Characterization of Bleomycin-Mediated Cleavage of a Hairpin DNA Library

Abstract

A study of BLM A5 was conducted using a previously isolated library of hairpin DNAs found to bind strongly to metal free BLM. The ability of Fe(II)•BLM to effect cleavage on both the 3' and 5'-arms of the hairpin DNAs was characterized. The strongly bound DNAs were found to be efficient substrates for Fe•BLM A5-mediated hairpin DNA cleavage. Surprisingly, the most prevalent site of BLM-mediated cleavage was found to be the 5′-AT-3′ dinucleotide sequence. This dinucleotide sequence, and other sequences generally not cleaved well by BLM when examined using arbitrarily chosen DNA substrates, were apparent when examining the library of ten hairpin DNAs. In total, 132 sites of DNA cleavage were produced by exposure of the hairpin DNA library to Fe•BLM A5. The existence of multiple sites of cleavage on both the 3′- and 5′-arms of the hairpin DNAs suggested that some of these might be double-strand cleavage events. Accordingly, an assay was developed with which to test the propensity of the hairpin DNAs to undergo double-strand DNA damage. One hairpin DNA was characterized using this method, and gave results consistent with earlier reports of double-strand DNA cleavage, but with a sequence selectivity different from those reported previously.

The Interaction of the Metallo-Glycopeptide Anti-Tumour Drug Bleomycin with DNA

“…Bleomycin preferentially cleaves DNA at particular dinucleotide sequences. DNA sequence specificity studies with purified plasmid sequences have indicated that the dinucleotides 5′-GT* and 5′-GC* are preferentially cleaved by bleomycin [ 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 ]. For ease of discussion in this review, the bleomycin cleavage site nucleotides are numbered with respect to the cleavage site at nucleotide position 0, and the positions discussed are from −3 to +2 (see Table 1 ).…”

Interaction of Zn(II)bleomycin-A2 and Zn(II)peplomycin with a DNA hairpin containing the 5′-GT-3′ binding site in comparison with the 5′-GC-3′ binding site studied by NMR spectroscopy

“…Previous studies have focused on the two known DNA cleavage sites that BLMs are known to interact with, mainly 5’-GC-3’ and 5’-GT-3’[24], however a preference of cleavage site is not well understood [19,25]. The interactions of different MBLMs with DNA including Fe(II)- [26–28], Zn(II)- [17,18], Co(III)- [6,20–23,29–34], and Cu(I)-bound BLMs [35], have been examined. However the possibility of different coordination geometries of these metal centers to BLM and their repercussions on the final structure of the MBLM-DNA triad have received little attention.…”

Invertebrates living in polluted environments are potential source of novel anticancer agents

“…The anticancer mechanisms of each of these molecules are however different from each other. While actinomycin D triggers p53-independent apoptosis in cells [21] and bleomycin exhibit oxygen and metal ion-dependent cleavage of the DNA [22]. Doxorubicin, in turn, inhibits DNA and RNA replication and transcription and causes oxidative stress in tumor cells, triggering membrane, protein and DNA damage [23].…”