Real-time monitoring of enzymatic DNA hydrolysis by electrospray ionization mass spectrometry.
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.
van den Heuvel
Robert H H
RH
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. r.h.h.vandenheuvel@chem.uu.nl
Gato
Sara
S
Versluis
Cees
C
Gerbaux
Pascal
P
Kleanthous
Colin
C
Heck
Albert J R
AJ
D003087:Colicins / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000032:analysis / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D006868:Hydrolysis; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity; D013997:Time Factors
D003087:Colicins; D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; C488859:immE9 protein, E coli; D004706:Endodeoxyribonucleases