DNA Conformational analysis in solution by uranyl mediated photocleavage

Nielsen, Peter E.; Møllegaard, Niels Erik; Jeppesen, Claus

doi:10.1093/nar/18.13.3847

Cited by 38 publications

(43 citation statements)

References 16 publications

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“…Therefore, we chose to use the kinetoplast DNA to further analyse whether polyamines preferentially bind to or distort the structure of curved A-tracts as was suggested by the result of the tyr T fragment. As expected in the absence of polyamine, the uranyl cleavage intensity in each A-tract of this fragment is increasing from the 5′ end towards the 3′ end (40,42) (Figure 2A). However, in the presence of spermidine the strongly cleaved positions in the 3′ end of each A-tract are dramatically reduced.…”

Section: Resultssupporting

confidence: 80%

“…Uranyl photo-probing can be employed for analysing protein–DNA interactions and drug–DNA interactions (36,37), for the detection of metal-ion-binding sites in nucleic acids (38,39), as well as for probing the structure and conformation of double-stranded DNA, including A-tract-induced DNA bending (40–43). This makes uranyl photo-probing more versatile in comparison with the other probing techniques.…”

Section: Introductionmentioning

confidence: 99%

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Polyamines preferentially interact with bent adenine tracts in double-stranded DNA

Lindemose

Nielsen

Møllegaard

2005

Nucleic Acids Research

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Polyamines, such as putrescine, spermidine and spermine, have indirectly been linked with the regulation of gene expression, and their concentrations are typically increased in cancer cells. Although effects on transcription factor binding to cognate DNA targets have been demonstrated, the mechanisms of the biological action of polyamines is poorly understood. Employing uranyl photo-probing we now demonstrate that polyamines at submillimolar concentrations bind preferentially to bent adenine tracts in double-stranded DNA. These results provide the first clear evidence for the sequence-specific binding of polyamines to DNA, and thereby suggest a mechanism by which the cellular effects of polyamines in terms of differential gene transcriptional activity could, at least partly, be a direct consequence of sequence-specific interactions of polyamines with promoters at the DNA sequence level.

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Section: Resultssupporting

confidence: 80%

Section: Introductionmentioning

confidence: 99%

Polyamines preferentially interact with bent adenine tracts in double-stranded DNA

Lindemose

Nielsen

Møllegaard

2005

Nucleic Acids Research

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“…In addition to the nucleases and metal chelates mentioned in the Results we also attempted to use uranyl nitrate as a photofootprinting probe. Uranyl-mediated photocleavage represents a sensitive method for studying protein-DNA phosphate contacts (14,45), DNA conformation ( 46,47), triple-helix formation ( 48) and interaction with minor groove binding drugs such as mithramycin ( 49) and SN6999 (Bailly and Waring, unpublished). However, the uranyl probe failed to footprint echinomycin in our hands (as it also failed for others: 49).…”

Section: Discussionmentioning

confidence: 99%

Comparison of Different Footprinting Methodologies for Detecting Binding Sites for a Small Ligand on DNA

Bailly

Waring

1995

Journal of Biomolecular Structure and Dynamics

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In order to assess the utility of different methods of footprinting applied to the study of sequence-selective small molecule-DNA interaction we have performed a homologous series of experiments on the binding of echinomycin, a bis-intercalator, to a 133 base pair DNA restriction fragment containing a small number of discrete binding sites. Two of those sites each contained a pair of closely clustered CpG steps, the cognate dinucleotide sequence which is the common denominator of sites recognised by echinomycin. DNAse I was found to be much the best enzyme for footprinting in terms of sensitivity, accuracy, and ease of handling. DNAase II and micrococcal nuclease were of limited value. Excellent results were recorded with methidiumpropyl-EDTA.FeII which picked up strong binding sites and yielded sharp footprints from which a parsimonious estimate of site size could be determined. Orthophenanthroline.CuI proved to be a very suitable, sensitive chemical nuclease but hydroxyl radical footprinting with EDTA.FeII was only partially successful. Positive footprinting with conformation-sensitive probes diethylpyrocarbonate, osmium tetroxide and potassium permanganate yielded information to complement that afforded by the enzymic and chemical nucleases. Evidence of binding to both CpG steps in the clustered pair was obtained, with indications of possible cooperativity.

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“…Ten fmol of TIF-IB in HEG 10 buffer (20 mM HEPES, pH 7.5, 0.1 mM EDTA, and 10% glycerol) or buffer alone was added to the reaction and incubated for 20 min at 25°C. Conditions for the UO 2 (NO 3 ) 2 cutting reactions and precipitations were modified from published procedures (41,42 (43,44). After the addition of 30 l of 1 mg/ml proteinase K and 0.1% SDS solution, samples were incubated at 50°C for 30 min.…”

Section: Methodsmentioning

confidence: 99%

The Fundamental Ribosomal RNA Transcription Initiation Factor-IB (TIF-IB, SL1, Factor D) Binds to the rRNA Core Promoter Primarily by Minor Groove Contacts

Geiss¹,

Radebaugh

Paule

1997

Journal of Biological Chemistry

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Acanthamoeba castellanii transcription initiation factor-IB (TIF-IB) is the TATA-binding protein-containing transcription factor that binds the rRNA promoter to form the committed complex. Minor groove-specific drugs inhibit TIF-IB binding, with higher concentrations needed to disrupt preformed complexes because of drug exclusion by bound TIF-IB. TIF-IB/DNA interactions were mapped by hydroxyl radical and uranyl nitrate footprinting. TIF-IB contacts four minor grooves in its binding site. TIF-IB and DNA wrap around each other in a right-handed superhelix of high pitch, so the upstream and downstream contacts are on opposite faces of the helix. Dimethyl sulfate protection assays revealed limited contact with a few guanines in the major groove. This detailed analysis suggests significant DNA conformation dependence of the interaction.Extensive in vitro and in vivo analyses have shown that efficient transcription of the rRNA gene by RNA polymerase I requires formation of a stable transcription factor-promoter DNA complex, the committed complex (for review, see Refs. 1 and 2). In most species, two regions of the rRNA promoter, the core promoter element (CPE) 1 and upstream promoter element (UPE), participate in expression of the rRNA gene and formation of the committed complex. The CPE is located between Ϫ50 and ϩ10 relative to the transcription start site (position ϩ1), is absolutely required in all organisms, and is sufficient in some for initiation. It is the primary binding site for the TBPcontaining transcription factor. The UPE is located farther upstream, from approximately Ϫ150 to Ϫ110, and acts primarily to stimulate transcription. It has a variable requirement between species; at one end of the spectrum, Acanthamoeba castellanii has no UPE discernible in vitro (3). We (4) and others (5) have shown recently that the CPE can be subdivided into an element that interacts intimately with TIF-IB and an element that functions like the initiator element (Inr) of some RNA polymerase II promoters, interacting with a specific TAF I .The UPE and CPE are important because they serve as the binding sites for two RNA polymerase I transcription factors, UBF and TIF-IB (also known as SL1, factor D, Rib1, or corebinding factor) respectively. These two proteins interact, by mechanisms that are still unclear but apparently involve DNA looping (6), to form a stable initiation complex capable of directing specific RNA polymerase I recruitment through multiple rounds of transcription. The need for UBF is variable between species; UBF is required in human and Xenopus (7-9), but is dispensable in rat (10), mouse (11), yeast (12-14), and A. castellanii (15). Therefore, TIF-IB is, by elimination, the fundamental transcription factor for rRNA genes, recruiting RNA polymerase I in the next step of the initiation process (16).TIF-IB has recently been purified to homogeneity from several eukaryotic species, including human (17), mouse (18), yeast (12,14), and A. castellanii.2 Biochemical analysis has shown that TIF-IB consists of TBP an...

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DNA Conformational analysis in solution by uranyl mediated photocleavage

Cited by 38 publications

References 16 publications

Polyamines preferentially interact with bent adenine tracts in double-stranded DNA

Polyamines preferentially interact with bent adenine tracts in double-stranded DNA

Comparison of Different Footprinting Methodologies for Detecting Binding Sites for a Small Ligand on DNA

The Fundamental Ribosomal RNA Transcription Initiation Factor-IB (TIF-IB, SL1, Factor D) Binds to the rRNA Core Promoter Primarily by Minor Groove Contacts

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