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Nucleic Acids Research 2005 33(10):e96; doi:10.1093/nar/gni099
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Published online 13 June 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oupjournals.org

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Real-time monitoring of enzymatic DNA hydrolysis by electrospray ionization mass spectrometry

Robert H. H. van den Heuvel*, Sara Gato, Cees Versluis, Pascal Gerbaux, Colin Kleanthous1 and Albert J. R. Heck

Department of Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands 1Department of Biology, University of York York YO10 5YW, UK

*To whom correspondence should be addressed. Tel: +31302536805; Fax: +31302518219; Email: r.h.h.vandenheuvel{at}chem.uu.nl

Received April 27, 2005. Revised June 2, 2005. Accepted June 2, 2005.

A fast and direct method for the monitoring of enzymatic DNA hydrolysis was developed using electrospray ionization mass spectrometry. We incorporated the use of a robotic chip-based electrospray ionization source for increased reproducibility and throughput. The mass spectrometry method allows the detection of DNA fragments and intact non-covalent protein–DNA complexes in a single experiment. We used the method to monitor in real-time single-stranded (ss) DNA hydrolysis by colicin E9 DNase and to characterize transient non-covalent E9 DNase–DNA complexes present during the hydrolysis reaction. The mass spectra showed that E9 DNase interacts with ssDNA in the absence of a divalent metal ion, but is strictly dependent on Ni2+ or Co2+ for ssDNA hydrolysis. We demonstrated that the sequence selectivity of E9 DNase is dependent on the ratio protein:ssDNA or the ssDNA concentration and that only 3'-hydroxy and 5'-phosphate termini are produced. It was also shown that the homologous E7 DNase is reactive with Zn2+ as transition metal ion and that this DNase displays a different sequence selectivity. The method described is of general use to analyze the reactivity and specificity of nucleases.

Present address: Pascal Gerbaux, Organic Chemistry Laboratory, University of Mons-Hainaut, 19 Avenue Maistriau, 7000 Mons, Belgium


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