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...
| Main Authors: | , , , , , |
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| Format: | Journal article |
| Language: | English |
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2005
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| _version_ | 1826288264966832128 |
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| author | van den Heuvel, R Gato, S Versluis, C Gerbaux, P Kleanthous, K Heck, A |
| author_facet | van den Heuvel, R Gato, S Versluis, C Gerbaux, P Kleanthous, K Heck, A |
| author_sort | van den Heuvel, R |
| collection | OXFORD |
| description | 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. © The Author 2005. Published by Oxford University Press. All rights reserved. |
| first_indexed | 2024-03-07T02:11:06Z |
| format | Journal article |
| id | oxford-uuid:a0ad4536-7ab8-429e-b6fa-ba34ff80af80 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T02:11:06Z |
| publishDate | 2005 |
| record_format | dspace |
| spelling | oxford-uuid:a0ad4536-7ab8-429e-b6fa-ba34ff80af802022-03-27T02:07:15ZReal-time monitoring of enzymatic DNA hydrolysis by electrospray ionization mass spectrometryJournal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:a0ad4536-7ab8-429e-b6fa-ba34ff80af80EnglishSymplectic Elements at Oxford2005van den Heuvel, RGato, SVersluis, CGerbaux, PKleanthous, KHeck, AA 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. © The Author 2005. Published by Oxford University Press. All rights reserved. |
| spellingShingle | van den Heuvel, R Gato, S Versluis, C Gerbaux, P Kleanthous, K Heck, A Real-time monitoring of enzymatic DNA hydrolysis by electrospray ionization mass spectrometry |
| title | Real-time monitoring of enzymatic DNA hydrolysis by electrospray ionization mass spectrometry |
| title_full | Real-time monitoring of enzymatic DNA hydrolysis by electrospray ionization mass spectrometry |
| title_fullStr | Real-time monitoring of enzymatic DNA hydrolysis by electrospray ionization mass spectrometry |
| title_full_unstemmed | Real-time monitoring of enzymatic DNA hydrolysis by electrospray ionization mass spectrometry |
| title_short | Real-time monitoring of enzymatic DNA hydrolysis by electrospray ionization mass spectrometry |
| title_sort | real time monitoring of enzymatic dna hydrolysis by electrospray ionization mass spectrometry |
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