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

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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.

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Abbreviations

CE-LIF:

Capillary electrophoresis with laser-induced fluorescence

dNTP:

Deoxynucleotide triphosphates

Exo-SAP:

Exonuclease I and shrimp alkaline phosphatase

FAM-dUTP:

Aminoallyl-dUTP-6-FAM

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Acknowledgements

Support of this work by the University of New South Wales, Science Faculty Research Grant Scheme is gratefully acknowledged. SDG was funded by an Australian Postgraduate Award.

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Authors and Affiliations

  1. School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW, 2052, Australia

    Shweta D. Gautam, Jon K. Chen & Vincent Murray

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  1. Shweta D. Gautam
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  2. Jon K. Chen
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  3. Vincent Murray
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Correspondence to Vincent Murray.

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Gautam, S.D., Chen, J.K. & Murray, V. The DNA sequence specificity of bleomycin cleavage in a systematically altered DNA sequence. J Biol Inorg Chem 22, 881–892 (2017). https://doi.org/10.1007/s00775-017-1466-z

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  • DOI: https://doi.org/10.1007/s00775-017-1466-z

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