Sequence-dependent reactivity of linear DNA to chemical cleavage by Cu(II):thiol combinations including cysteine or glutathione.
The chemical cleavage of linear DNA by Cu(II):thiol combinations was investigated using end-labelled double-stranded restriction fragments. Single-strand cleavage of the target DNA occurred with apparent sequence preference, but there appeared to be no apparent consensus sequence for such preferentially cleaved sites based on studies of three different restriction fragments. For any given restriction fragment, the observed Cu(II):thiol DNA-cleavage sequence preference was found to be independent of the nature of the thiol used. Cleavage of 3'-end-labelled DNA generated fragments bearing 5'-phosphoryl termini, and cleavage of 5'-end-labelled DNA gave rise to fragments bearing 3'-phosphoryl and 3'-'phosphatase-inert' termini in equal proportions. No appreciable amounts of fragments bearing 3'- or 5'-hydroxy termini were detected. This pattern of cleavage products is similar to that observed using the hydroxyl-radical-generating systems, 60Co gamma-irradiation or methidium propyl EDTA:Fe(II), but is dissimilar to that found with the Cu(II):phenanthroline or bleomycin systems. Cu(II):thiol cleavage of DNA fragments containing inserts of repeated dinucleotide sequence showed alternating patterns of relative cleavage intensities within the insert-sequence regions which were not seen in the sequences flanking the insert sequence. The assembled experimental data indicate that a reaction product of the Cu(II):thiol interaction, probably the hydroxyl radical, causes DNA cleavage. The Cu(II):thiol interaction may occur free in solution or via a copper species which is bound non-specifically to DNA. The observed low sequence-dependence of the cleavage reaction probably reflects the nature of the DNA structure itself, the Cu(II):thiol system serving as a structural probe of sequence-dependent structural variations in the DNA structure.
Department of Pharmacy, University of Manchester, U.K.
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John
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Douglas
D001483:Base Sequence; D003300:Copper; D003545:Cysteine; D004247:DNA / Q000737:chemistry*; D004262:DNA Restriction Enzymes; D003850:Deoxyribonuclease I; D005978:Glutathione; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds; D013997:Time Factors
D013438:Sulfhydryl Compounds; D003300:Copper; D004247:DNA; D004262:DNA Restriction Enzymes; D003850:Deoxyribonuclease I; D005978:Glutathione; D003545:Cysteine