Repair of DNA Alkylation Damage by the Escherichia coli Adaptive AlkB as Studied by ESI-TOF Mass Spectrometry
DNA alkylation can cause mutations, epigenetic changes, and even cell death. All living organisms have evolved enzymatic and non-enzymatic strategies for repairing such alkylation damage. AlkB, one of the Escherichia coli adaptive response proteins, uses an α-ketoglutarate/Fe(II)-dependent mechani...
| Main Authors: | , , , , , , |
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SAGE-Hindawi Access to Research
2011
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| Online Access: | http://hdl.handle.net/1721.1/64788 https://orcid.org/0000-0001-5486-2755 https://orcid.org/0000-0001-6159-0778 https://orcid.org/0000-0002-2196-5691 |
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| author | Li, Deyu Delaney, James C. Page, Charlotte M. Chen, Alvin S. Wong, Cintyu Essigmann, John M. Drennan, Catherine L |
| author2 | Massachusetts Institute of Technology. Center for Environmental Health Sciences |
| author_facet | Massachusetts Institute of Technology. Center for Environmental Health Sciences Li, Deyu Delaney, James C. Page, Charlotte M. Chen, Alvin S. Wong, Cintyu Essigmann, John M. Drennan, Catherine L |
| author_sort | Li, Deyu |
| collection | MIT |
| description | DNA alkylation can cause mutations, epigenetic changes, and even cell death. All living organisms have evolved enzymatic and non-enzymatic strategies for repairing such alkylation damage. AlkB, one of the Escherichia coli adaptive response proteins, uses an α-ketoglutarate/Fe(II)-dependent mechanism that, by chemical oxidation, removes a variety of alkyl lesions from DNA, thus affording protection of the genome against alkylation. In an effort to understand the range of acceptable substrates for AlkB, the enzyme was incubated with chemically synthesized oligonucleotides containing alkyl lesions, and the reaction products were analyzed by electrospray ionization time-of-flight (ESI-TOF) mass spectrometry. Consistent with the literature, but studied comparatively here for the first time, it was found that 1-methyladenine, 1,N6-ethenoadenine, 3-methylcytosine, and 3-ethylcytosine were completely transformed by AlkB, while 1-methylguanine and 3-methylthymine were partially repaired. The repair intermediates (epoxide and possibly glycol) of 3,N4-ethenocytosine are reported for the first time. It is also demonstrated that O6-methylguanine and 5-methylcytosine are refractory to AlkB, lending support to the hypothesis that AlkB repairs only alkyl lesions attached to the nitrogen atoms of the nucleobase. ESI-TOF mass spectrometry is shown to be a sensitive and efficient tool for probing the comparative substrate specificities of DNA repair proteins in vitro. |
| first_indexed | 2024-09-23T10:46:44Z |
| format | Article |
| id | mit-1721.1/64788 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T10:46:44Z |
| publishDate | 2011 |
| publisher | SAGE-Hindawi Access to Research |
| record_format | dspace |
| spelling | mit-1721.1/647882022-09-27T14:56:18Z Repair of DNA Alkylation Damage by the Escherichia coli Adaptive AlkB as Studied by ESI-TOF Mass Spectrometry Li, Deyu Delaney, James C. Page, Charlotte M. Chen, Alvin S. Wong, Cintyu Essigmann, John M. Drennan, Catherine L Massachusetts Institute of Technology. Center for Environmental Health Sciences Massachusetts Institute of Technology. Department of Biological Engineering Massachusetts Institute of Technology. Department of Biology Massachusetts Institute of Technology. Department of Chemistry Drennan, Catherine L. Li, Deyu Delaney, James C. Page, Charlotte M. Chen, Alvin S. Wong, Cintyu Drennan, Catherine L. Essigmann, John M. DNA alkylation can cause mutations, epigenetic changes, and even cell death. All living organisms have evolved enzymatic and non-enzymatic strategies for repairing such alkylation damage. AlkB, one of the Escherichia coli adaptive response proteins, uses an α-ketoglutarate/Fe(II)-dependent mechanism that, by chemical oxidation, removes a variety of alkyl lesions from DNA, thus affording protection of the genome against alkylation. In an effort to understand the range of acceptable substrates for AlkB, the enzyme was incubated with chemically synthesized oligonucleotides containing alkyl lesions, and the reaction products were analyzed by electrospray ionization time-of-flight (ESI-TOF) mass spectrometry. Consistent with the literature, but studied comparatively here for the first time, it was found that 1-methyladenine, 1,N6-ethenoadenine, 3-methylcytosine, and 3-ethylcytosine were completely transformed by AlkB, while 1-methylguanine and 3-methylthymine were partially repaired. The repair intermediates (epoxide and possibly glycol) of 3,N4-ethenocytosine are reported for the first time. It is also demonstrated that O6-methylguanine and 5-methylcytosine are refractory to AlkB, lending support to the hypothesis that AlkB repairs only alkyl lesions attached to the nitrogen atoms of the nucleobase. ESI-TOF mass spectrometry is shown to be a sensitive and efficient tool for probing the comparative substrate specificities of DNA repair proteins in vitro. National Institutes of Health (U.S.) (grant CA080024) National Institutes of Health (U.S.) (grant CA26731) National Institutes of Health (U.S.) (grant ES02109) 2011-07-13T16:21:23Z 2011-07-13T16:21:23Z 2010-09 2010-08 Article http://purl.org/eprint/type/JournalArticle 2090-0201 2090-021X http://hdl.handle.net/1721.1/64788 Deyu Li, James C. Delaney, Charlotte M. Page, et al., “Repair of DNA Alkylation Damage by the Escherichia coli Adaptive Response Protein AlkB as Studied by ESI-TOF Mass Spectrometry,” Journal of Nucleic Acids, vol. 2010, Article ID 369434, 9 pages, 2010. https://orcid.org/0000-0001-5486-2755 https://orcid.org/0000-0001-6159-0778 https://orcid.org/0000-0002-2196-5691 en_US http://dx.doi.org/10.4061/2010/369434 Journal of Nucleic Acids Creative Commons Attribution http://creativecommons.org/licenses/by/2.0/ application/pdf SAGE-Hindawi Access to Research Hindawi |
| spellingShingle | Li, Deyu Delaney, James C. Page, Charlotte M. Chen, Alvin S. Wong, Cintyu Essigmann, John M. Drennan, Catherine L Repair of DNA Alkylation Damage by the Escherichia coli Adaptive AlkB as Studied by ESI-TOF Mass Spectrometry |
| title | Repair of DNA Alkylation Damage by the Escherichia coli Adaptive AlkB as Studied by ESI-TOF Mass Spectrometry |
| title_full | Repair of DNA Alkylation Damage by the Escherichia coli Adaptive AlkB as Studied by ESI-TOF Mass Spectrometry |
| title_fullStr | Repair of DNA Alkylation Damage by the Escherichia coli Adaptive AlkB as Studied by ESI-TOF Mass Spectrometry |
| title_full_unstemmed | Repair of DNA Alkylation Damage by the Escherichia coli Adaptive AlkB as Studied by ESI-TOF Mass Spectrometry |
| title_short | Repair of DNA Alkylation Damage by the Escherichia coli Adaptive AlkB as Studied by ESI-TOF Mass Spectrometry |
| title_sort | repair of dna alkylation damage by the escherichia coli adaptive alkb as studied by esi tof mass spectrometry |
| url | http://hdl.handle.net/1721.1/64788 https://orcid.org/0000-0001-5486-2755 https://orcid.org/0000-0001-6159-0778 https://orcid.org/0000-0002-2196-5691 |
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