Biomedical subjects

K Waterloh

Publications and source records attributed to K Waterloh.

6 recordsLinked to original sources

PubMed1992

Secondary (non-GpC) binding sites for actinomycin on DNA.

Actinomycin D has long been known to bind selectively to the dinucleotide step GpC. We have investigated its ability to bind to other non-canonical sequences using a series of synthetic DNA fragments. DNase I footprinting experiments reveal that actinomycin can also bind well to GG (CC). Binding to this sequence and the canonical GC site is potentiated by flanking regions of (GT)n.(AC)n. Weaker but specific binding to GT and AC is also evident and appears to be cooperative.

Base Sequence↗

PubMed1992

Bifunctional intercalator [N-MeCys3,N-MeCys7]TANDEM binds to the dinucleotide TpA.

The binding of [N-MeCys3,N-MeCys7]TANDEM has been examined by DNase I footprinting and diethyl pyrocarbonate modification of several synthetic DNA fragments containing AT-rich regions. DNase I footprinting reveals that at low concentrations the ligand binds preferentially to the center of (AT)n regions. A fragment containing the tetranucleotide AATT was unaffected by the ligand. Diethyl pyrocarbonate modification of several fragments containing blocks of (AT)n revealed a pattern in which alternate adenines were rendered more reactive in the presence of the ligand. These reactive adenines were staggered across the two DNA strands in the 3'-direction, consistent with ligand binding to the dinucleotide TpA. In sequences of the type (TAA)n.(TTA)n, binding of [N-MeCys3,N-MeCys7]TANDEM resulted in strong modification of the second adenine in the sequence TAA, i.e., the base on the 3'-side of the ligand binding site. Data for binding to (AT)n are best explained by suggesting that the adenines sandwiched between the quinoxaline chromophores are rendered most reactive to diethyl pyrocarbonate.

Base Sequence↗

PubMed1991

Interaction of echinomycin with An.Tn. and (AT)n regions flanking its CG binding site.

We have prepared DNA fragments containing the sequences A15CGT15, T15CGA15 and T(AT)8CG(AT)15 cloned within the SmaI site of the pUC19 polylinker. These have been used as substrates in footprinting experiments with DNase I and diethylpyrocarbonate probing the effects of echinomycin, binding to the central CG, on the structure of the surrounding sequences. No clear DNase I footprints are seen with T15CGA15 though alterations in the nuclease susceptibility of surrounding regions suggest that the ligand is binding, albeit weakly at this site. All the other fragments show the expected footprints around the CG site. Regions of An and Tn are rendered much more reactive to DNase I and adenines on the 3'-side of the CG become hyperreactive to diethylpyrocarbonate. Regions of alternating AT show unusual changes in the presence of the ligand. At low concentrations (5 microM) cleavage of TpA is enhanced, whereas at higher concentrations a cleavage pattern with a four base pair repeat is evident. A similar pattern is seen with micrococcal nuclease. Modification by diethylpyrocarbonate is strongest at alternate adenines which are staggered in the 5'-direction across the two strands. We interpret these changes by suggesting secondary drug binding within regions of alternating AT, possibly to the dinucleotide ApT. DNase I footprinting experiments performed at 4 degrees C revealed neither enhancements nor footprints for flanking regions of homopolymeric A and T suggesting that the conformational changes are necessary consequence of drug binding.

Amino Acid Sequence↗

PubMed1991

Echinomycin binding to alternating AT.

We have studied the binding of echinomycin to DNA fragments containing GC-rich regions flanked by blocks of alternating AT by DNase I footprinting and diethylpyrocarbonate modification. Regions of alternating AT flanking the sequences CCCG, CCGC, CGGC and GG show a four base pair DNase I cleavage pattern and reaction of alternate adenines with diethylpyrocarbonate. This pattern is strongest when the AT-block is immediately adjacent to the CpG ligand binding site. We explain these phenomena by suggesting that echinomycin binds to the dinucleotide step ApT in a cooperative fashion. The cooperative effects can be transmitted through the dinucleotide step GC but not CC or AA. No such repetitive patterns are seen with surrounding regions of (ATT).(AAT). Evidence is presented for secondary drug binding sites at CpC and TpG with weaker interaction at the CpG site within the hexanucleotide TTCGAA.

Base Sequence↗

PubMed1991

The effects of actinomycin on the structure of dAn.dTn and (dA-dT)n regions surrounding its GC binding site. A footprinting study.

The effect of actinomycin on the structure of DNA fragments containing the sequences (AT)5GC(AT)5, (TA)5GC(TA)5, A9GCT9, and T9GCA9, cloned into the SmaI site of pUC19, has been studied by footprinting analysis using a variety of probes known to be sensitive to DNA structure. In each case clear footprints are found around the central GC sites. DNase I cleavage of fragments containing alternating AT shows much greater cutting at ApT than TpA; in the presence of actinomycin, although this preference is retained, there is a large increase in the cutting efficiency at the closest TpA steps. DNase I cleavage in homopolymeric regions of A and T, which is normally very poor, is greatly enhanced by drug binding. With T9GCA9 the enhancements are propagated in both directions, whereas changes are only found to the 5'-side of the GC site in A9GCT9. The results are confirmed by similar experiments with micrococcal nuclease and DNase II. Small increases in sensitivity to diethylpyrocarbonate are found at adenines proximal to GC. Experiments performed at 4 degrees C suggest that conformational changes are a necessary consequence of drug binding.

Base Sequence↗

PubMed1990

Effect of actinomycin on a (TA)6 plasmid insert.

The effect of actinomycin on DNA structure has been studied by nuclease digestion of a DNA fragment containing the sequence AAGCT(TA)6AGCTT. The drug enhances DNase I cleavage at ApT and TpA bonds closest to the drug binding site (GC). Large changes in the relative susceptibility of bonds to cleavage by micrococcal nuclease and DNase II are also observed. The changes suggest that actinomycin alters DNA structure around its binding site, facilitating the formation of an alternating DNA structure. When a similar TA sequence was inserted further from the actinomycin binding site no changes in nuclease susceptibility were observed.

Autoradiography↗