Laurence Hurley

PMID: 3030397;Abstract:

The design, construction, and characterization of a site-directed CC-1065-N3-adenine adduct in a 117 base pair segment of M13mpI DNA are described. CC-1065 is an extremely potent antitumor antibiotic produced by Streptomyces zelensis. Previous studies have demonstrated that the cyclopropyl ring of CC-1065 reacts quite specifically with N3 of adenine in double-stranded DNA to form a CC-1065-DNA adduct. Following alkylation, the drug molecule lies snugly within the minor groove of DNA, overlapping with five base pairs for which a marked sequence preference exists [Hurley, L. H., Reynolds, V. R., Swenson, D. H., Petzold, G. L., & Scahill, T. A. (1984) Science (Washington, D.C.) 226, 843-844]. On the basis of the unique characteristics of the reaction of CC-1065 with DNA and the structure of the resulting DNA adduct, we have designed a general strategy to construct a site-directed CC-1065-DNA adduct in a restriction fragment. The presence of unique AluI and HaeIII restriction enzymes sites on each side of a high-affinity CC-1065 binding sequence (5′-GATTA) permitted the preparation of a partial duplex DNA molecule containing the CC-1065 binding sequence in the duplex DNA region. Since CC-1065 only binds to duplex DNA, potential CC-1065 binding sequences in the long single-stranded regions were protected from drug binding during the construction process. After purification of the CC-1065 partial duplex DNA adduct by differential melting of the modified and unmodified partial duplex DNA, DNA polymerase I was used to generate the full duplex DNA molecule, which contained a single site-directed CC-1065-N3-adenine adduct at adenine 6229 of the 117 base pair MspI-BstNI DNA restriction fragment of the Escherichia coli lac insert of M13mpI DNA. A CC-1065 thermal strand scission assay was used to confirm the unique binding site on the covalently modified strand. Methidiumpropyl-EDTA-iron(II) [MPE-Fe(II)] digestions were used to locate the binding site and the orientation of CC-1065 in the minor groove of DNA. MPE-Fe(11) footprinting revealed a slight enhancement of digestion on both DNA strands, but just to one side of the CC-1065-DNA adduct. © 1986 American Chemical Society.

PMID: 6494915;Abstract:

Sequence-dependent variations in DNA revealed by x-ray crystallographic studies have suggested that certain DNA-reactive drugs may react preferentially with defined sequences in DNA. Drugs that wind around the helix and reside within one of the grooves of DNA have perhaps the greatest chance of recognizing sequence-dependent features of DNA. The antitumor antibiotic CC-1065 covalently binds through N-3 of adenine and resides within the minor groove of DNA. This drug overlaps with five base pairs for which a high sequence specificity exists.

PMID: 6406412;Abstract:

The biosynthesis is the antitumor antibiotic, CC-1065, has been investigated by radioactive isotope techniques, in combination with chemical degradation of CC-1065. Tyrosine, dopa, serine and methionine (S-CH3 group) have been shown to be precursors of CC-1065. Tyrosine is proposed to be a precursor of all three benzodipyrrole subunits, while dopa is only apparently incorporated into subunits B and C. Serine is postulated to contribute three 2C units, with loss of C-1, to all three subunits of CC-1065. The S-CH3 group of methionine probably contributes four C-1 units to CC-1065 of which one is incorporated with considerable loss of tritium, most probably into the cyclopropane ring of subunit A.

In this review, the authors describe a novel mechanism for control of MYC expression that involves a four-stranded DNA structure, termed a G-quadruplex, amenable to small molecule targeting. The DNA element involved in this mechanism, the nuclease hypersensitive element III(1) (NHE III(1)), is just upstream of the P1 promoter and is subjected to dynamic stress (negative superhelicity) resulting from transcription. This is sufficient to convert the duplex DNA to a G-quadruplex on the purine-rich strand and an i-motif of the pyrimidine-rich strand, which displaces the activating transcription factors to silence gene expression. Specific proteins have been identified, NM23-H2 and nucleolin, that resolve and fold the G-quadruplex to activate and silence MYC expression, respectively. Inhibition of the activity of NM23-H2 molecules that bind to the G-quadruplex silences gene expression, and redistribution of nucleolin from the nucleolus to the nucleoplasm is expected to inhibit MYC. The authors also describe the mechanism of action of Quarfloxin, a first-in-class G-quadruplex-interactive compound that involves the redistribution of nucleolin from the nucleolus to the nucleoplasm. G-quadruplexes have been best known as test-tube oddities for more than four decades. However, during the past decade, they have emerged as likely players in a number of important biological processes, including transcriptional control. Only time will tell if these odd DNA structures will assume the role of an established receptor class, but it is clear from the scientific literature that there is a dramatic increase in interest in this little-known area in the past few years.