The solution structure determination of DNA molecules has been an important part of structural biology. NMR solution structures are a complement to structures solved via X-ray crystallography; the two methods are the only ways of obtaining three dimensional coordinates of macromolecules. Because of the nature of the molecule, the solution structure determination of DNA has been a challenging task. Assignments are the first and most important part of NMR structures, and can be simplified for DNA with the use of the rotating frame Overhauser spectroscopy (ROESY) experiment. The ROESY technique can be used for unambiguous assignments of H2' and H2" protons and for distinguishing the three main forms of DNA duplexes: A-form, B-form and Z-form. Many types of DNA have been examined using NMR spectroscopy, including drug-bound DNA complexes. Most previous studies of complexes of the anti-cancer drug Actinomycin D (ActD) and DNA used self- complementary sequences to identify stabilizing features. The studies presented in this thesis use non-self-complementary DNA hexamers to identify the two orientations in the binding of the asymmetric ActD drug. The largest preference of asymmetric binding was found for the d(CCGCCG)•d(CGGCGG) sequence; however, NMR spectral complications prevented the structure elucidation of this complex. Instead the solution structure was determined for the complex with the next largest orientational preference, ActD:d(CTGCGG)•d(CCGCAG), which has 67% of ActD molecules intercalated with the benzenoid side of ActD in the first strand. The solved structure identifies unusual DNA features, which could be due to the bound drug inducing structural changes to the B-DNA duplex or the presence of conformational motion. For seven of the eight sequences, the orientation of ActD intercalation within the DNA duplex was identified. The largest preference occurs when the benzenoid intercalation site is followed by a guanine. When this guanine is replaced by an inosine, a reduction in the asymmetric binding of ActD is observed, indicating that the guanine NH₂ group plays a role in the intermolecular contacts. Thus, the two orientations of ActD binding are not present in equal concentrations although their structures are similar, and the preference of orientation is influenced by the asymmetric DNA sequence flanking the intercalation site. / Graduation date: 2002

Actinomycin D in low concentrations was suggested to inhibit ribosomal RNA (rRNA) transcription via an extranucleolar mechanism. Actinomycin D was proposed to inhibit unique messenger RNAs (mRNAs) coding for proteins needed for the maintenance of rRNA transcription. According to this hypothesis actinomycin D would bind to specific nonribosomal DNA with high affinity. This hypothesis was investigated by isolating high molecular weight rat liver DNA, digesting it with restriction endonuclease EcoRI, adding [³H] actinomycin D in low concentration, performing RPC-5 chromatography to separate the restriction fragments and subsequent hybridization to rRNA. It was observed that actinomycin D bound to nonribosomal DNA with high affinity. The same experiment was performed with nucleolar DNA. High affinity actinomycin D binding was not observed in nucleolar DNA. Discrete high affinity binding DNA for actinomycin in rat liver DNA was also observed when another restriction endonuclease BamHI was used to cleave rat liver DNA. However, with rat liver DNA digested with restriction endonuclease HindIII, such a high affinity actinomycin D binding DNA was not observed. Actinomycin D was also demonstrated to bind to discrete site(s) in at least four more eukaryotic species (salmon, calf, herring and human) after DNA from these species were digested by EcoRI, labeled actinomycin D added, and RPC-5 chromatography performed. Labeled actinomycin D bound to its high affinity binding DNA was displaced by unlabeled actinomycin D in a concentration range of biological significance. However, six other antitumor agents, (doxorubicin, aclacinomycin, carminomycin, marcellomycin, musettamycin and pyrromycin) which also intercalate into DNA, did not significantly displace labeled actinomycin D from its high affinity binding DNA. Since this high affinity actinomycin D binding DNA is hypothesized to be involved in the inhibition of rRNA transcription, the actinomycin D binding DNA could have a role in the regulation of rRNA transcription. To date this is the first time that a probable regulatory DNA has been characterized by selective drug binding.

Thesis (doctoral)--München, 1983.

No description available.

No description available.

The binding of low concentrations of actinomycin D (Act D) to fixed human metaphase chromosomes was studied using both autoradiographic and immunofluorescent techniques. At the concentration range of 0.001 - 0.1 μg/ml Act D is known to selectively inhibit rRNA synthesis. Although it was previously suggested that at these low concentrations Act D would selectively bind to the ribosomal cistrons, evidence also exists that the drug binds to non-ribosomal DNA, and inhibits rRNA transcription in an indirect fashion. Because of the conflicting data on Act D binding and a lack of focus on biologically relevant concentrations of drug, it was decided to systematically investigate the distribution of the drug binding in low concentrations to chromosomes from 72-hr human lymphocyte cultures. Autoradiographic detection of [³H]Act D bound to chromosomes showed no selective binding of the drug at concentrations that maximally inhibit rRNA synthesis. A new technique was employed using Formvar and potassium chromium sulfate as a pretreatment to autoradiography. This technique permitted simultaneous detection of silver grains and chromosome identification by G-banding. With autoradiographic exposure times of 1 and 7 days, there was a positive correlation of autoradiographic grains with chromosome length. To increase sensitivity in detection of Act D bound to chromosomes, a specific anti-Act D antibody was generated in rabbits. Antibody avidity was evaluated on the basis of a rapid charcoal assay. This charcoal assay was then used in development of a radioimmunoassay for Act D which is sensitive in quantitating the drug down to 0.005 μg/ml. The anti-Act D antibody was characterized to be IgG, and was shown to be specific for the pentapeptide lactone portion of the Act D molecule. Indirect immunofluorescence of Protein A-purified IgG containing anti-Act D was used to detect drug bound to fixed human chromosomes. The antibody was shown to be specific for drug bound to chromatin. When 0.1 μg/ml Act D was bound to chromosomes, the drug was observed bound throughout the genome, with no selective binding at the ribosomal cistrons. This confirms the autoradiographic data and supports the model of extranucleolar regulation of rRNA synthesis. Preliminary results suggest that Act D binds to GC-rich DNA, since an R-banding pattern was observed in 5% of the immunofluorescent metaphases examined.

Actinomycins are a class of compounds consisting of phenoxazinone-like core attached to two peptidolactone rings, denoted as α and β. A unique component of a few families—actinomycins G, Y, and Z—is a chlorinated β-ring threonine residue. Families G and Y also contained an actinomycin that possess a β-ring heterocycle (actinomycins G5 and Y5, respectively); prior to this work, no β-ring heterocycle-containing actinomycins were reported for the Z family. Unlike other actinomycin derivatives, Y5’s cytotoxicity was abolished while still maintaining some antibacterial potency. We constructed a model compound to probe the physical properties of the actinomycin core to test conditions under which heterocycle formation would occur. We also analyzed the gene clusters of these actinomycin producers for gene candidates to from this structural motif. We found the the actinomycin core aniline to have pKa values of 2.976 and 8.429 and a significant shift in UV absorption between 300-310nm when the group becomes charged. We also found cyclization conditions and no obvious gene candidates to form the β-ring heterocycle based on our gene cluster analysis. We hypothesize that the familial diversity of the actinomycin G, Y and Z familes is due to the reactivity of the phenoxazinone-like core.

No description available.

Myotonic dystrophy (DM) is an inherited disease characterized by myotonia, insulin resistance, cardiomyopathy, and cognitive deficiencies. DM is a triplet repeat disorder, meaning that affected individuals carry anywhere between 50 and thousands of CTG/CCTG repeats in their genetic makeup. When transcribed into RNA, these repeats become “toxic” in the sense that they serve to bind and sequester important RNA binding proteins. One such family of proteins, the Muscleblind-like (MBNL) family, is important in the regulation of alternative mRNA splicing, and thus the sequestration of MBNL proteins leads to a number of mis-splicing events. Many of these events are directly correlated to DM symptoms. While there is no known cure for DM, the use of small molecules to treat symptoms is a well-characterized therapeutic tactic with immense promise. Pentamidine is a small molecule that was found to reverse mis-splicing in both DM cell and mouse models. Mechanistically, this molecule is particularly unique because unlike many small molecules, which physically displace MBNL from the toxic CUG RNA, pentamidine reduces CUG RNA levels, possibly through inhibition of CTG transcription. Chapter I summarizes alternative splicing mechanisms and regulation, defines MBNL protein structure and function, describes DM pathophysiology and molecular mechanism, and finally provides an overview of pentamidine characterization as a small molecule therapeutic. Chapter II reports the development of an in vitro T7 transcription assay, which allowed us to compare the relative efficacy by which pentamidine is able to inhibit the transcription of various repeat and non-repeat DNA sequences. This chapter further reports the characterization of a series of methylene linker analogues of pentamidine, which were also characterized through the T7 transcription assay. Chapter III details our thorough structure-activity relationship investigation of bisbenzamidine analogues of pentamidine, both in in vivo and in vitro models. Chapter IV describes our characterization of actinomycin D, a known transcription inhibitor and chemotherapeutic, within the DM disease framework. Chapter V summarizes these data, which ultimately serve as a proof of concept for the potential of CTG transcription inhibition in therapeutic contexts and broadly describe their application in other repeat diseases. This dissertation contains previously published and unpublished co-authored material. / 10000-01-01

MDMX has emerged as a negative regulator of p53 transcriptional activity following DNA damage, loss of ribosomal integrity, and aberrant mitogenic signaling. Disruption of rRNA biogenesis by ribosomal stress activates p53 by releasing ribosomal proteins from nucleoli which bind MDM2 and inhibit p53 degradation. We found that p53 activation by ribosomal stress requires degradation of MDMX by MDM2. This occurs by L11 binding to the acidic domain of MDM2 which promotes its E3 ligase function preferentially towards MDMX. Further, unlike DNA damage which regulates MDMX stability through ATM-dependent phosphorylation events, ribosomal stress does not require MDMX phosphorylation suggesting p53 may be more sensitive to suppression by MDMX under these conditions. Indeed, we find that tumor cells overexpressing MDMX are less sensitive to ribosomal stress-induced growth arrest by the addition of actinomycin D due to formation of inactive p53-MDMX complexes that fail to transcriptionally activate downstream targets such as p21. Knockdown of MDMX increases sensitivity to actinomycin D, whereas MDMX overexpression abrogates p53 activation. Furthermore, MDMX expression promotes resistance to the chemotherapeutic agent 5-fluorouracil (5-FU), which at low concentrations activates p53 by inducing ribosomal stress without significant DNA damage signaling. Knockdown of MDMX abrogates HCT116 tumor xenograft formation in nude mice. MDMX overexpression does not accelerate tumor growth but increases resistance to 5-FU treatment in vivo. In addition to MDMX regulation at the protein level, we found that regulation of cellular MDMX levels, like MDM2, can occur at the transcriptional level by inducing the Ras/Raf/MEK/ERK pathway. We found MDMX levels in tumor cell lines closely correlate with promoter activity and mRNA level. Activated K-Ras and growth factor IGF-1 induce MDMX expression at the transcriptional level through mechanisms that involve the MAPK kinase and c-Ets-1 transcription factors. Pharmacological inhibition of MEK results in down-regulation of MDMX in tumor cell lines. MDMX overexpression is detected in ~50% of human colon tumors and showed strong correlation with increased Erk phosphorylation. Taken together, the data show that MDMX has multiple modes of regulation, which ultimately determine the overall extent of p53 activation.