Anthramycin inhibition of restriction endonuclease cleavage and its use as a reversible blocking agent in DNA constructions

Sumner, Walton; Bennett, George N.

doi:10.1093/nar/9.9.2105

Cited by 25 publications

(20 citation statements)

References 20 publications

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“…Alkaline phosphatase, DNAase I, and polynucleotide kinase were obtained from Boehringer Mannheim, Indianapolis, IN. The BspR I preparation was reported previously (20). E. coli RNA polymerase was prepared by the procedures of Burgess and Jendrisak (21) and Lowe,et al (22).…”

Section: Methodsmentioning

confidence: 99%

Characterization of the β-lactamase promoter of pBR322

Russell

Bennett

1981

Nucl Acids Res

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The beta-lactamase promoter of pBR322, derived from Tn3, has been characterized using several techniques. The transcription initiation site is located 35 base pairs from the translation initiation codon of beta-lactamase. The mRNA produced in vitro has a 5' pppGpA terminus. RNA polymerase bound at this start site protects a region from about -50 to +20 from DNase I cleavage using the footprinting technique. RNA polymerase binds rapidly to the beta-lactamase promoter. The half-time of association is less than one-half minute. The half-time of dissociation is approximately 6 hr. A study of the binding of RNA polymerase at different temperatures showed a large change between 11 degrees and 15 degrees C. Comparison of these parameters with those reported for other promoters is discussed.

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Section: Methodsmentioning

confidence: 99%

Characterization of the β-lactamase promoter of pBR322

Russell

Bennett

1981

Nucl Acids Res

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“… The N 4 ‐acyl‐modified dNTPs used in this study: N 4 ‐acetyl‐2′‐deoxycytidine 5′‐triphosphate (dC Ac TP), N 4 ‐hexanoyl‐2′‐deoxycytidine 5′‐triphosphate (dC Hex TP), N 4 ‐nicotinoyl‐2′‐deoxycytidine 5′‐triphosphate (dC Nic TP), N 4 ‐(2‐acetylbenzoyl)‐2′‐deoxycytidine 5′‐triphosphate (dC AcBz TP) and N 4 ‐(2‐benzoylbenzoyl)‐2′‐deoxycytidine 5′‐triphosphate (dC BzBz TP). dC Hex TP, dC Nic TP, dC AcBz TP and dC BzBz TP were synthesised and reported previously …”

Section: Resultsmentioning

confidence: 99%

“…Precise control over the demodification process is required and results in two major concerns: only labile modifications can be attached to predetermined positions of a nucleobase and the rest of the nucleic acid molecule must remain intact. The first example of reversible DNA protection against cleavage by restriction enzymes was based on the antibiotic anthramycin, a minor groove DNA binder, which covalently bound to the exocyclic amino group of guanine, and was shown to provide resistance to digestion by a number of REs . Shifting the pH to an acidic environment removes anthramycin, which leaves the DNA susceptible to cleavage.…”

Section: Introductionmentioning

confidence: 99%

Transient N⁴‐Acyl‐DNA Protection against Cleavage by Restriction Endonucleases

2019

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A set of five N4‐acyl‐modified 2′‐deoxycytidine 5′‐triphosphates were incorporated into modified DNA by using phi29 DNA polymerase, and cleavage by selected restriction endonucleases was studied. Modified DNA containing N4‐acyl functional groups in either one or both strands of a DNA molecule was resistant to the majority of restriction enzymes tested, whereas modifications outside of the recognition sequences were well tolerated. The N4‐acylated cytidine derivatives were subjected to competitive nucleotide incorporation by using phi29 DNA polymerase, showing that a high‐fidelity phi29 DNA polymerase efficiently used the modified analogues in the presence of its natural counterpart. These N4 modifications were also demonstrated to be easily removed in an aqueous ethanolamine solution, in which all steps, including primer extension, demodification, and cleavage by restriction endonuclease, could be performed in a one‐pot procedure that eliminated additional purification stages. It is suggested that N4‐modified nucleotides are promising building blocks for a programmable; transient; and, most importantly, straightforward DNA protection against specific endonucleases.

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“…It is also known that distamycin, netropsin and sibiromycin prevent cleavage of DNA by a number of restriction enzymes (52,53).…”

Section: Discussionmentioning

confidence: 99%

Interaction of Lambda cro Repressor with Synthetic Operator 0_R3 Studied by Competition Binding with Minor Groove Binders

Gursky

Surovaya

Kurochkin

et al. 1992

Journal of Biomolecular Structure and Dynamics

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In the present work, we employ a combination of CD spectroscopy and gel retardation technique to characterize thermodynamically the binding of lambda phage cro repressor to a 17 base pair operator OR3. We have found that three minor groove-binding antibiotics, distamycin A, netropsin and sibiromycin, compete effectively with the cro for binding to the operator OR3. Among these antibiotics, sibiromycin binds covalently to DNA in the minor groove at the NH2 of guanine, whereas distamycin A and netropsin interact preferentially with runs of AT base pairs and avoid DNA regions containing guanine bases in the two polynucleotide strands. Only subtle DNA conformation changes are known to take place upon binding of these antibiotics. Both the CD spectral profiles and the results of the gel retardation experiments indicate that distamycin A and netropsin can displace cro repressor from the operator OR3. The binding of cro repressor to the OR3 is accompanied by considerable changes in CD in the far-UV region which appear to be attributed to a DNA-dependent structural transition in the protein. Spectral changes are also induced in the wavelength region of 270-290 nm. The CD spectral profile of the cro-OR3 mixture in the presence of distamycin A can be represented as a sum of the CD spectrum of the repressor-operator complex and spectrum of distamycin-DNA complex at the appropriate molar ratio of the bound antibiotic to the operator DNA (r). When r tends to the saturation level of binding the CD spectrum in the region of 270-360 nm approaches a CD pattern typical of complexes of the antibiotic with the free DNA oligomer. This suggests that simultaneous binding of cro repressor and distamycin A to the same DNA oligomer is not possible and that distamycin A and netropsin can be used to determine the equilibrium affinity constant of cro repressor to the synthetic operator from competition-type experiments. The binding constant of cro repressor to the OR3 is found to be (6 +/- 1).10(6)M-1 at 20 degrees C in 10 mM sodium cacodylate buffer (pH 7.0) in the presence of 0.1 M NH4F.

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Anthramycin inhibition of restriction endonuclease cleavage and its use as a reversible blocking agent in DNA constructions

Cited by 25 publications

References 20 publications

Characterization of the β-lactamase promoter of pBR322

Characterization of the β-lactamase promoter of pBR322

Transient N⁴‐Acyl‐DNA Protection against Cleavage by Restriction Endonucleases

Interaction of Lambda cro Repressor with Synthetic Operator 0_R3 Studied by Competition Binding with Minor Groove Binders

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Anthramycin inhibition of restriction endonuclease cleavage and its use as a reversible blocking agent in DNA constructions

Cited by 25 publications

References 20 publications

Characterization of the β-lactamase promoter of pBR322

Characterization of the β-lactamase promoter of pBR322

Transient N4‐Acyl‐DNA Protection against Cleavage by Restriction Endonucleases

Interaction of Lambda cro Repressor with Synthetic Operator 0R3 Studied by Competition Binding with Minor Groove Binders

Contact Info

Product

Resources

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Transient N⁴‐Acyl‐DNA Protection against Cleavage by Restriction Endonucleases

Interaction of Lambda cro Repressor with Synthetic Operator 0_R3 Studied by Competition Binding with Minor Groove Binders