Electrospray ionization mass spectrometric characterization of photocrosslinked DNA-EcoRI DNA methyltransferase complexes
We describe a novel strategy combining photocrosslinking and HPLC-based electrospray ionization mass spectrometry to identify UV crosslinked DNA-protein complexes. EcoRI DNA methyltransferase modifies the second adenine within the recognition sequence GAATTC. Substitution of 5-iodouracil for the thy...
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| Veröffentlicht in: | Nucleic acids research 1998-01, Vol.26 (2), p.645-649 |
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| container_title | Nucleic acids research |
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| creator | Wong, David L. Pavlovich, James G. Reich, Norbert O. |
| description | We describe a novel strategy combining photocrosslinking and HPLC-based electrospray ionization mass spectrometry to identify UV crosslinked DNA-protein complexes. EcoRI DNA methyltransferase modifies the second adenine within the recognition sequence GAATTC. Substitution of 5-iodouracil for the thymine adjacent to the target base (GAATTC) does not detectably alter the DNA-protein complex. Irradiation of the 5-iodouracil-substituted DNA-protein complex at various wavelengths was optimized, with a crosslinking yield >60% at 313 nm after 1 min. No protein degradation was observed under these conditions. The crosslinked DNA-protein complex was further analyzed by electrospray ionization mass spectrometry. The total mass is consistent with irradiation-dependent covalent bond formation between one strand of DNA and the protein. These preliminary results support the possibility of identifying picomole quantities of crosslinked peptides by similar strategies. |
| doi_str_mv | 10.1093/nar/26.2.645 |
| format | Article |
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EcoRI DNA methyltransferase modifies the second adenine within the recognition sequence GAATTC. Substitution of 5-iodouracil for the thymine adjacent to the target base (GAATTC) does not detectably alter the DNA-protein complex. Irradiation of the 5-iodouracil-substituted DNA-protein complex at various wavelengths was optimized, with a crosslinking yield >60% at 313 nm after 1 min. No protein degradation was observed under these conditions. The crosslinked DNA-protein complex was further analyzed by electrospray ionization mass spectrometry. The total mass is consistent with irradiation-dependent covalent bond formation between one strand of DNA and the protein. 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EcoRI DNA methyltransferase modifies the second adenine within the recognition sequence GAATTC. Substitution of 5-iodouracil for the thymine adjacent to the target base (GAATTC) does not detectably alter the DNA-protein complex. Irradiation of the 5-iodouracil-substituted DNA-protein complex at various wavelengths was optimized, with a crosslinking yield >60% at 313 nm after 1 min. No protein degradation was observed under these conditions. The crosslinked DNA-protein complex was further analyzed by electrospray ionization mass spectrometry. The total mass is consistent with irradiation-dependent covalent bond formation between one strand of DNA and the protein. These preliminary results support the possibility of identifying picomole quantities of crosslinked peptides by similar strategies.</description><subject>Base Sequence</subject><subject>Binding Sites</subject><subject>Chromatography, High Pressure Liquid</subject><subject>Cross-Linking Reagents</subject><subject>DNA - chemistry</subject><subject>DNA - metabolism</subject><subject>Electrophoresis, Polyacrylamide Gel</subject><subject>Mass Spectrometry - methods</subject><subject>Oligodeoxyribonucleotides - chemistry</subject><subject>Oligodeoxyribonucleotides - metabolism</subject><subject>Photochemistry</subject><subject>Site-Specific DNA-Methyltransferase (Adenine-Specific) - chemistry</subject><subject>Site-Specific DNA-Methyltransferase (Adenine-Specific) - metabolism</subject><subject>Substrate Specificity</subject><subject>Ultraviolet Rays</subject><subject>Uracil - analogs & derivatives</subject><subject>Uracil - chemistry</subject><issn>0305-1048</issn><issn>1362-4962</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1998</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkc1v1DAQxS0EKkvhxhUpJ05k62_HBw5VWWjFClABqeJizToTNjSJUzuLuvz1uN1lBSdOY-n93uh5HiHPGZ0zasXJAPGE6zmfa6kekBkTmpfSav6QzKigqmRUVo_Jk5R-UMokU_KIHFnJmeLVjGwXHfophjRG2BZtGNpfMOVR9JBSkcZ7sccptr7wa4jgJ4x_mNAU4zpMwWd_6trhGuvizYfTcuHD5cXdq8jO9babIgypwQgJCx_6scNbTE_Jowa6hM_285h8fbv4cnZeLj--uzg7XZZeSjOVFiisbINe4IqaShuquKitrFBxkFoZlBZEo0BZJhgIapQ2TNYrJo2vGyuOyevd3nGz6rH2OOQ4nRtj20PcugCt-1cZ2rX7Hn66vIBbmv0v9_4YbjaYJte3yWPXwYBhk5zJp66okf8FmeZc5cAZfLUD7-8WsTmEYdTdVepypY5rx12uNOMv_v7AAd53mPVyp7dpwtuDDPHaaSOMcudX39wn-n6pruhndyl-A7pWr_8</recordid><startdate>19980115</startdate><enddate>19980115</enddate><creator>Wong, David L.</creator><creator>Pavlovich, James G.</creator><creator>Reich, Norbert O.</creator><general>Oxford University Press</general><scope>BSCLL</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QL</scope><scope>7TM</scope><scope>C1K</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>19980115</creationdate><title>Electrospray ionization mass spectrometric characterization of photocrosslinked DNA-EcoRI DNA methyltransferase complexes</title><author>Wong, David L. ; Pavlovich, James G. ; Reich, Norbert O.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c447t-9a0ab9fec3eb078670523d948e52a4657e49a3f5a59131a30756714db147cdf93</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1998</creationdate><topic>Base Sequence</topic><topic>Binding Sites</topic><topic>Chromatography, High Pressure Liquid</topic><topic>Cross-Linking Reagents</topic><topic>DNA - chemistry</topic><topic>DNA - metabolism</topic><topic>Electrophoresis, Polyacrylamide Gel</topic><topic>Mass Spectrometry - methods</topic><topic>Oligodeoxyribonucleotides - chemistry</topic><topic>Oligodeoxyribonucleotides - metabolism</topic><topic>Photochemistry</topic><topic>Site-Specific DNA-Methyltransferase (Adenine-Specific) - chemistry</topic><topic>Site-Specific DNA-Methyltransferase (Adenine-Specific) - metabolism</topic><topic>Substrate Specificity</topic><topic>Ultraviolet Rays</topic><topic>Uracil - analogs & derivatives</topic><topic>Uracil - chemistry</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Wong, David L.</creatorcontrib><creatorcontrib>Pavlovich, James G.</creatorcontrib><creatorcontrib>Reich, Norbert O.</creatorcontrib><collection>Istex</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Nucleic Acids Abstracts</collection><collection>Environmental Sciences and Pollution Management</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Nucleic acids research</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Wong, David L.</au><au>Pavlovich, James G.</au><au>Reich, Norbert O.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Electrospray ionization mass spectrometric characterization of photocrosslinked DNA-EcoRI DNA methyltransferase complexes</atitle><jtitle>Nucleic acids research</jtitle><addtitle>Nucleic Acids Research</addtitle><date>1998-01-15</date><risdate>1998</risdate><volume>26</volume><issue>2</issue><spage>645</spage><epage>649</epage><pages>645-649</pages><issn>0305-1048</issn><eissn>1362-4962</eissn><abstract>We describe a novel strategy combining photocrosslinking and HPLC-based electrospray ionization mass spectrometry to identify UV crosslinked DNA-protein complexes. EcoRI DNA methyltransferase modifies the second adenine within the recognition sequence GAATTC. Substitution of 5-iodouracil for the thymine adjacent to the target base (GAATTC) does not detectably alter the DNA-protein complex. Irradiation of the 5-iodouracil-substituted DNA-protein complex at various wavelengths was optimized, with a crosslinking yield >60% at 313 nm after 1 min. No protein degradation was observed under these conditions. The crosslinked DNA-protein complex was further analyzed by electrospray ionization mass spectrometry. The total mass is consistent with irradiation-dependent covalent bond formation between one strand of DNA and the protein. These preliminary results support the possibility of identifying picomole quantities of crosslinked peptides by similar strategies.</abstract><cop>England</cop><pub>Oxford University Press</pub><pmid>9421528</pmid><doi>10.1093/nar/26.2.645</doi><tpages>5</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | Nucleic acids research, 1998-01, Vol.26 (2), p.645-649 |
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| source | Oxford Journals Open Access Collection; MEDLINE; PubMed Central; Free Full-Text Journals in Chemistry |
| subjects | Base Sequence Binding Sites Chromatography, High Pressure Liquid Cross-Linking Reagents DNA - chemistry DNA - metabolism Electrophoresis, Polyacrylamide Gel Mass Spectrometry - methods Oligodeoxyribonucleotides - chemistry Oligodeoxyribonucleotides - metabolism Photochemistry Site-Specific DNA-Methyltransferase (Adenine-Specific) - chemistry Site-Specific DNA-Methyltransferase (Adenine-Specific) - metabolism Substrate Specificity Ultraviolet Rays Uracil - analogs & derivatives Uracil - chemistry |
| title | Electrospray ionization mass spectrometric characterization of photocrosslinked DNA-EcoRI DNA methyltransferase complexes |
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