Structural homology among calf thymus α-polymerase polypeptides

Albert, Waltraud; Grummt, Friedrich; Hübscher, Ulrich; Wilson, Samuel H.

doi:10.1093/nar/10.3.935

Cited by 37 publications

(15 citation statements)

References 19 publications

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“…This enzyme and the natural enzyme purified from virions were found to be indistinguishable in reaction properties.2 DNA polymerase a! was purified from calf thymus (Albert et al, 1982). DNA polymerase y, purified from pig liver by using DEAE-Sephadex, phosphocellulose, and heparin-agarose chromatography, was a generous gift of D. Mosbaugh.…”

Section: Methodsmentioning

confidence: 99%

Stepwise mechanism of HIV reverse transcriptase: primer function of phosphorothioate oligodeoxynucleotide

Majumdar

Stein²,

Cohen³

et al. 1989

Biochemistry

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194

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Primer recognition by purified HIV reverse transcriptase has been investigated. Earlier we found that the reaction pathway for DNA synthesis is ordered, with template-primer and free enzyme combining to form the first complex in the reaction sequence (Majumdar et al., 1988). We now find that d(C)28 is a linear competitive inhibitor of DNA synthesis against poly[r(A)].oligo[d(T)] as template.primer, indicating that d(C)28 and the template.primer combine with the same form of the enzyme in the reaction scheme, i.e., the free enzyme. The phosphorothioate oligodeoxynucleotide Sd(C)28 also is a linear competitive inhibitor against template.primer. However, the Ki for inhibition (approximately 2.8 nM) is approximately 200-fold lower than the Ki for inhibition by d(C)28. Since the inhibition is linear competitive, the dissociation constant is equal to the Ki for inhibition. Filter binding assays confirmed high-affinity binding between Sd(C)28 and the enzyme and yielded a KD similar to the Ki for inhibition. Substrate kinetic studies of DNA synthesis using Sd(C)28 as primer, and poly[r(I)] as template, revealed that the Km for Sd(C)28 is 24 nM. The Km for this primer is, therefore, 8-fold higher than the KD for enzyme-primer binding (2.8 nM). These results enable calculation of real time rate values for the enzyme-primer association (kon = 5.7 x 10(8) M-1 s-1) and dissociation (koff = 1.6 s-1).

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Section: Methodsmentioning

confidence: 99%

Stepwise mechanism of HIV reverse transcriptase: primer function of phosphorothioate oligodeoxynucleotide

Majumdar

Stein²,

Cohen³

et al. 1989

Biochemistry

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194

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“…Several polymerases (1)(2)(3)(4)(5)(6)(7) have been isolated as multisubunit proteins consisting of a catalytic core with an approximate Mr of 150,000 in association with three or four smaller subunits whose Mrs range from 40,000 to 60,000. However, other polymerase preparations are lacking a high molecular weight subunit (8,9).…”

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confidence: 99%

Isolation of an intact DNA polymerase-primase from embryos of Drosophila melanogaster.

Kaguni¹,

Rossignol²,

Conaway³

et al. 1983

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

131

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A procedure has been devised for the purification of intact DNA polymerase a from early embryos of Drosophila melanogaster. The purified enzyme consists of at least three polypeptides with Mrs of 182,000, 60,000, and 50,000. These are related antigenically to the a (Mr 148,000), .l (Mr 58,000), and Y (Mr 46,000) subunits, respectively, of the DNA polymerase described previously [Banks, G. R., Boezi, J. A. & Lehman, I. R. (1979)J. Biol. Chem. 254, 9886-9892]. The-a subunit (Mr 182,000) has a molecular weight indistinguishable from that observed in extracts of freshly harvested embryos and presumably present in vivo. As in the previous preparation, the a subunit is required for DNA polymerase activity and is very likely the catalytic subunit of the enzyme. The ratio of primase to polymerase remains constant throughout the purification. Thus, the primase is very likely an integral component of the Drosophila DNA polymerase a. The purified DNA polymerase-primase contains no detectable endoor exodeoxyribonuclease and has pH, MgCl2, (NH4)2SO4, and NaCI optima identical to those reported previously. In contrast, the Km for dTTP is 3.7 ,uM as compared with 17.5 ,uM for the previous enzyme. Sensitivities to aphidicolin and N-ethylmaleiimide and resistance to dideoxy TTP are unchanged.DNA polymerase a as purified from a variety of eukaryotes consists of two or more polypeptides. Several polymerases (1-7) have been isolated as multisubunit proteins consisting of a catalytic core with an approximate Mr of 150,000 in association with three or four smaller subunits whose Mrs range from 40,000 to 60,000. However, other polymerase preparations are lacking a high molecular weight subunit (8,9). Our recent studies (10) and those of others (11)(12)(13)(14) have shown that a DNA primase activity is associated with the a polymerase and may be part of the multisubunit molecule.Uncontrolled proteolysis during purification has prevented the unambiguous determination of the subunit structure of DNA polymerase a and the structure-function relationships of its individual polypeptides. That proteolysis may yield an active but highly degraded form of the enzyme has been clearly demonstrated both in Drosophila melanogaster embryos and in calf thymus (4,(15)(16)(17) were prepared for use as described (19). Leupeptin was purchased from the Peptide Institute (Minoh-shi, Japan). Aphidicolin was prepared as a stock solution at 1 mg/ ml in 40% ethanol/10% dimethyl sulfoxide. N-Ethylmaleiimide (Schwarz/Mann) was dissolved in water immediately before use.Antisera. Antisera directed against Drosophila DNA polymerase a and its isolated a and J subunits were prepared 'as described (18). Purified IgG was prepared by ammonium sulfate precipitation and DEAE-cellulose chromatography (20).Enzymes. Escherichia coli DNA polymerase I and E. coli RNA polymerase were the generous gifts of J. Kelly and J. Kaguni, respectively, of this department. Pancreatic DNase I was purchased from Worthington.Nucleic acids. Calf thymus DNA (grade A, Calbiochem) wa...

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“…In a system developed for the detection of DNA polymerase a activity in NaDodSO4/ polyacrylamide gels, more than one subunit appears to contain DNA chain elongation activity (29), whereas the exact roles of the low molecular weight bands lacking chain elongation activity are not known. Closely related heavier subunits (125-150 kDa) in the DNA polymerase a complex may arise from the specific proteolysis of a high molecular weight subunit (30), whereas subunits of intermediate (75-110 kDa) and low molecular weight are the products either of further proteolytic cleavage (30,31,55) or of different gene loci (32). These small molecular weight proteins, noncovalently bound to the DNA polymerase complex, may play some regulatory rather than a direct catalytic role in the chain elongation process.…”

mentioning

confidence: 99%

Stimulation of human neuroblastoma DNA polymerase alpha and primase activities by a protein factor isolated from rat liver chromatin.

Takada¹,

Torres-Rosado²,

Ray³

et al. 1986

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

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Nuclear protein factor type 1 (NPF-1) thatsimulates IMR-32 primase-associated DNA polymerase a, and a2 activities has been purified from a high-salt extract of liver chromatin from 6-month-old rats. The final purified factor lacks DNA polymerase a, RNA polymerase, and DNA-unwinding or topoisomerase type I activities. The stimulatory activity is destroyed by trypsin (60 min at 37C), DNase II (60 min at 370C), and heat treatment (2 min at 68C). The '2I-labeled NPF-1 does not bind to activated calf thymus DNA or poly(dC). However, it forms a ternary complex with DNA in the presence of DNA polymerase a-primase complex (a, and a2). The ternary complex sediments on sucrose density gradient as a heavier band (11S). The NPF-1 also stimulates (2.5-fold) primase-catalyzed incorporation of GMP and dGMP from the corresponding triphosphates on poly(dC) template even in the presence of a high concentration of a-amanitin (400 ,tg/ml). 8.0/1 mM EDTA/6 mM KCI/2 mM MgCl2/1 mM 2-mercaptoethanol/0.1 mM phenylmethylsulfonyl fluoride/0.1% polyethylene glycol (PEG; 8 kDa) using a glass-Teflon homogenizer. The homogenate was centrifuged at 100,000 x g for 1 hr. The supernatant (1.5 ml) was applied to a DE 23 column (1.5 x 10 cm) equilibrated with 50 mM potassium phosphate, pH 7.6/1 mM MgCl2/1 mM 2-mercaptoethanol/0.1% PEG/ 0.1 mM phenylmethylsulfonyl fluoride (buffer C). After

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Structural homology among calf thymus α-polymerase polypeptides

Cited by 37 publications

References 19 publications

Stepwise mechanism of HIV reverse transcriptase: primer function of phosphorothioate oligodeoxynucleotide

Stepwise mechanism of HIV reverse transcriptase: primer function of phosphorothioate oligodeoxynucleotide

Isolation of an intact DNA polymerase-primase from embryos of Drosophila melanogaster.

Stimulation of human neuroblastoma DNA polymerase alpha and primase activities by a protein factor isolated from rat liver chromatin.

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