Real-time monitoring of enzymatic DNA hydrolysis by electrospray ionization mass spectrometry
van den Heuvel RHH., Gato S., Versluis C., Gerbaux P., Kleanthous C., Heck AJR.
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 Ni 2+ or Co 2+ 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 Zn 2+ 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. © The Author 2005. Published by Oxford University Press. All rights reserved.