and is associated with the 5'-terminal endonuclease Hae III fragments of both the viral and complementary strands of RF. The size of the protein is estimated to be 60,000-70,000 daltons from its effect on buoyant density of DNA. DNA with covalently bound protein has not been found in H-1 virions.

Centrifugation, Isopycnic↗
PubMed1979

Deoxyribonucleic acid synthesis in Saccharomyces cerevisiae cells permeabilized with ether.

Cells of Saccharomyces cerevisiae permeabilized by treatment with ether take up and incorporate exogenous deoxynucleoside triphosphate into deoxyribonucleic acid (DNA). With rho(+) strains, more than 95% of the product was mitochondrial DNA (mtDNA). This report characterizes ether-permeabilized yeast cells and describes studies on the mechanism of mtDNA synthesis with this system. The initial rate of in vitro mtDNA synthesis with one strain (X2180-1Brho(+)) was close to the rate of mtDNA replication in vivo. The extent of synthesis after 45 min was sufficient for the duplication of about 25% of the total mtDNA in the cells. The incorporated radioactivity resulting from in vitro DNA synthesis appeared in fragments that were an average of 30% mitochondrial genome size. Density-labeling experiments showed that continuous strands of at least 7 kilobases after denaturation, and up to 25 kilobase pairs before denaturation, were synthesized by this system. Pulse-chase experiments demonstrated that a large proportion of DNA product after short labeling times appeared in 0.25-kilobase fragments (after denaturation), which served as precursors of high-molecular-weight DNA. It is not yet clear whether the short pieces participate in a mechanism of discontinuous replication similar to that of bacterial and animal cell chromosomal DNA or whether they are related to the rapidly turning over, short initiation sequence of animal cell mtDNA. In rho(0) strains, which lack mtDNA, the initial rate of nuclear DNA synthesis in vitro was 1 to 2% of the average in vivo rate. With temperature-sensitive DNA replication mutants (cdc8), the synthesis of nuclear DNA was temperature sensitive in vitro as well, and in vitro DNA synthesis was blocked in an initiation mutant (cdc7) that was shifted to the restrictive temperature before the ether treatment.

DNA, Fungal↗