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Biomedical subjects

J T Millard

Publications and source records attributed to J T Millard.

11 recordsLinked to original sources
PubMed2004

DNA interstrand cross-linking by a mycotoxic diepoxide.

The diepoxide mycotoxin (2R, 3R, 8R, 9R)-4,6-decadiyne-2,3:8,9-diepoxy-1,10-diol (repandiol) was both isolated from the mushroom Hydnum repandum and synthesized de novo. Repandiol was found to form interstrand cross-links within a restriction fragment of DNA, linking deoxyguanosines on opposite strands primarily within the 5'-GNC and 5'-GNNC sequences preferred by diepoxyoctane. However, repandiol was a significantly less efficient cross-linker than either of the diepoxyalkanes (diepoxyoctane and diepoxybutane) to which it was compared.

Alkynes↗
PubMed2001

Diepoxybutane and diepoxyoctane interstrand cross-linking of the 5S DNA nucleosomal core particle.

Diepoxyalkanes form interstrand cross-links in DNA oligomers preferentially at 5'-GNC sites. We have examined cross-linking by 1,2,3,4-diepoxybutane (DEB) and 1,2,7,8-diepoxyoctane (DEO) within a fragment of the 5S RNA gene of Xenopus borealis in both the free and nucleosomal states. Sites and efficiencies of interstrand cross-linking were probed through denaturing polyacrylamide gel electrophoresis and quantitative phosphorimagery. Both agents targeted 5'-GNC sites for cross-linking in the restriction fragment in its free state, and DEO also targeted 5'-GNNC sites. Monoalkylation occurred at all deoxyguanosines. The sites for both monoalkylation and interstrand cross-linking were similar in nucleosomal and free DNA, and cross-linked DNA was cleanly incorporated into the core particle structure. These findings suggest that the 5S core particle is able to tolerate any structural abnormalities induced by diepoxide cross-linking.

Animals↗
PubMed2000

cis- and trans-diamminedichloroplatinum(II) interstrand cross-linking of a defined sequence nucleosomal core particle.

Interstrand cross-linking studies with the antitumor drug cis-diamminedichloroplatinum(II) and its clinically inactive isomer, trans-diamminedichloroplatinum(II), were performed on a fragment of the 5S rRNA gene of Xenopus borealis in the free and nucleosomal state. 5S nucleosomes were formed via histone octamer exchange from chicken erythrocyte core particles. Native polyacrylamide gel electrophoresis was used to probe the ability of platinated DNA to reconstitute into core particles. Both isomers negatively impacted reconstitution when histones were present during incubation with the drug. When histones were not present during the drug treatment, platinated DNA was successfully reconstituted into core particles. These results suggest that platination of histones impedes reconstitution of free DNA. However, already-formed core particles were not disrupted upon platination. Sites of interstrand cross-linking were probed through denaturing polyacrylamide gel electrophoresis and quantitative phosphorimagery. We found both site-specific enhancement and depression of cis-diamminedichloroplatinum(II) cross-linking in the nucleosomal samples relative to free DNA at both drug concentrations that were tested (0.01 and 0.0025 mM). trans-Diamminedichloroplatinum(II) exhibited no detectable differences in the interstrand cross-linking of free and nucleosomal samples.

Animals↗
PubMed1998

Effect of nucleosome structure on DNA interstrand cross-linking reactions.

Antitumor agents of the nitrogen mustard family and mitomycin C form interstrand cross-links in duplex DNA. To provide information about the cellular mechanism by which these compounds exert their cytotoxic effects, we examined cross-linking of a nucleosomal core particle formed on a fragment of the 5S RNA gene of Xenopus borealis. For the mustards mechlorethamine, chlorambucil, and melphalan, both sites of monoalkylation and interstrand cross-linking were similar in nucleosomal and free DNA. Some small (two- to three- fold) differences in intensity of cross-linking at some sites were apparent. However, these differences did not appear to correlate with rotational or translational positioning. For mitomycin C, cross-linking was inhibited five- to ten-fold at the nucleosomal dyad and showed attenuation of inhibition toward the ends. Furthermore, rotational positioning also appeared to be a factor, with sites facing inward in the nucleosome less accessible for mitomycin cross-linking. None of these agents demonstrated the 10-base pair periodicity exhibited by hydroxyl radical cleavage of nucleosomal DNA.

Animals↗
PubMed1996

DNA modifying agents as tools for studying chromatin structure.

DNA modification leads to mutagenicity, carcinogenicity, and cytotoxicity. Because the majority of cellular DNA exists as chromatin, histone and other proteins may mediate DNA binding and bonding reactions in vivo. Chromatin modification by a wide variety of agents, including intercalators, groove binders, cross-linkers, and strand cleavers, is reviewed. Although the majority of agents demonstrate a preference for linker over core DNA, the sequence preference appears to be retained in most cases, suggesting that both the major and minor grooves in chromatin are quite accessible to small molecules. Furthermore, only a handful of agents demonstrate the 10-11 base pair periodicity of hydroxyl radical, strongly supporting a dynamic model of nucleosome structure.

Animals↗
PubMed1996

1,2,5,6-Diepoxyhexane and 1,2,7,8-diepoxyoctane cross-link duplex DNA at 5'-GNC sequences.

The carcinogenicity of epoxide compounds has been attributed to covalent binding to DNA. Whereas monoepoxides form only monoadducts, diepoxides can form both monoadducts and interstrand cross-links. The latter are believed to be the more significant cytotoxic lesions as diepoxides are frequently more carcinogenic and mutagenic than their monoepoxide analogues. We therefore examined the relative DNA interstrand cross-linking capabilities of several diepoxides with respect to chain length, molecular flexibility, reported carcinogenic potential, and DNA sequences targeted. Using denaturing polyacrylamide gel electrophoresis, we found that 1,2,5,6-diepoxyhexane and 1,2,7,8-diepoxyoctane share the 5'-GNC target sequence previously found for 1,2,3,4-diepoxybutane [Millard, J.T., and White, M.M. (1993) Biochemistry 32, 2120-2124] and that the efficiency of cross-linking this sequence may reflect carcinogenicity, 1,2,5,6-Diepoxycyclooctane, the biologically inactive rigid analogue of 1,2,5,6-diepoxyhexane, was found to be a poor cross-linker of all DNA sequences examined. Moreover, increasing the diepoxyalkane chain length did not result in enhanced cross-linking ability.

Carcinogens↗
PubMed1993

Cytosine methylation enhances mitomycin C cross-linking.

Mitomycin C (MC) is a powerful antitumor agent that targets the DNA sequence CpG. Because it is likely that this dinucleotide will contain 5-methylcytosine in vivo, we have compared the cross-linking efficiency of MC for DNA containing either 5-methylcytosine or normal cytosine embedded in random-sequence DNA oligomers. We have found that mitomycin C displays a small but significant preference for methylated DNA. Recognition of an abnormal methylation pattern in the DNA of transformed cells may therefore be one mechanism by which MC exerts its chemotherapeutic effects.

5-Methylcytosine↗
PubMed1993

Diepoxybutane cross-links DNA at 5'-GNC sequences.

Epoxides are cancer-causing agents chemically analogous to the nitrogen mustards, a family of powerful antitumor drugs. We found that the DNA interstrand cross-linking sequence preference of diepoxybutane is the same as that of the mustard mechlorethamine: 5'-GNC. Therefore, the genomic site of cross-linking alone cannot explain why some interstrand cross-linkers act as antitumor agents whereas others are deadly toxins.

Base Sequence↗
PubMed1993

Site-specific metal-induced damage of mitomycin C-crosslinked DNA fragments in the presence of sodium dithionite.

Purified singly mitomycin C-crosslinked DNA duplex fragments were treated with Cu(II) in the presence of sodium dithionite. Cleavage products were analyzed through single-nucleotide resolving denaturing polyacrylamide gel electrophoresis. In addition to the previously described non-specific cleavage, which may be attributed to the production of hydroxyl radical, a novel site-specific cleavage in the immediate vicinity of the MC-crosslink was observed. Possible mechanisms of this reaction are discussed.

Base Sequence↗
PubMed1991

Sequence preferences of DNA interstrand crosslinking agents: quantitation of interstrand crosslink locations in DNA duplex fragments containing multiple crosslinkable sites.

A general approach to the quantitative study of the sequence specificity of DNA interstrand crosslinking agents in synthetic duplex DNA fragments is described. In the first step, a DNA fragment previously treated with an interstrand crosslinking agent is subjected to denaturing PAGE. Not only does this distinguish crosslinked from native or monoadducted DNA, it is shown herein that isomeric crosslinked DNAs differing in position of the crosslink can in some cases be separated. In the second stage, the now fractionated crosslinked DNAs isolated from denaturing PAGE are subjected to fragmentation using iron(II)/EDTA. For those fractions which are structurally homogeneous, analysis of the resulting fragment distribution has previously been shown to reveal the crosslink position at nucleotide resolution. It is shown herein that in fractions which are structurally heterogeneous due to differences in position of crosslink, this analysis quantifies the relative extent of crosslinking at distinct sites. Using this method it is shown that reductively activated mitomycin C crosslinks the duplex sequences 5'-GCGC and 5'-TCGA with 3 +/- 1:1 relative efficiency.

Base Sequence↗