scholarly journals DNA recognition by quinoxaline antibiotics: use of base-modified DNA molecules to investigate determinants of sequence-specific binding of triostin A and TANDEM

1998 ◽  
Vol 330 (1) ◽  
pp. 81-87 ◽  
Author(s):  
Christian BAILLY ◽  
J. Michael WARING
Keyword(s):  
Cyclic Peptide ◽  
Hydrophobic Interactions ◽  
Specific Binding ◽  
Amino Group ◽  
Sequence Specificity ◽  
Bonding Interaction ◽  
Modified Dna ◽  
Antibiotics Use ◽  
Triostin A ◽  
Groove Structure

The methodology of DNAase I footprinting has been adapted to investigate the sequence-specific binding of two quinoxaline drugs to DNA fragments containing natural and modified bases. In order to help comprehend the molecular origin of selectivity in the bis-intercalation of triostin A and TANDEM at CpG and TpA sites respectively, we have specifically examined the effect of the 2-amino group of guanine on their sequence specificity by using DNA in which that group has been either removed from guanine, added to adenine or both. Previous studies suggested that the recognition of particular nucleotide sequences by these drugs might be dependent upon the placement of the purine 2-amino group, serving as a positive or a negative effector for triostin A and TANDEM respectively. However, the footprinting data reported here indicate that this is not entirely correct, since they show that the 2-amino group of guanine is important for the binding of triostin A to DNA but has absolutely no influence on the interaction of TANDEM with TpA steps. Apparently the binding of triostin A to CpG sites is primarily due to hydrogen bonding interaction between the cyclic peptide of the antibiotic and the 2-amino group of guanine residues, whereas the selective binding of TANDEM to TpA sites is not hydrogen-bond driven and probably originates mainly from steric and/or hydrophobic interactions, perhaps involving indirect recognition of a suitable minor groove structure.

scholarly journals Quantitative Analysis of the Binding of Simian Virus 40 Large T Antigen to DNA

2007 ◽  
Vol 81 (17) ◽  
pp. 9162-9174 ◽  
Author(s):  
Amélie Fradet-Turcotte ◽  
Caroline Vincent ◽  
Simon Joubert ◽  
Peter A. Bullock ◽  
Jacques Archambault
Keyword(s):  
Specific Binding ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Sequence Specificity ◽  
Large T Antigen ◽  
Sv40 Large T Antigen ◽  
Viral Origin ◽  
Single Stranded Dna ◽  
Terminal Domain

ABSTRACT SV40 large T antigen (T-ag) is a multifunctional protein that successively binds to 5′-GAGGC-3′ sequences in the viral origin of replication, melts the origin, unwinds DNA ahead of the replication fork, and interacts with host DNA replication factors to promote replication of the simian virus 40 genome. The transition of T-ag from a sequence-specific binding protein to a nonspecific helicase involves its assembly into a double hexamer whose formation is likely dictated by the propensity of T-ag to oligomerize and its relative affinities for the origin as well as for nonspecific double- and single-stranded DNA. In this study, we used a sensitive assay based on fluorescence anisotropy to measure the affinities of wild-type and mutant forms of the T-ag origin-binding domain (OBD), and of a larger fragment containing the N-terminal domain (N260), for different DNA substrates. We report that the N-terminal domain does not contribute to binding affinity but reduces the propensity of the OBD to self-associate. We found that the OBD binds with different affinities to its four sites in the origin and determined a consensus binding site by systematic mutagenesis of the 5′-GAGGC-3′ sequence and of the residue downstream of it, which also contributes to affinity. Interestingly, the OBD also binds to single-stranded DNA with an ∼10-fold higher affinity than to nonspecific duplex DNA and in a mutually exclusive manner. Finally, we provide evidence that the sequence specificity of full-length T-ag is lower than that of the OBD. These results provide a quantitative basis onto which to anchor our understanding of the interaction of T-ag with the origin and its assembly into a double hexamer.

scholarly journals Bifunctional intercalation and sequence specificity in the binding of quinomycin and triostin antibiotics to deoxyribonucleic acid

1978 ◽  
Vol 173 (1) ◽  
pp. 115-128 ◽  
Author(s):  
J S Lee ◽  
M J Waring
Keyword(s):  
Binding Constant ◽  
Binding Constants ◽  
Sequence Specificity ◽  
Peptide Antibiotics ◽  
Naturally Occurring ◽  
Binding Isotherms ◽  
Triostin A ◽  
Specificity Pattern ◽  
Intercalating Agents ◽  
Binding Curves

Quinomycin C, triostin A and triostin C are peptide antibiotics of the quinoxaline family, of which echinomycin (quinomycin A) is also a member. They all remove and reverse the supercoiling of closed circular duplex DNA from bacteriophage PM2 in the fashion characteristic of intercalating drugs, and the unwinding angle at I 0.01 is, in all cases, almost twice that of ethidium. Thus, as with echinomycin, they can be characterized as bifunctional intercalating agents. For the triostins this conclusion has been confirmed by measurements of changes in the viscosity of sonicated rod-like DNA fragments; the helix extension was found to be almost double that expected for a simple monofunctional intercalation process. For triostin A, further evidence for bifunctionality was derived from the cross-over point of binding isotherms to nicked circular and closed circular bacteriophage-PM2DNA. Binding curves for the interaction of quinomycin C and triostin A with a variety of synthetic and naturally occurring nucleic acids were determined by solvent-partition analysis, but triostin C was too insoluble in aqueous solution to make this method applicable. For quinomycin C the highest binding constant was found with Micrococcus lysodeikticus DNA, and its pattern of specificity among natural DNA species was broadly similar to that of echinomycin, although the binding constants were 2–6 times as large. For triostin A the highest binding constant was again found for M. lysodeikticus DNA, but the specificity pattern was quite different from that of the quinomycins. In particular, triostin A bound better to poly(dA-dT) than to the poly(dG-dC) whereas this order was reversed for quinomycin C. There was also evidence that the binding to poly(dA-dT) might be co-operative in nature. No significant interaction could be detected with poly(dA).poly(dT) or with RNA from Escherichia coli. Poly(dG).poly(dC) gave variable results, depending on the source of the polymer. The different patterns of specificity displayed by the quinomycins and triostins are tentatively ascribed to differences in their conformations in solution.

scholarly journals Sequence specificity in DNA binding is determined by association rather than dissociation

2021 ◽  
Author(s):  
Emil Marklund ◽  
Guanzhong Mao ◽  
Sebastian Deindl ◽  
Johan Elf
Keyword(s):  
Dna Binding ◽  
Genetic Information ◽  
Specific Binding ◽  
Theoretical Framework ◽  
Simple Equation ◽  
Target Sequence ◽  
Sequence Specificity ◽  
Target Search ◽  
Target Binding ◽  
Site Recognition

AbstractSequence-specific binding of proteins to DNA is essential for accessing genetic information. Here, we derive a simple equation for target-site recognition, which uncovers a previously unrecognized coupling between the macroscopic association and dissociation rates of the searching protein. Importantly, this relationship makes it possible to recover the relevant microscopic rates from experimentally determined macroscopic ones. We directly test the equation by observing the binding and unbinding of individual lac repressor (LacI) molecules during target search. We find that LacI dissociates from different target sequences with essentially identical microscopic dissociation rates. Instead, sequence specificity is determined by the efficiency with which the protein recognizes different targets, effectively reducing its risk of being retained on a non-target sequence. Our theoretical framework also accounts for the coupling between off-target binding and unbinding of the catalytically inactive Cas9 (dCas9), showing that the binding pathway can be obtained from macroscopic data.One Sentence SummaryAssociation and dissociation rates are anti-correlated for reactions that include a nonspecific probing step.

scholarly journals Novel DNA Bis-intercalation by MLN944, a Potent Clinical Bisphenazine Anticancer Drug

2004 ◽  
Vol 279 (44) ◽  
pp. 46096-46103 ◽  
Author(s):  
Jixun Dai ◽  
Chandanamalie Punchihewa ◽  
Prakash Mistry ◽  
Aik Teong Ooi ◽  
Danzhou Yang
Keyword(s):  
Dna Binding ◽  
Anticancer Drug ◽  
Topoisomerase I ◽  
Specific Binding ◽  
Binding Mode ◽  
Dependent Manner ◽  
Sequence Specificity ◽  
Phase I Clinical Trials ◽  
Mobility Shift ◽  
Dna Sequence Specificity

The new bisphenazine anticancer drug MLN944 is a novel cytotoxic agent with exceptional anti-tumor activity against a range of human and murine tumor models both invitroand invivo. MLN944 has recently entered Phase I clinical trials. Despite the structural similarity with its parent monophenazine carboxamide and acridine carboxamide anticancer compounds, MLN944 appears to work by a distinct mechanism of inhibiting DNA transcription rather than the expected mechanism of topoisomerase I and II inhibition. Here we present the first NMR structure of MLN944 complexed with d(ATGCAT)2DNA duplex, demonstrating a novel binding mode in which the two phenazine rings bis-intercalate at the 5′-TpG site, with the carboxamide amino linker lying in the major groove of DNA. The MLN944 molecule adopts a significantly unexpected conformation and side chain orientation in the DNA complex, with the N10 on the phenazine ring protonated at pH 7. The phenazine chromophore of MLN944 is very well stacked with the flanking DNA base pairs using the parallel base-stacking intercalation binding mode. The DNA sequence specificity and the groove recognition of MLN944 binding is determined by several site-specific hydrogen bond interactions with the central G:C base pair as well as the favorable stacking interactions with the 5′-flanking thymine. The specific binding site of MLN944 is known to be recognized by a number of important transcription factors. Our electrophoretic gel mobility shift assay results demonstrated that the c-Jun DNA binding to the AP-1 site is significantly inhibited by MLN944 in a dose-dependent manner. Thus, the exceptional biological activity of MLN944 may be due to its novel DNA binding mode leading to a unique mechanism of action.

scholarly journals A quantitative binding model for the Apl protein, the dual purpose recombination-directionality factor and lysis-lysogeny regulator of bacteriophage 186

2020 ◽  
Vol 48 (16) ◽  
pp. 8914-8926
Author(s):  
Erin E Cutts ◽  
J Barry Egan ◽  
Ian B Dodd ◽  
Keith E Shearwin
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Attachment Site ◽  
Binding Energies ◽  
Sequence Specificity ◽  
Binding Studies ◽  
Binding Model ◽  
Specific Binding Sites

Abstract The Apl protein of bacteriophage 186 functions both as an excisionase and as a transcriptional regulator; binding to the phage attachment site (att), and also between the major early phage promoters (pR-pL). Like other recombination directionality factors (RDFs), Apl binding sites are direct repeats spaced one DNA helix turn apart. Here, we use in vitro binding studies with purified Apl and pR-pL DNA to show that Apl binds to multiple sites with high cooperativity, bends the DNA and spreads from specific binding sites into adjacent non-specific DNA; features that are shared with other RDFs. By analysing Apl's repression of pR and pL, and the effect of operator mutants in vivo with a simple mathematical model, we were able to extract estimates of binding energies for single specific and non-specific sites and for Apl cooperativity, revealing that Apl monomers bind to DNA with low sequence specificity but with strong cooperativity between immediate neighbours. This model fit was then independently validated with in vitro data. The model we employed here is a simple but powerful tool that enabled better understanding of the balance between binding affinity and cooperativity required for RDF function. A modelling approach such as this is broadly applicable to other systems.

scholarly journals The use of radiolabelled triostin antibiotics to measure low levels of binding to deoxyribonucleic acid

1983 ◽  
Vol 211 (3) ◽  
pp. 543-551 ◽  
Author(s):  
K R Fox ◽  
A Cornish ◽  
R C Williams ◽  
M J Waring
Keyword(s):  
Cyclic Peptide ◽  
Tight Binding ◽  
Binding Constants ◽  
Biologically Relevant ◽  
Low Concentrations ◽  
Binding Isotherms ◽  
Triostin A ◽  
Ranking Order ◽  
Partition Method ◽  
First Time

Triostin antibiotics, which contain a cyclic peptide with a disulphide bridge, have been prepared by growing Streptomyces triostinicus in the presence of inorganic [35S]-sulphate. The labelled triostin A has been shown to behave in all respects similarly to the authentic natural product and to enable a much more sensitive radiochemical adaptation of the solvent-partition method for determining antibiotic binding to DNA. By this means, binding isotherms at low, biologically relevant levels (down to one antibiotic molecule per gene) have been measured. The results indicate the existence of some tight binding sites in natural DNA species that are preferentially occupied at low concentrations. No evidence has been found for any allosteric transitions provoked by interaction between these antibiotics and natural DNA species, though there is evidence for co-operativity in the binding of triostin A to poly(dA-dT). For the first time accurate isotherms have been determined for the binding of triostin C to DNA; its binding constants for a variety of polydeoxynucleotides are uniformly tighter than those of triostin A but fall into the same ranking order when different species of natural DNA are compared.

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