The Molecular Weight and Aggregation of DNA

Cavalieri, Liebe F.; Deutsch, J.F.; Rosenberg, Barbara Hatch

doi:10.1016/s0006-3495(61)86890-2

Cited by 33 publications

(23 citation statements)

References 6 publications

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“…It is of course possible that the subunits separating at the completion of denaturation are paired double helices (18), but since the separation appears to be related to the dissociation of base pairs (see also (1)) we presently prefer the postulate of strand separation.…”

Section: Discussionmentioning

confidence: 99%

Physicochemical Studies on the Reaction between Formaldehyde and DNA

Freifelder¹,

Davison²

1963

Biophysical Journal

140

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The reaction between formaldehyde and phage T7 DNA has been studied by optical absorbance and sedimentation measurements. Through the course of denaturation, OD(200) and s(20, w) rise; after the attainment of full hyperchromicity the s(20, w) falls sharply, suggesting a decrease in molecular weight. Conditions in which formaldehyde causes cross-linking are defined. Some experimental applications of the denaturation technique are given. Evidence which suggests that preformed single-strand interruptions may exist in phage DNA is briefly discussed.

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Section: Discussionmentioning

confidence: 99%

Physicochemical Studies on the Reaction between Formaldehyde and DNA

Freifelder¹,

Davison²

1963

Biophysical Journal

140

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“…The mixtures were then allowed to cool slowly to room temperature. The preparation of calf-thymus DNA has been described (5). Activated DNA was prepared according to the procedure of Aposhian and Kornberg (6).…”

Section: Methodsmentioning

confidence: 99%

Evidence for Allosterism in In Vitro DNA Synthesis on RNA Templates

Cavalieri¹,

Modak²,

Marcus³

1974

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

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Hemoglobin mRNA and (rA)n. (dT)lo have been used as primer-templates in a kinetic study of DNA synthesis with Escherichia coli DNA polymerase I (DNA nucleotidyl transferase, EC 2.7.7.7) and Mason-Pfizer monkey virus reverse transcriptase (RNA-directed DNA polymerase). The rate versus enzyme concentration curve is sigmoidal and is consistent with a cooperative phenomenon. The results could be interpreted in terms of the formation of an active complex containing enzyme dimers (or oligomers) on the primer-template. We have also observed sigmoidal kinetics in rate versus deoxynucleotide triphosphate concentration. These results are consistent with an allosteric mechanism in which the triphosphates act as both modifiers and DNA precursors. In the critical range, a 6-to 8-fold increase in both enzyme and triphosphate concentrations can lead to a 1500-fold increase in the rate of synthesis on an RNA template. Thus, small changes in enzyme and precursor concentrations could play a regulatory role in vivo.The complexity of the process of polymerization by various DNA polymerases (DNA nucleotidyl transferases, EC 2.7.-7.7) is only partially understood. There are at least five components to the system, namely, primer, template, enzyme, deoxynucleoside triphosphates, and metal ions. Studies of physical interactions between the enzyme and other individual components of the reaction, such as the work of Loeb and coworkers (1) on Escherichia coli DNA polymerase I-Mn++ complexes, and of Kornberg and coworkers (2-4) on the binding sites of E. coli DNA polymerase I, have contributed to an understanding of the system. Such studies are necessarily limited in their bearing on the synthetic process as a whole. In order to learn more about the nature of the interactions which take place during DNA synthesis, all components of the reaction must be present. We have therefore undertaken a kinetic study of DNA synthesis using DNA and RNA templates with E. coli DNA polymerase I. We report in this paper the effect of both polymerase and deoxynucleoside triphosphate concentrations on the rate of DNA synthesis. Rather than simple saturation kinetics we have observed sigmoid curves with RNA primer-templates (as opposed to DNA templates), suggesting a cooperative mechanism. MATERIALS AND METHODSUnlabeled deoxynucleoside-5'-triphosphates, (rA)0-(dT)lo, (dA)n (dT)lo, and (dC)0. (dG)12 were purchased from P-L Biochemical Laboratories; labeled triphosphates (18 Ci/mmol) were purchased from Schwarz-Mann. The homopolymer pairs were reannealed by heating at 55-60' for (rA)n0 (dT)lo and (dA).* (dT)lo and at 800 for (dCn*v (dG)12 for 15 min, in 0.05 M Tris, pH 7.8. The mixtures were then allowed to cool slowly to room temperature. The preparation of calf-thymus DNA has been described (5). Activated DNA was prepared according to the procedure of Aposhian and Kornberg (6). Hemo

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“…When the light scattering measurements are carried out immediately after denaturation, no aggregation is observed in the concentration range 0.04 to 0.15 mg/ml (19); consequently, the apparent constancy of molecular weight cannot very readily be attributed to aggregation. In addition, a study of the de crease in molecular weight as a function of the number of bonds hydrolyzed by DNase II indicated that there was no change in the number of strands per molecule upon denaturation [Cavalieri & Rosenberg (69)].…”

Section: Denaturationmentioning

confidence: 97%

“…A wide range of particle weights by light scattering has been reported, but the values have centered around 6 to 8 million, with some as high as 16 million [Shooter (13) ; Hermans (14) ; Fleischman (15)]. The problem of protein and metal ion contamination has been recognized [Butler,Robins & Shooter (17) ; Kirby (18)1 and its role in molecular-weight determination has been emphasized recently by Cavalieri, Deutsch & Rosenberg (19). In a systematic study of molecular weights of DNA s a mples from seven sources, these authors have shown that the weights, after exhaustive disaggregation treatment, lie in the range 0.6 to 2 million.…”

Section: Molecular Weight and Aggregationmentioning

confidence: 99%

“…In contrast, light scattering shows that the molecular weight is unchanged by denaturation [Rice & Doty (34) ; Cavalieri, Deutsch & Rosenberg (19)], even when measured above the transition [Sadron (12a)]. When the light scattering measurements are carried out immediately after denaturation, no aggregation is observed in the concentration range 0.04 to 0.15 mg/ml (19); consequently, the apparent constancy of molecular weight cannot very readily be attributed to aggregation.…”

Section: Denaturationmentioning

confidence: 99%

See 1 more Smart Citation

Nucleic Acids: Molecular Biology of DNA

Lf¹,

Bh²

1962

Annu. Rev. Biochem.

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The Molecular Weight and Aggregation of DNA

Cited by 33 publications

References 6 publications

Physicochemical Studies on the Reaction between Formaldehyde and DNA

Physicochemical Studies on the Reaction between Formaldehyde and DNA

Evidence for Allosterism in In Vitro DNA Synthesis on RNA Templates

Nucleic Acids: Molecular Biology of DNA

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