Fidelity of fractionated deoxyribonucleic acid polymerases from human placenta

Krauss, Sharon Wald; Linn, Stuart

doi:10.1021/bi00542a033

Cited by 33 publications

(26 citation statements)

References 48 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…CMV DNA polymerase and HeLa DNA polymerase a were partially purified from nuclear extracts through DEAEcellulose by published procedures (21). HeLa DNA polymerase a was further purified by phosphocellulose and DNA cellulose chromatography (21,28).…”

Section: Methodsmentioning

confidence: 99%

Ganciclovir-resistant cytomegalovirus clinical isolates: mode of resistance to ganciclovir

Stanat

Reardon

Erice

et al. 1991

Antimicrob Agents Chemother

166

View full text Add to dashboard Cite

Cytomegalovirus strains with reduced in vitro susceptibilities to ganciclovir have been recovered from patients who failed long-term ganciclovir therapy. The ganciclovir-resistant clinical isolates in this study were unable to induce ganciclovir phosphorylation in virus-infected cells. The viral DNA polymerase function appeared unaltered in one genetically pure ganciclovir-resistant strain, compared with that of its wild-type ganciclovir-sensitive counterpart. All nine of the ganciclovir-resistant strains were susceptible to foscarnet. Moreover, these strains were sensitive to inhibition both by vidarabine and 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine (FIAC), antiviral agents that are activated by cellular enzymes, and by (S)-1(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC), which is a monophosphate nucleoside analog. The in vitro resistance to ganciclovir of the ganciclovir-resistant clinical isolates studied was attributed to the inability of the cells infected with these isolates to phosphorylate ganciclovir; the virally encoded DNA polymerase did not appear to play a role in this ganciclovir resistance.

show abstract

Section: Methodsmentioning

confidence: 99%

Ganciclovir-resistant cytomegalovirus clinical isolates: mode of resistance to ganciclovir

Stanat

Reardon

Erice

et al. 1991

Antimicrob Agents Chemother

166

View full text Add to dashboard Cite

show abstract

“…Moreover, the parental DNA pol a, similarly purified, has the same properties except for aphidicolin sensitivity. By using DE-52 and gradient elution with potassium phosphate buffer, multiple forms of pol a have been found (33,34). All reported forms of DNA pol a, however, including a1 and a2 (34), 8 (35, 36), and A2 and C (24), exhibit similar sensitivity to aphidicolin.…”

mentioning

confidence: 98%

Mammalian mutator mutant with an aphidicolin-resistant DNA polymerase alpha.

Liu

Chang

Trosko

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The Chinese hamster V79 cell mutant aphr4-2, selected for its resistance to aphidicolin, a specific inhibitor of DNA polymerase a (DNA nucleotidyltransferase, EC 2.7.7.7), is characterized by slow growth, UV sensitivity, and hypersensitivity to UV-induced mutation. DNA polymerase a has been purified from mitochondria-free crude extracts of the mutant and its parental wild-type cells by sequential column chromatography on DEAE-cellulose and phosphocellulose. The major DNA polymerase activity from both cell lines was found to have characteristics of the a-type polymerase: sensitivity to 0.2 M KCI, resistance to heat denaturation (450C for 15 min), an apparent Km of 5 FM for dATP, and an ability to copy poly(dT)(rA)10 but not poly(rA)J(dT)12.The crude extracts and purified DNA polymerase a from the mutant cells are not inhibited by aphidicolin (>0.6 pAM). The apparent Km for dCTP with DNA polymerase a is 1.0 ± 0.4 pM (mean ± SD) for the mutant enzyme. The polymerase from the parental cells, similarly purified, is sensitive to aphidicolin and has an apparent K. for dCTP of 10 ± 4 FM. The spontaneous mutation rate (per cell per division), determined by fluctuation analysis at the Na+/K+-ATPase (EC 3.& 1.8) locus, is higher for mutant cells (42-73 x 10-8) than for parental cells (3-16 x 10-8). These data suggest a mechanism for aphidicolin resistance of the mutant-i.e., a decrease in the Km for dCTP. The results also indicate that an altered DNA polymerase a may be intrinsically mutagenic during normal semiconservative replicative as well as during UV-induced repair syntheses.Three generic DNA polymerases in mammalian cells have been purified, characterized, and designated as a, A, and y (1). The roles of these enzymes in cellular metabolism have been difficult to identify unambiguously in the absence of mutant polymerases. Conversely, analysis of the phenotype of cells containing mutant polymerases might help to establish the contribution of DNA polymerases to the fidelity of DNA replication and repair and to determine the involvement of each polymerase in spontaneous and induced mutagenesis.Evidence accumulated over the past two decades suggests that DNA polymerase a (pol a) is primarily associated with DNA replication. (2). DNA pol a is the major activity present in replicating cells, increases during the DNA synthetic period of the cell cycle (3), and is induced when quiescent cells are stimulated to undergo DNA replication (4). Furthermore, at the time of DNA synthesis, there is evidence for the translocation of pol a from the cytoplasm to the nucleus (5, 6).Aphidicolin, a tetracyclic diterpenoid mycotoxin, specifically inhibits DNA pol a but not polymerase ( or y. The inhibition of pol a in vitro by aphidicolin correlates positively with the inhibition of DNA replication in vivo (7). An additional role for pol a in DNA repair cannot be excluded (8-11). There is controversy, however, concerning whether or not aphidicolin also inhibits DNA repair synthesis (12-14).We have previously reported (15,16) the s...

show abstract

“…Assay 1, which detects pol y (and pol A), (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) mCi/Amol). In an alternative procedure, assay 4b, ATP, and phosphoenolpyruvate were replaced by 40 mM NaOAc and MgCl2 was reduced from 10 mM to 7 mM.…”

Section: Methodsmentioning

confidence: 99%

“…The 3'-terminal foldback could provide a base-paired priming point for synthesis of the complementary strand (1, 2, 6-10), and it is assumed that this functions as the initiation site for synthesis of parental RF in vivo. Although evidence for the viral DNA to RF reaction in vivo has been reported (11,12), this step has been generally difficult to study, in part due to high particle/ infectivity ratios.In (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) mCi/Amol). In an alternative procedure, assay 4b, ATP, and phosphoenolpyruvate were replaced by 40 mM NaOAc and MgCl2 was reduced from 10 mM to 7 mM.…”

mentioning

confidence: 99%

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

Kollek¹,

Goulian²

1981

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

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 y.H-1 is an autonomous (helper-independent) member ofthe parvovirus group (1, 2). Its genome consists of a linear singlestranded (ss) DNA (==5 kilobases) with stable hairpin structures at both ends. After attachment to a susceptible cell and uptake of viral DNA, infecting viral ss DNA must first be converted to the double-stranded (ds) replicative form (RF) by synthesis of the complementary strand. The RF undergoes replication, probably by a continuous (as opposed to semidiscontinuous) 5' --3' displacement mechanism on both strands (1, 3-5), followed by synthesis and packaging of viral ss DNA. Given the small size of the genome and the known coding requirements for viral capsid polypeptides, it is assumed that replication must depend almost entirely on host functions, as is the case for several other prokaryotic and eukaryotic small DNA viruses.We are studying parvoviral DNA replication as a model for eukaryotic DNA replication and describe here studies on the enzymatic requirements for synthesis of parental RF from infecting viral ss DNA. Relatively little is known from in vivo experiments about this initial step in parvoviral DNA replication. The 3'-terminal foldback could provide a base-paired priming point for synthesis of the complementary strand (1, 2, 6-10), and it is assumed that this functions as the initiation site for synthesis of parental RF in vivo. Although evidence for the viral DNA to RF reaction in vivo has been reported (11,12), this step has been generally difficult to study, in part due to high particle/ infectivity ratios.In (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) mCi/Amol). In an alternative procedure, assay 4b, ATP, and phosphoenolpyruvate were replaced by 40 mM NaOAc and MgCl2 was reduced from 10 mM to 7 mM. All assay mixtures also contained 1 mM dithiothreitol and bovine plasma albumin (0.2 mg/ml).Results for response to inhibitors represent % activity remaining in the presence ofinhibitor compared with 100% in the absence of inhibitor. The concentration of 2',3'-dideoxythymidine-5'-triphosphate (ddTTP) was the same as that of dTJP; aphidicolin (gift of A. H. Todd, Imperial Chemical Industries, London) was at 3 Ag/ml; N-ethylmaleimide (MalNEt) was at 6 mM. All assays were carried out in a total volume of 30 ILI, at 370C, for 30 min (assays 1, 2, and 3) or 60 min (assay 4) (unless stated otherwise). DNA synthesis was determined by incorporation of radioactivity from dNTP into acid-insoluble material. One unit of polymerase activity = 1 nmol of total dNMP incorporated in 60 min at 37C.Fractionation of NB Cell Extracts. Cytosol was prepared from uninfected NB cells by disruption with a Dounce homogenizer followed by centrifugation to remove nuclei.Whole cell...

show abstract

Fidelity of fractionated deoxyribonucleic acid polymerases from human placenta

Cited by 33 publications

References 48 publications

Ganciclovir-resistant cytomegalovirus clinical isolates: mode of resistance to ganciclovir

Ganciclovir-resistant cytomegalovirus clinical isolates: mode of resistance to ganciclovir

Mammalian mutator mutant with an aphidicolin-resistant DNA polymerase alpha.

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

Contact Info

Product

Resources

About