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 Kleanthous; Albert J R Heck

doi:10.1093/nar/gni099

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 Kleanthous, Albert J R Heck

Biology

Research output: Contribution to journal › Article › peer-review

Abstract

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.

Original language	English
Pages (from-to)	e96
Journal	Nucleic Acids Research
Volume	33
Issue number	10
DOIs	https://doi.org/10.1093/nar/gni099
Publication status	Published - 2005

Keywords

Colicins
DNA, Single-Stranded
Endodeoxyribonucleases
Escherichia coli Proteins
Hydrolysis
Spectrometry, Mass, Electrospray Ionization
Substrate Specificity
Time Factors

Access to Document

10.1093/nar/gni099

Cite this

@article{0e8b9a3a16ea445fba395e7b6b9a5123,

title = "Real-time monitoring of enzymatic DNA hydrolysis by electrospray ionization mass spectrometry",

abstract = "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.",

keywords = "Colicins, DNA, Single-Stranded, Endodeoxyribonucleases, Escherichia coli Proteins, Hydrolysis, Spectrometry, Mass, Electrospray Ionization, Substrate Specificity, Time Factors",

author = "{van den Heuvel}, {Robert H H} and Sara Gato and Cees Versluis and Pascal Gerbaux and Colin Kleanthous and Heck, {Albert J R}",

year = "2005",

doi = "10.1093/nar/gni099",

language = "English",

volume = "33",

pages = "e96",

journal = "Nucleic Acids Research",

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T1 - Real-time monitoring of enzymatic DNA hydrolysis by electrospray ionization mass spectrometry

AU - van den Heuvel, Robert H H

AU - Gato, Sara

AU - Versluis, Cees

AU - Gerbaux, Pascal

AU - Kleanthous, Colin

AU - Heck, Albert J R

PY - 2005

Y1 - 2005

N2 - 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.

AB - 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.

KW - Colicins

KW - DNA, Single-Stranded

KW - Endodeoxyribonucleases

KW - Escherichia coli Proteins

KW - Hydrolysis

KW - Spectrometry, Mass, Electrospray Ionization

KW - Substrate Specificity

KW - Time Factors

U2 - 10.1093/nar/gni099

DO - 10.1093/nar/gni099

M3 - Article

C2 - 15956101

VL - 33

SP - e96

JO - Nucleic Acids Research

JF - Nucleic Acids Research

IS - 10

ER -