The DNA sequence specificity of bleomycin cleavage in a systematically altered DNA sequence

Abstract

Bleomycin is an anti-tumour agent that is clinically used to treat several types of cancers. Bleomycin cleaves DNA at specific DNA sequences and recent genome-wide DNA sequencing specificity data indicated that the sequence 5'-RTGT*AY (where T* is the site of bleomycin cleavage, R is G/A and Y is T/C) is preferentially cleaved by bleomycin in human cells. Based on this DNA sequence, we constructed a plasmid clone to explore this bleomycin cleavage preference. By systematic variation of single nucleotides in the 5'-RTGT*AY sequence, we were able to investigate the effect of nucleotide changes on bleomycin cleavage efficiency. We observed that the preferred consensus DNA sequence for bleomycin cleavage in the plasmid clone was 5'-YYGT*AW (where W is A/T). The most highly cleaved sequence was 5'-TCGT*AT and, in fact, the seven most highly cleaved sequences conformed to the consensus sequence 5'-YYGT*AW. A comparison with genome-wide results was also performed and while the core sequence was similar in both environments, the surrounding nucleotides were different.

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

“…More recently, updated technology using capillary electrophoresis with laser-induced fluorescence (CE-LIF) and automated DNA sequencers, has permitted a more accurate and precise determination of the DNA sequence specificity of bleomycin DNA damage in longer purified DNA sequences [ 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 ]. In addition, the use of separate 5′- and 3′-end-labelling in a DNA sequence specificity study contributes to the precision of the process by greatly reducing end-label bias [ 78 , 79 ]. It should be noted that experiments with end-labelled DNA mainly detect single-strand breaks (see below).…”

“…It was observed that the preferred nucleotide sequence for high intensity bleomycin cleavage was 5′-YYGT*AW (where W is A or T) ( Table 1 ). The DNA sequence that had the highest intensity of bleomycin cleavage was 5′-TCGT*AT and the seven highest intensity bleomycin cleavage sites conformed to the 5′-YYGT*AW consensus sequence [ 79 ]. This study permitted a precise evaluation of crucial neighbouring nucleotides that produced high intensity bleomycin DNA cleavage sites.…”

“…This study permitted a precise evaluation of crucial neighbouring nucleotides that produced high intensity bleomycin DNA cleavage sites. This approach of systematically altering the nucleotides around bleomycin cleavage sites is a powerful method of analysis because every possible nucleotide is included in the analysis and no variant is omitted [ 79 ]. This is in contrast to an analysis of random DNA sequences where some nucleotide variants may not be present in the analysis.…”

“…This sequence along with systematically altered nucleotide variations, was placed into a plasmid construct called the RTGT*AY plasmid. The consensus DNA sequence derived from the most highly cleaved bleomycin cleavage sites with this plasmid was 5′-YYGT*AW [ 79 ]; while from the genome-wide data, it was 5′-TGT*AW for purified genomic DNA from the individual nucleotide data; and 5′-TGT*AT for purified genomic DNA from the complete sequence data ( Table 1 ). There was a consistent core of 5′-GT*A in these consensus sequences.…”

Investigating Nucleosome Accessibility for MNase, FeII Peplomycin, and Duocarmycin B2 by Using Capillary Electrophoresis

“…The same reaction affinity was observed in our study (marked in Figure B), with two hotspots containing more 5′‐GT‐3′ and 5′‐GC‐3′ exhibiting high frequency of cleavage (P3–P6, P15–P18). P10 and P11 targeting of 5′‐ATGTA‐3′, which was also considered a hot sequence for the peplomycin approach, was further enhanced in the nucleosomal DNA structure, as shown by the observed increased reactivity of P11. In addition, except for P10 and P11, although some very low intensity peaks showed up in the nucleosomal core region for peplomycin⋅Fe II ‐mediated cleavage, about 5.6‐fold (47.1–8.4 %) suppression was observed, which was highly consistent with previous reports of bleomycin…”