Adenylate oligomers in single- and double-strand conformation

Brahms, J.; Michelson, A.M.; Holde, Kensal E. van

doi:10.1016/s0022-2836(66)80122-5

Cited by 404 publications

(213 citation statements)

References 26 publications

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“…The decrease of the intensity of the CD signal of poly(A) is similar to that observed when the temperature is increased [6,7] . This change in the CD spectrum corresponds to a change of the polynucleotide conformation which clearly indicates that the lac repressor binds to the polynucleotide.…”

Section: Resultssupporting

confidence: 76%

“…This spectrum is characteristic of the single strand ordered conformation of poly(A) [6,7] Addition of Zac repressor to poly(A) leads to an important decrease of the CD signal without any change in shape. As larger quantities of repressor are added one can notice a small shift of the maximum towards 265 nm ( fig.1).…”

Section: Resultsmentioning

confidence: 95%

“…Therefore it was of interest to know whether lac repressor binds to single stranded nucleic acids and how the conformation of single strands is affected upon lac repressor binding. We chose poly(A) as a model because its conformation is very well known and is characterized by a large CD spectrum [6,7] , and also because this polynucleotide has been used as a model compound in the study of nucleic acid-protein interactions. One could then try Poly (A) was purchased from Miles.…”

Section: Introductionmentioning

confidence: 99%

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Lac repressor binding to single‐stranded polyadenylic acid

Maurizot

Charlier

1977

FEBS Letters

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Section: Resultssupporting

confidence: 76%

Section: Resultsmentioning

confidence: 95%

Section: Introductionmentioning

confidence: 99%

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Lac repressor binding to single‐stranded polyadenylic acid

Maurizot

Charlier

1977

FEBS Letters

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“…This is due to the fact that the first cleavage events produce intermediates which do not give rise to a hyperchromic effect or acid-soluble products (Bernardi, 1971). Changes in hyperchromicity, for example, are only observed when the DNA has been degraded such that oligodeoxynucleotides smaller than 10 nucleotides in length are produced (Brahms, 1966). For the evaluation of the steady-state parameters from hyperchromicity data we took the maximal slope of an A,,, versus time plot as a substitute for the initial velocity of the reaction.…”

Section: Resultsmentioning

confidence: 99%

Kinetic Analysis of the Cleavage of Natural and Synthetic Substrates by the Serratia Nuclease

Friedhoff

Meiß

Kolmes

et al. 1996

European Journal of Biochemistry

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The extracellular nuclease from Serratia marcescens is a non-specific endonuclease that hydrolyzes double-stranded and single-stranded DNA and RNA with high specific activity. Steady-state and presteady-state kinetic cleavage experiments were performed with natural and synthetic DNA and RNA substrates to understand the mechanism of action of the Serrutia nuclease. Most of the natural substrates are cleaved with similar k,,, and K,,, values, the k,,,lK,,, ratios being comparable to that of staphylococcal nuclease. Substrates with extreme structural features, like poly(dA) . poly(dT) or poly(dG) . poly(dC), are cleaved by the Serratia nuclease with a 50 times higher or 10 times lower K,,,, respectively, as salmon testis DNA. Neither with natural DNA or RNA nor synthetic oligodeoxynucleotide substrates did we observe substrate inhibition for the Serratia nuclease as reported recently. Experiments with short oligodeoxynucleotides confirmed previous results that for moderately good cleavage activity the substrate should contain at least five phosphate residues. Shorter substrates are still cleaved by the Serratia nuclease, albeit at a rate reduced by a factor of more than 100. Cleavage experiments with oligodeoxynucleotides substituted by a single phosphorothioate group showed that the negative charge of the pro-R,-oxygen of the phosphate group 3' adjacent to the scissile phosphodiester bond is essential for cleavage, as only the R,-phosphorothioate supports cleavage at the 5' adjacent phosphodiester bond. Furthermore, the modified bond itself is only cleaved in the R,-diastereomer, albeit 1000 times more slowly than the corresponding unmodified phosphodiester bond, which offers the possibility to determine the stereochemical outcome of cleavage. Pre-steady-state cleavage experiments demonstrate that it is not dissociation of products but association of enzyme and substrate or the cleavage of the phosphodiester bond that is the rate-limiting step of the reaction. Finally, it is shown that Serratia nuclease accepts thymidine 3',5'-bis(p4trophenyl)phosphate as a substrate and cleaves it at its 5'-end to produce nitrophenol and thymidine 3'-@-nitrophenylphosphate) 5-phosphate. The rate of cleavage of this artificial substrate, however, is 6-7 orders of magnitude smaller than the rate of cleavage of macromolecular DNA or RNA.

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“…Incubation, 60 min at 31 "C bovine ribonuclease A, preliminary experiments indicate that in this case too the phenomenon might be structure-dependent. Under conditions (slightly alkaline pH, very low ionic strength, 37 "C) where no double-stranded structure of poly(A) is present [43] and only some degree of stacking interactions should be possible, the action of pancreatic ribonuclease A is actually higher than that of ribonuclease BS-1 [44] and ribonuclease A dimers (Palmieri and Libonati, unpublished). This, on the other hand, may be not surprising since ribonuclease BS-1 [10,11] and aggregates of ribonuclease A [16,35] have, in terms of Kunitz units [45] with yeast RNA as substrate, about half the specific activity of pancreatic ribonuclease A.…”

Section: Guinea Pig a Pancreliicmentioning

confidence: 99%

Basic Charges on Mammalian R bonuclease Molecules and the Ability to Attack Double‐Stranded. RNA

Libonati

Furia

Beintema

1976

European Journal of Biochemistry

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A ribonuclease recently isolated from the pancreas of the lesser rorqual or pike whale (Baluenoptera acutorostrata), definitely more basic than bovine ribonuclease A, degrades very efficiently doublestranded RNA under conditions where the action of bovine pancreatic ribonuclease is minimal.On the contrary, a ribonuclease from the pancreas of reindeer, definitely less basic than bovine ribonuclease A, is also significantly less active than the bovine enzyme at degrading the doublehelical RNA.Two ribonucleases (from the pancreas of guinea pig, the A component, and red deer), with a net charge and a number of basic amino acids similar to those of bovine pancreatic ribonuclease, show an activity on double-stranded RNA which is indistinguishable from that of bovine ribonuclease A.Finally, a ribonuclease from the pancreas of rat, having a net charge equal to that of bovine ribonuclease A, but three basic residues more than the latter enzyme, shows a modest activity towards the double-helical RNA.These results are compatible with the idea that the action on secondary structures of RNA by ribonucleases non-specific for double-stranded RNA may be related to the number (and possibly the position) of basic charges on the protein molecules. Double-stranded RNA's are specifically degraded by ribonuclease 111 from Escherichiu coli [l -41, whereas the action on them of bovine pancreatic ribonuclease A is negligible, provided the ionic strength is high enough to maintain the secondary structure of the nucleic acids [5,6]. Resistance of RNA to digestion by ribonuclease A under standard conditions [7] is generally assumed, in fact, as one of the criteria to assess that the RNA has a stable secondary structure.Other nucleases have been described as capable of degrading double-stranded as well as single-stranded RNA [S, 91. Among them, bovine seminal ribonuclease (ribonuclease BS-l), a basic (PI = 10.3), dimeric protein [lo-121, in many respects similar to ribonuclease A [lo, 113, definitely degrades double-helical polyribonucleotides [13].Abbreviations. NaCl/Cit, 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7; poly(A), polyadenylate; poly(A) . poly(U) and poly(G) . poly(C), the complexes formed between polyadenylate and polyuridylate, and between polyguanylate and polycytidylate, respectively; poly(rUj . poly(dA), the complex formed by polyribouridylate and polydeoxyriboadenylate.Enzymes. Ribonuclease TI (EC 3.1.4.8); bovine pancreatic ribonuclease A (EC 3.1.4.22); ribonuclease 111 (EC 3.1.4.24). ~-Artificial dimerization [14-161 of bovine ribonuclease A makes this enzyme also capable of significantly depolymerizing double-stranded RNA's [13, 15-171. The dimeric nature of a ribonuclease active on double-stranded RNA, with the simultaneous availability of two active sites on its molecule, was consequently thought to be responsible for the activity towards double-helical polyribonucleotides [ 3 8,191. However, recent results (degradation of DNA . RNA hybrids by ribonuclease , and by aggregates of ribonuclease A [21], the main...

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Adenylate oligomers in single- and double-strand conformation

Cited by 404 publications

References 26 publications

Lac repressor binding to single‐stranded polyadenylic acid

Lac repressor binding to single‐stranded polyadenylic acid

Kinetic Analysis of the Cleavage of Natural and Synthetic Substrates by the Serratia Nuclease

Basic Charges on Mammalian R bonuclease Molecules and the Ability to Attack Double‐Stranded. RNA

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