Laurence Hurley

Abstract:

In this contribution we demonstrate that Bizelesin can translocate along the minor groove of DNA from a kinetically favored monoalkylation site to a thermodynamically favored cross-linking site. This translocation is prevented in compounds that have a charged guanidino linkage substituting for the ureadiyl linkage. Furthermore, the manipulative interplay of Bizelesin and the target sequence 5'-TAATTA (Seaman, F.C.; Chu, J.; Hurley, L.H. J. Am. Chem. Soc. 1996, 118, 5383-5395) that is required to produce a suitably rearranged product for cross-linking is prevented by the substitution of a guanidino for the ureadiyl linkage. A structural basis involving hydrogen bonding of the guanidino linkage with phosphates on the backbone of DNA is proposed to account for the absence of translocation, the slow conversion of monoalkylated to cross-linked species, and the non-rearranged cross-linked product.

The recently discovered role of the BCL2 (B-cell lymphoma 2 gene) promoter i-motif DNA in modulation of gene expression via interaction with the ribonucleoprotein hnRNP L-like (hnRNP LL) has prompted a more detailed study of the nature of this protein-DNA interaction. The RNA recognition motifs (RRMs) of hnRNP LL were expressed individually, and both RRM1 and RRM2 were found to bind efficiently to the BCL2 i-motif DNA, as well as being critical for transcriptional activation, whereas RRM3-4 bound only weakly to this DNA. Binding was followed by unfolding of the DNA as monitored by changes in the CD spectrum. Mutational analysis of the i-motif DNA revealed that binding involved primarily the lateral loops of the i-motif. The kinetics of binding of the DNA with RRM1 was explored by recording CD spectra at predetermined times following admixture of the protein and DNA. The change in molar ellipticity was readily apparent after 30 s and largely complete within 1 min. A more detailed view of protein-DNA interaction was obtained by introducing the fluorescence donor 6-CNTrp in RRM1 at position 137, and the acceptor 4-aminobenzo[g]quinazoline-2-one (Cf) in lieu of cytidine22 in the i-motif DNA. The course of binding of the two species was monitored by FRET, which reflected a steady increase in energy transfer over a period of several minutes. The FRET signal could be diminished by the further addition of (unlabeled) RRM2, no doubt reflecting competition for binding to the i-motif DNA. These experiments using the individual RRM domains from hnRNP LL confirm the role of this transcription factor in activation of BCL2 transcription via the i-motif in the promoter element.

PMID: 1085163;Abstract:

11-Demethyltomaymycin, an antitumor antibiotic produced by Streptomyces achromogenes, and its biologically inactive metabolite oxotomaymycin are biosynthesized from L-tyrosine, DL-tryptophan, and L-methionine. The anthranilate part of 11-demethyltomaymycin is derived from tryptophan probably via the kynurenine pathway. The predominant loss of tritium from DL-[5-³H]tryptophan, during its conversion to 11-demethyltomaymycin and oxotomaymycin is interpreted to mean by NIH shift rules, that the main pathway to the 5-methoxy-4-hydroxy anthranilate moiety is through hydroxylation at C-8 prior to hydroxylation at C-7. The methoxy carbon is derived from the S-methyl group of methionine by transfer of an intact methyl group. The ethylideneproline moiety of 11-demethyltomaymycin is biosynthesized from tyrosine, without a 1-carbon unit from methionine. The results of biosynthetic feeding experiments with L-[1-¹⁴C, 3- or 5-3H] tyrosine are consistent with a "meta" or extradiol cleavage of 6, 7-dihydroxycyclodopa as has also been demonstrated previously for anthramycin and lincomycin A. An experiment in which L-[1-¹⁴C, Ala-2,3-³H]tyrosine was fed showed that both of the β hydrogens of this amino acid are retained in 11-demethyltomaymycin. It has been demonstrated in cultures and washed cell preparations that 11-demethyltomaymycin is enzymatically converted to oxotomaymycin by an intracellular constitutive enzyme. Conversion of oxotomaymycin to 11-demethyltomaymycin by these same preparations could not be demonstrated. The enzymatic activity associated with the conversion of 11 -demethyltomaymycin to oxotomaymycin is not limited to the 11-demethyltomaymycin production phase, since trophophase cells and even cells from 11-demethyltomaymycin nonproducing cultures of S. achromogenes were equally active in converting 11-demethyltomaymycin to oxotomaymycin.

PMID: 16890804;Abstract:

In this review, we describe the evidence for a parallel-stranded G-quadruplex in the purine-rich strand of the nuclease hypersensitivity element III1 (NHE III1) of the promoter of c-MYC upstream of the P1 and P2 promoters. This biologically relevant G-quadruplex is a mixture of four loop isomers. The folding pattern of a nuclear magnetic resonance (NMR)-derived structure for the predominant loop isomer of this G-quadruplex has been obtained. This G-quadruplex has been demonstrated to be a silencer element, and the cationic porphyrin TMPyP4 has been shown to stabilize this G-quadruplex. Furthermore, TMPyP4 has been shown to repress c-MYC expression, and this effect is mediated through the silencer element. Last, the in vivo activity of TMPyP4 in xenograph models is presented. © 2006 Elsevier Inc. All rights reserved.