Definitive Identification of Mammalian 5-Hydroxymethyluracil DNA N-Glycosylase Activity as SMUG1
Purification from calf thymus of a DNA N-glycosylase activity (HMUDG) that released 5-hydroxymethyluracil (5hmUra) from the DNA of Bacillus subtilis phage SPO1 was undertaken. Analysis of the most purified fraction by SDS-polyacrylamide gel electrophoresis revealed a multiplicity of protein species...
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| Veröffentlicht in: | The Journal of biological chemistry 2001-11, Vol.276 (45), p.41991-41997 |
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| container_end_page | 41997 |
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| container_issue | 45 |
| container_start_page | 41991 |
| container_title | The Journal of biological chemistry |
| container_volume | 276 |
| creator | Boorstein, Robert J. Archie, Cummings Marenstein, Dina R. Chan, Michael K. Ma, Yuliang Neubert, Thomas A. Brown, Stuart M. Teebor, George W. |
| description | Purification from calf thymus of a DNA N-glycosylase activity (HMUDG) that released 5-hydroxymethyluracil (5hmUra) from the DNA of Bacillus subtilis phage SPO1 was undertaken. Analysis of the most purified fraction by SDS-polyacrylamide gel electrophoresis revealed a multiplicity of protein species making it impossible to identify HMUDG by inspection. Therefore, we renatured the enzyme after SDS-polyacrylamide gel electrophoresis and assayed slices of the gel for DNA N-glycosylase activity directed against 5hmUra. Maximum enzymatic activity was identified between molecular mass markers 30 and 34 kDa. Protein was extracted from gel slices and subjected to tryptic digestion and analysis by mass spectrometry. Analysis revealed the presence of 11 peptides that were homologous or identical to the sequence of the recently characterized human single-stranded monofunctional uracil DNA N-glycosylase (hSMUG1). The cDNA of hSMUG1 was isolated and expressed as a recombinant glutathione S-transferase fusion protein that was shown to release 5hmUra with 20× the specific activity of the most purified bovine fraction. We conclude that hSMUG1 and HMUDG are the same protein. |
| doi_str_mv | 10.1074/jbc.M106953200 |
| format | Article |
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Analysis of the most purified fraction by SDS-polyacrylamide gel electrophoresis revealed a multiplicity of protein species making it impossible to identify HMUDG by inspection. Therefore, we renatured the enzyme after SDS-polyacrylamide gel electrophoresis and assayed slices of the gel for DNA N-glycosylase activity directed against 5hmUra. Maximum enzymatic activity was identified between molecular mass markers 30 and 34 kDa. Protein was extracted from gel slices and subjected to tryptic digestion and analysis by mass spectrometry. Analysis revealed the presence of 11 peptides that were homologous or identical to the sequence of the recently characterized human single-stranded monofunctional uracil DNA N-glycosylase (hSMUG1). The cDNA of hSMUG1 was isolated and expressed as a recombinant glutathione S-transferase fusion protein that was shown to release 5hmUra with 20× the specific activity of the most purified bovine fraction. We conclude that hSMUG1 and HMUDG are the same protein.</description><identifier>ISSN: 0021-9258</identifier><identifier>EISSN: 1083-351X</identifier><identifier>DOI: 10.1074/jbc.M106953200</identifier><identifier>PMID: 11526119</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Amino Acid Sequence ; Animals ; Bacillus subtilis ; Cattle ; DNA glycosylase ; DNA Glycosylases ; DNA, Complementary - isolation & purification ; Electrophoresis, Polyacrylamide Gel ; Molecular Sequence Data ; N-Glycosyl Hydrolases - chemistry ; N-Glycosyl Hydrolases - genetics ; N-Glycosyl Hydrolases - isolation & purification ; Pentoxyl - analogs & derivatives ; Pentoxyl - metabolism ; Phage SPO1 ; Uracil-DNA Glycosidase</subject><ispartof>The Journal of biological chemistry, 2001-11, Vol.276 (45), p.41991-41997</ispartof><rights>2001 © 2001 ASBMB. Currently published by Elsevier Inc; originally published by American Society for Biochemistry and Molecular Biology.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c477t-9e9475f07b4c30e64a034b8b1b2424dabb7496e629aca5884ce278961a4d76913</citedby><cites>FETCH-LOGICAL-c477t-9e9475f07b4c30e64a034b8b1b2424dabb7496e629aca5884ce278961a4d76913</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,777,781,27905,27906</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/11526119$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Boorstein, Robert J.</creatorcontrib><creatorcontrib>Archie, Cummings</creatorcontrib><creatorcontrib>Marenstein, Dina R.</creatorcontrib><creatorcontrib>Chan, Michael K.</creatorcontrib><creatorcontrib>Ma, Yuliang</creatorcontrib><creatorcontrib>Neubert, Thomas A.</creatorcontrib><creatorcontrib>Brown, Stuart M.</creatorcontrib><creatorcontrib>Teebor, George W.</creatorcontrib><title>Definitive Identification of Mammalian 5-Hydroxymethyluracil DNA N-Glycosylase Activity as SMUG1</title><title>The Journal of biological chemistry</title><addtitle>J Biol Chem</addtitle><description>Purification from calf thymus of a DNA N-glycosylase activity (HMUDG) that released 5-hydroxymethyluracil (5hmUra) from the DNA of Bacillus subtilis phage SPO1 was undertaken. Analysis of the most purified fraction by SDS-polyacrylamide gel electrophoresis revealed a multiplicity of protein species making it impossible to identify HMUDG by inspection. Therefore, we renatured the enzyme after SDS-polyacrylamide gel electrophoresis and assayed slices of the gel for DNA N-glycosylase activity directed against 5hmUra. Maximum enzymatic activity was identified between molecular mass markers 30 and 34 kDa. Protein was extracted from gel slices and subjected to tryptic digestion and analysis by mass spectrometry. Analysis revealed the presence of 11 peptides that were homologous or identical to the sequence of the recently characterized human single-stranded monofunctional uracil DNA N-glycosylase (hSMUG1). The cDNA of hSMUG1 was isolated and expressed as a recombinant glutathione S-transferase fusion protein that was shown to release 5hmUra with 20× the specific activity of the most purified bovine fraction. We conclude that hSMUG1 and HMUDG are the same protein.</description><subject>Amino Acid Sequence</subject><subject>Animals</subject><subject>Bacillus subtilis</subject><subject>Cattle</subject><subject>DNA glycosylase</subject><subject>DNA Glycosylases</subject><subject>DNA, Complementary - isolation & purification</subject><subject>Electrophoresis, Polyacrylamide Gel</subject><subject>Molecular Sequence Data</subject><subject>N-Glycosyl Hydrolases - chemistry</subject><subject>N-Glycosyl Hydrolases - genetics</subject><subject>N-Glycosyl Hydrolases - isolation & purification</subject><subject>Pentoxyl - analogs & derivatives</subject><subject>Pentoxyl - metabolism</subject><subject>Phage SPO1</subject><subject>Uracil-DNA Glycosidase</subject><issn>0021-9258</issn><issn>1083-351X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2001</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkD1PwzAQQC0EglJYGZEnthSf48T2WPFRkCgMgMRmHOcijPIBdorIvydVKzEhTifd8u4Nj5ATYDNgUpy_F262BJbrLOWM7ZAJMJUmaQYvu2TCGIdE80wdkMMY39k4QsM-OQDIeA6gJ-T1Eivf-t5_Ib0tse195Z3tfdfSrqJL2zS29ralWXIzlKH7Hhrs34Z6FazzNb28n9P7ZFEProtDbSPSuRtVvh-ojfRx-byAI7JX2Tri8fZOyfP11dPFTXL3sLi9mN8lTkjZJxq1kFnFZCFcyjAXlqWiUAUUXHBR2qKQQueYc22dzZQSDrlUOgcrSplrSKfkbOP9CN3nCmNvGh8d1rVtsVtFIznPxhX_gqBAMdBrcLYBXehiDFiZj-AbGwYDzKzjmzG--Y0_PpxuzauiwfIX39YeAbUBcAzx5TGY6Dy2Dksf0PWm7Pxf7h9trJG9</recordid><startdate>20011109</startdate><enddate>20011109</enddate><creator>Boorstein, Robert J.</creator><creator>Archie, Cummings</creator><creator>Marenstein, Dina R.</creator><creator>Chan, Michael K.</creator><creator>Ma, Yuliang</creator><creator>Neubert, Thomas A.</creator><creator>Brown, Stuart M.</creator><creator>Teebor, George W.</creator><general>Elsevier Inc</general><scope>6I.</scope><scope>AAFTH</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>7TM</scope><scope>7X8</scope></search><sort><creationdate>20011109</creationdate><title>Definitive Identification of Mammalian 5-Hydroxymethyluracil DNA N-Glycosylase Activity as SMUG1</title><author>Boorstein, Robert J. ; Archie, Cummings ; Marenstein, Dina R. ; Chan, Michael K. ; Ma, Yuliang ; Neubert, Thomas A. ; Brown, Stuart M. ; Teebor, George W.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c477t-9e9475f07b4c30e64a034b8b1b2424dabb7496e629aca5884ce278961a4d76913</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2001</creationdate><topic>Amino Acid Sequence</topic><topic>Animals</topic><topic>Bacillus subtilis</topic><topic>Cattle</topic><topic>DNA glycosylase</topic><topic>DNA Glycosylases</topic><topic>DNA, Complementary - isolation & purification</topic><topic>Electrophoresis, Polyacrylamide Gel</topic><topic>Molecular Sequence Data</topic><topic>N-Glycosyl Hydrolases - chemistry</topic><topic>N-Glycosyl Hydrolases - genetics</topic><topic>N-Glycosyl Hydrolases - isolation & purification</topic><topic>Pentoxyl - analogs & derivatives</topic><topic>Pentoxyl - metabolism</topic><topic>Phage SPO1</topic><topic>Uracil-DNA Glycosidase</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Boorstein, Robert J.</creatorcontrib><creatorcontrib>Archie, Cummings</creatorcontrib><creatorcontrib>Marenstein, Dina R.</creatorcontrib><creatorcontrib>Chan, Michael K.</creatorcontrib><creatorcontrib>Ma, Yuliang</creatorcontrib><creatorcontrib>Neubert, Thomas A.</creatorcontrib><creatorcontrib>Brown, Stuart M.</creatorcontrib><creatorcontrib>Teebor, George W.</creatorcontrib><collection>ScienceDirect Open Access Titles</collection><collection>Elsevier:ScienceDirect:Open Access</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Nucleic Acids Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>The Journal of biological chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Boorstein, Robert J.</au><au>Archie, Cummings</au><au>Marenstein, Dina R.</au><au>Chan, Michael K.</au><au>Ma, Yuliang</au><au>Neubert, Thomas A.</au><au>Brown, Stuart M.</au><au>Teebor, George W.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Definitive Identification of Mammalian 5-Hydroxymethyluracil DNA N-Glycosylase Activity as SMUG1</atitle><jtitle>The Journal of biological chemistry</jtitle><addtitle>J Biol Chem</addtitle><date>2001-11-09</date><risdate>2001</risdate><volume>276</volume><issue>45</issue><spage>41991</spage><epage>41997</epage><pages>41991-41997</pages><issn>0021-9258</issn><eissn>1083-351X</eissn><abstract>Purification from calf thymus of a DNA N-glycosylase activity (HMUDG) that released 5-hydroxymethyluracil (5hmUra) from the DNA of Bacillus subtilis phage SPO1 was undertaken. Analysis of the most purified fraction by SDS-polyacrylamide gel electrophoresis revealed a multiplicity of protein species making it impossible to identify HMUDG by inspection. Therefore, we renatured the enzyme after SDS-polyacrylamide gel electrophoresis and assayed slices of the gel for DNA N-glycosylase activity directed against 5hmUra. Maximum enzymatic activity was identified between molecular mass markers 30 and 34 kDa. Protein was extracted from gel slices and subjected to tryptic digestion and analysis by mass spectrometry. Analysis revealed the presence of 11 peptides that were homologous or identical to the sequence of the recently characterized human single-stranded monofunctional uracil DNA N-glycosylase (hSMUG1). The cDNA of hSMUG1 was isolated and expressed as a recombinant glutathione S-transferase fusion protein that was shown to release 5hmUra with 20× the specific activity of the most purified bovine fraction. 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| language | eng |
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| source | MEDLINE; EZB-FREE-00999 freely available EZB journals; Alma/SFX Local Collection |
| subjects | Amino Acid Sequence Animals Bacillus subtilis Cattle DNA glycosylase DNA Glycosylases DNA, Complementary - isolation & purification Electrophoresis, Polyacrylamide Gel Molecular Sequence Data N-Glycosyl Hydrolases - chemistry N-Glycosyl Hydrolases - genetics N-Glycosyl Hydrolases - isolation & purification Pentoxyl - analogs & derivatives Pentoxyl - metabolism Phage SPO1 Uracil-DNA Glycosidase |
| title | Definitive Identification of Mammalian 5-Hydroxymethyluracil DNA N-Glycosylase Activity as SMUG1 |
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