Ethidium binding sites on plasmid DNA determined by photoaffinity labeling.

Hardwick, J. Marie; Sprecken, R.Suzanne von; Yielding, K. Lemone; Yielding, Lerena W.

doi:10.1016/s0021-9258(18)90626-5

Cited by 23 publications

(4 citation statements)

References 27 publications

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“…The inhibition of Mbol activity on B-form BZ-III by actinomycin, ametantrone, and ethidium is expected and is similar to that observed previously with various restriction enzymes and DNA-binding molecules (Hardwick et al, 1984;Ushay et al, 1981; Barton & Paranawithana, 1986; Mallamaci et al, 1991). Inhibition by these agents of Mbol cleavage of BZ-III containing both B and Z conformations is also observed.…”

Section: Discussionsupporting

confidence: 86%

Enhanced reactivity of a B-Z junction for cleavage by the restriction enzyme MboI

Winkle

Aloyo²,

Morales³

et al. 1991

Biochemistry

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We have been investigating the structure, dynamics, and ligand-binding properties of the interface that exists between a right-handed conformation and a left-handed conformation (i.e., a B-Z junction) in synthetic DNA oligomers. Since exo- and endonuclease activity is known to be sensitive to the conformation of the template DNA, we have designed and synthesized a DNA oligonucleotide of 20 base pairs (designated as BZ-III) with an MboI recognition site (GATC) at the location of a potential B-Z junction. The activity of the MboI enzyme toward this molecule and DNA oligomers that contain multiple MboI sites located at B-Z junctions was monitored in the absence and presence of the Z-conformation-inducing reagent cobalt hexaammine. In all cases, the activity of the enzyme was enhanced in the presence of cobalt hexaammine. The activity of MboI toward BZ-III, in the presence and absence of cobalt hexaammine, was also examined when the DNA oligomer is also in the presence of the DNA binding drugs actinomycin D, ametantrone, or ethidium bromide. In all cases, the activity of the enzyme was inhibited in the presence of drug. The results suggest that B-Z junctions are structurally unique and that this uniqueness may alter nuclease activity at sites in or near the junction.

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Section: Discussionsupporting

confidence: 86%

Enhanced reactivity of a B-Z junction for cleavage by the restriction enzyme MboI

Winkle

Aloyo²,

Morales³

et al. 1991

Biochemistry

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“…The most likely explanation for this difference is twofold: drug destabilization of the NaeI-L43K-DNA complex and the transient nature of the covalent NaeI-L43K-DNA intermediate. Intercalating drugs destabilize the interaction of restriction enzymes with DNA (Goppelt et al, 1981;Hardwick et al, 1984). DNA relaxation reactions without inhibitor present (Jo & Topal, 1995).…”

Section: Resultsmentioning

confidence: 99%

Changing a Leucine to a Lysine Residue Makes NaeI Endonuclease Hypersensitive to DNA Intercalative Drugs

Topal

1996

Biochemistry

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A single amino acid change transforms restriction enzyme NaeI to a topoisomerase and recombinase (NaeI-L43K) that shows no sequence similarity to these protein families. This transformation appears to result from coupled endonuclease and ligase domains. To further elucidate the relationship between NaeI-L43K and the topoisomerase protein family, we studied the effect of the topoisomerase inhibitors on NaeI-L43K activity. The intercalative drugs amsacrine, ellipticine, and daunorubicin inhibited NaeI-L43K, whereas the nonintercalating drugs camptothecin, VP-16, and oxolinic acid did not. Ethidium bromide also inhibited NaeI-L43K, implying that intercalation is responsible for its inhibition. The effects of the intercalative drugs on the DNA cleavage steps of NaeI and NaeI-L43K were compared. The drugs hardly inhibited DNA cleavage by wild type NaeI but completely inhibited DNA cleavage by NaeI-L43K. This difference in inhibition demonstrates that the L43K amino acid change sensitized NaeI to these drugs. Low concentrations of the intercalative drugs, except for ethidium bromide, enhance production of topoisomerase--DNA covalent intermediates but inhibited production of the NaeI-L43K--DNA covalent intermediate. These results imply some unique differences between DNA relaxation by NaeI-L43K and DNA topoisomerase. Concomitant with studying inhibition of the cleavage intermediate, NaeI-L43K was found to covalently bond with the 5' end of the cleaved DNA strand.

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“…In a clear concentration-dependent fashion, increasing amounts of ligand resulted in the binding of multiple units of ethidium bromide and mitoxantrone. For these ligands, a maximum stoichiometry of three units was observed, consistent with the number of possible binding sites present between adjacent GC pairs, which are particularly favorable to intercalation (21,22). The minor groove binder distamycin A afforded lower stoichiometries and reduced affinity, which is not surprising as the Ty3 PPT lacks contiguous AT base pairs preferred by this class (23).…”

Section: Resultsmentioning

confidence: 57%

Probing Anomalous Structural Features in Polypurine Tract-Containing RNA−DNA Hybrids with Neomycin B

et al. 2009

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During (−)-strand DNA synthesis in retroviruses and Saccharomyces cerevisiae LTR retrotransposons, a purine rich region of the RNA template, known as the polypurine tract (PPT), is resistant to RNase H-mediated hydrolysis and subsequently serves as a primer for (+)-strand, DNAdependent DNA synthesis. Although HIV-1 and Ty3 PPT sequences share no sequence similarity beyond the fact that both include runs of purine ribonucleotides, it has been suggested that these PPTs are processed by their cognate reverse transcriptases (RTs) through a common molecular mechanism. Here, we have used the aminoglycoside neomycin B (NB) to examine which structural features of the Ty3 PPT contribute to specific recognition and processing by its cognate RT. Using high-resolution NMR, direct infusion FTICR mass spectrometry, and isothermal titration calorimetry, we show that NB binds preferentially and selectively adjacent to the Ty3 3′ PPT-U3 cleavage junction and in an upstream 5′ region where the thumb subdomain of Ty3 RT putatively grips the substrate. Regions highlighted by NB on the Ty3 PPT are similar to those previously identified on the HIV-1 PPT sequence that are implicated as contact points for substrate binding by its RT. Our findings thus support the notion that common structural features of lentiviral and LTRretrotransposon PPTs facilitate the interaction with their cognate RT.

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Ethidium binding sites on plasmid DNA determined by photoaffinity labeling.

Cited by 23 publications

References 27 publications

Enhanced reactivity of a B-Z junction for cleavage by the restriction enzyme MboI

Enhanced reactivity of a B-Z junction for cleavage by the restriction enzyme MboI

Changing a Leucine to a Lysine Residue Makes NaeI Endonuclease Hypersensitive to DNA Intercalative Drugs

Probing Anomalous Structural Features in Polypurine Tract-Containing RNA−DNA Hybrids with Neomycin B

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