Laurence Hurley

The proximal promoter region of many human growth-related genes contains a polypurine/polypyrimidine tract that serves as multiple binding sites for Sp1 or other transcription factors. These tracts often contain a guanine-rich sequence consisting of four runs of three or more contiguous guanines separated by one or more bases, corresponding to a general motif known for the formation of an intramolecular G-quadruplex. Recent results provide strong evidence that specific G-quadruplex structures form naturally within these polypurine/polypyrimidine tracts in many human promoter regions, raising the possibility that the transcriptional control of these genes can be modulated by G-quadruplex-interactive agents. In this chapter, we describe three general biochemical methodologies, electrophoretic mobility shift assay (EMSA), dimethylsulfate (DMS) footprinting, and the DNA polymerase stop assay, which can be useful for initial characterization of G-quadruplex structures formed by G-rich sequences.

PMID: 9862977;PMCID: PMC148212;Abstract:

We have developed and characterized an assay for G-quadruplex-interactive compounds that makes use of the fact that G-rich DNA templates present obstacles to DNA synthesis by DNA polymerases. Using Taq DNA polymerase and the G-quadruplex binding 2,6-diamidoanthraquinone BSU-1051, we find that BSU-1051 leads to enhanced arrest of DNA synthesis in the presence of K⁺ by stabilizing an intramolecular G-quadruplex structure formed by four repeats of either TTGGGG or TTAGGG in the template strand. The data provide additional evidence that BSU-1051 modulates telomerase activity by stabilization of telomeric G-quadruplex DNA and point to a polymerase arrest assay as a sensitive method for screening for G-quadruplex-interactive agents with potential clinical utility.

PMID: 7217948;Abstract:

Reaction of anthramycin 11-methyl ether (AME) with trifluoroacetic acid results in formation of (1,11a)-didehydroanhydroanthramycin (DAA). Anthramycin biosynthetically labelled from DL-[3′RS(3′-³H)]; DL-[3′S(3′-³H)] and DL-[3′R(3′-³H)]tyrosine each lose approximately 50% of their tritium during this conversion to DAA confirming the labelling pattern of 3′-tritiated species of tyrosine in AME. As expected negligible losses of tritium occurred from AME biosynthetically labelled from L-[2- or 6-³H] or L-[3- or 5-³H]tyrosine. DAA did not form a stable adduct with DNA in accord with the postulated mechanism of action of anthramycin.

Abstract:

Anthramycin is a member of the pyrrolo[1,4]benzodiazepine group of antitumor antibiotics. Previous studies have demonstrated that anthramycin binds covalently through N-2 of guanine within the minor groove of DNA, resulting in a relatively nondistortive DNA adduct. From the nuclear Overhauser effect spectroscopy (NOESY) proton NMR spectra of the anthramycin-d(ATGCAT)2 adduct, we have obtained results that unambiguously assign the orientation of the drug molecule in the minor groove of DNA. Four sets of NOE cross-peaks between anthramycin protons and nucleotide protons on either the covalently or the noncovalently modified strands reveal that the drug is specifically oriented with the aromatic ring to the 3′-side of the covalently modified guanine. Unequivocal assignment of the geometry at the site of attachment of anthramycin to d(ATGCAT)2 cannot be made by J-correlated spectroscopy (COSY). However, when combined with the results of modeling with the molecular mechanics program AMBER, an 11S stereochemistry at this site can be confidently predicted. ³¹P NMR studies show that two of the resonance signals in the anthramycin-d(ATGCAT)2 adduct have moved significantly downfield. Both downfield ³¹P NMR signals have been assigned by ¹⁷O isotopic labeling and ¹H-³¹P two-dimensional J-correlation experiments and shown to correspond to the phosphates on the 5′-sides of the covalently modified deoxyguanine and the deoxycytosine on the opposite strand. Assignment of resonance signals of nonexchangeable base and sugar protons of the anthramycin-d(ATGCAT)2 has been made with two-dimensional Fourier transform NMR methods (COSY and NOESY). Conformational details about the sugar puckers, the glycosidic dihedral angles, and the effect of anthramycin bonding on secondary structure of the duplex have been obtained from the relative intensities of cross-peaks in the two-dimensional NMR spectra in aqueous solution. All of the sugars that are amenable to this analysis possess a conformation consistent with B-type DNA. Molecular mechanics calculations with AMBER are predictive of the orientation and stereochemistry of anthramycin bound to d(ATGCAT)2. The species having an 11S stereochemistry at the covalent bonding site and oriented with the aromatic ring of anthramycin to the 3′-side of the covalently modified guanine of anthramycin-d(ATGCAT)2 appears to be favored over the three other possible species. This is because of the greater intermolecular binding for this species rather than lower helix distortion energies. The molecular modeling is also in accord with the experimentally determined nondistortive nature of the anthramycin-d-(ATGCAT)2 adduct.