PubMed · 2121209
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M Markman. 1990. Supportive care.. https://pubmed.ncbi.nlm.nih.gov/2121209/
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We have examined the kinetics of dissociation of echinomycin from CpG sites in several DNA fragments containing synthetic DNA inserts by a variation of the footprinting technique. Complexes of the ligand with radiolabeled DNA fragments were dissociated by adding an excess of unlabeled calf thymus DNA. Samples were removed from this mixture at subsequent time intervals and subjected to DNase I footprinting. The rate of disappearance of the footprints varied considerably between the various CpG sites. At 20 degrees C, echinomycin dissociates more slowly from CpG sites flanked by (AT)n (t1/2 approximately 40 min) and (CA)n.(TG)n (t1/2 approximately 11 min) than by An.Tn (t1/2 < 3 min). In each sequence context the dissociation from ACGT is slower than that from TCGA. (TAA)4CG(TTA)4 also represents a very good binding site (t1/2 approximately 35 min), which is less sensitive to changes in temperature than most other sites. Within sequences (AT)10(G/C)4(AT)10, the dissociation from CGGC is slower than that from CCCG or CCGC.
Defects in DNA, e.g., unpaired/bulged nucleotides, are repaired by specific repair enzymes. Understanding the dynamics and structure of DNA defects is important. Two DNA heptamers, CTb-GTACG and CGTACTbG, each containing a bulged T nucleotide embedded in the CpG step, have been studied by NMR. Both duplexes are significantly destabilized, and the bulged T remains intrahelical. Binding of the anthracycline antitumor antibiotic nogalamycin (Ng) to these two heptamers stabilizes the duplex structure. The solution structures of the 2:1 complexes of Ng-d(CTbGTACG) and Ng-d(CGTACTbG) have been determined by the NOE-restrained refinement procedure. In both structures the elongated aglycon of Ng is intercalated between base pairs, and the nogalose and aminoglucose lie in the minor and major grooves, respectively. The bulged T behaves differently upon the binding of Ng. In Ng-CTbGTACG wobble G6:Tb base pairs are formed, leaving two dangling 5'-C1 nucleotides; whereas in Ng-CGTACTbG weak C1:Tb base pairs are formed, leaving two dangling 3'-G6 nucleotides. Thus Ng induces the bulged T and the opposing base in the duplex to stack on the aglycon and causes the base next to Tb to unpair, mimicking a "frame-shift". Such structural rearrangement of a bulged DNA site due to the binding of an intercalator drug may perturb the recognition of DNA defects by repair enzymes or may cause mutation during replication.
The WAF1/CIP1 protein has been identified as a downstream mediator of the tumor suppressor p53 in regulating cell cycle progression through a G1-phase check-point. Recent work has implicated the functional status of p53 as a critical determinant in the apoptotic response of certain cell lines to DNA damaging agents. By using human T-cell leukemia virus type I-transformed lymphoid cell lines that differ in their level and function of wild-type p53, we investigated the induction of WAF1/CIP1 and apoptosis after exposure to Adriamycin, a genotoxic agent. We found that regardless of the p53 status in these cell lines, WAF1/CIP1 RNA was rapidly induced in response to Adriamycin treatment. An elevated level of WAF1/CIP1 protein was observed as well. Additionally, we demonstrated that apoptosis was induced in all cell lines analyzed despite some having functionally inactive p53 protein. Our data suggest that a p53-independent pathway may play a role in the apoptotic response observed in some cell lines after exposure to DNA damaging agents.