Methylation by a mutant T2 DNA [N(6)-adenine] methyltransferase expands the usage of RecA-assisted endonuclease (RARE) cleavage
Properties of a mutant bacteriophage T2 DNA [N:(6)-adenine] methyltransferase (T2 Dam MTase) have been investigated for its potential utilization in RecA-assisted restriction endonuclease (RARE) cleavage. Steady-state kinetic analyses with oligonucleotide duplexes revealed that, compared to wild-typ...
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| Veröffentlicht in: | Nucleic acids research 2001-04, Vol.29 (7), p.1484-1490 |
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| creator | Minko, I Hattman, S Lloyd, R S Kossykh, V |
| description | Properties of a mutant bacteriophage T2 DNA [N:(6)-adenine] methyltransferase (T2 Dam MTase) have been investigated for its potential utilization in RecA-assisted restriction endonuclease (RARE) cleavage. Steady-state kinetic analyses with oligonucleotide duplexes revealed that, compared to wild-type T4 Dam, both wild-type T2 Dam and mutant T2 Dam P126S had a 1.5-fold higher k(cat) in methylating canonical GATC sites. Additionally, T2 Dam P126S showed increased efficiencies in methylation of non-canonical GAY sites relative to the wild-type enzymes. In agreement with these steady-state kinetic data, when bacteriophage lambda DNA was used as a substrate, maximal protection from restriction nuclease cleavage in vitro was achieved on the sequences GATC, GATN and GACY, while protection of GACR sequences was less efficient. Collectively, our data suggest that T2 Dam P126S can modify 28 recognition sequences. The feasibility of using the mutant enzyme in RARE cleavage with BCL:I and ECO:RV endonucleases has been shown on phage lambda DNA and with BCL:I and DPN:II endonucleases on yeast chromosomal DNA embedded in agarose. |
| doi_str_mv | 10.1093/nar/29.7.1484 |
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Steady-state kinetic analyses with oligonucleotide duplexes revealed that, compared to wild-type T4 Dam, both wild-type T2 Dam and mutant T2 Dam P126S had a 1.5-fold higher k(cat) in methylating canonical GATC sites. Additionally, T2 Dam P126S showed increased efficiencies in methylation of non-canonical GAY sites relative to the wild-type enzymes. In agreement with these steady-state kinetic data, when bacteriophage lambda DNA was used as a substrate, maximal protection from restriction nuclease cleavage in vitro was achieved on the sequences GATC, GATN and GACY, while protection of GACR sequences was less efficient. Collectively, our data suggest that T2 Dam P126S can modify 28 recognition sequences. The feasibility of using the mutant enzyme in RARE cleavage with BCL:I and ECO:RV endonucleases has been shown on phage lambda DNA and with BCL:I and DPN:II endonucleases on yeast chromosomal DNA embedded in agarose.</description><identifier>ISSN: 1362-4962</identifier><identifier>ISSN: 0305-1048</identifier><identifier>EISSN: 1362-4962</identifier><identifier>DOI: 10.1093/nar/29.7.1484</identifier><identifier>PMID: 11266550</identifier><identifier>CODEN: NARHAD</identifier><language>eng</language><publisher>England: Oxford Publishing Limited (England)</publisher><subject>Bacteriophage lambda - genetics ; Base Sequence ; Binding Sites ; DNA adenine-(N6) methyltransferase ; DNA Methylation ; DNA Restriction Enzymes - metabolism ; DNA, Fungal - genetics ; DNA, Fungal - metabolism ; DNA, Viral - genetics ; DNA, Viral - metabolism ; Mutation ; Oligonucleotides - genetics ; Oligonucleotides - metabolism ; Phage ^l ; Phage T2 ; Rec A Recombinases - genetics ; Rec A Recombinases - metabolism ; Saccharomyces cerevisiae - genetics ; Site-Specific DNA-Methyltransferase (Adenine-Specific) - genetics ; Site-Specific DNA-Methyltransferase (Adenine-Specific) - metabolism ; Viral Proteins</subject><ispartof>Nucleic acids research, 2001-04, Vol.29 (7), p.1484-1490</ispartof><rights>Copyright Oxford University Press(England) Apr 1, 2001</rights><rights>Copyright © 2001 Oxford University Press 2001</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c439t-ef59981c651a9837ab96bfbeb3976e0dda42e5e2afe559a8a824d39fcb6cc3c63</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC31273/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC31273/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,727,780,784,885,27924,27925,53791,53793</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/11266550$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Minko, I</creatorcontrib><creatorcontrib>Hattman, S</creatorcontrib><creatorcontrib>Lloyd, R S</creatorcontrib><creatorcontrib>Kossykh, V</creatorcontrib><title>Methylation by a mutant T2 DNA [N(6)-adenine] methyltransferase expands the usage of RecA-assisted endonuclease (RARE) cleavage</title><title>Nucleic acids research</title><addtitle>Nucleic Acids Res</addtitle><description>Properties of a mutant bacteriophage T2 DNA [N:(6)-adenine] methyltransferase (T2 Dam MTase) have been investigated for its potential utilization in RecA-assisted restriction endonuclease (RARE) cleavage. Steady-state kinetic analyses with oligonucleotide duplexes revealed that, compared to wild-type T4 Dam, both wild-type T2 Dam and mutant T2 Dam P126S had a 1.5-fold higher k(cat) in methylating canonical GATC sites. Additionally, T2 Dam P126S showed increased efficiencies in methylation of non-canonical GAY sites relative to the wild-type enzymes. In agreement with these steady-state kinetic data, when bacteriophage lambda DNA was used as a substrate, maximal protection from restriction nuclease cleavage in vitro was achieved on the sequences GATC, GATN and GACY, while protection of GACR sequences was less efficient. Collectively, our data suggest that T2 Dam P126S can modify 28 recognition sequences. The feasibility of using the mutant enzyme in RARE cleavage with BCL:I and ECO:RV endonucleases has been shown on phage lambda DNA and with BCL:I and DPN:II endonucleases on yeast chromosomal DNA embedded in agarose.</description><subject>Bacteriophage lambda - genetics</subject><subject>Base Sequence</subject><subject>Binding Sites</subject><subject>DNA adenine-(N6) methyltransferase</subject><subject>DNA Methylation</subject><subject>DNA Restriction Enzymes - metabolism</subject><subject>DNA, Fungal - genetics</subject><subject>DNA, Fungal - metabolism</subject><subject>DNA, Viral - genetics</subject><subject>DNA, Viral - metabolism</subject><subject>Mutation</subject><subject>Oligonucleotides - genetics</subject><subject>Oligonucleotides - metabolism</subject><subject>Phage ^l</subject><subject>Phage T2</subject><subject>Rec A Recombinases - genetics</subject><subject>Rec A Recombinases - metabolism</subject><subject>Saccharomyces cerevisiae - genetics</subject><subject>Site-Specific DNA-Methyltransferase (Adenine-Specific) - genetics</subject><subject>Site-Specific DNA-Methyltransferase (Adenine-Specific) - metabolism</subject><subject>Viral Proteins</subject><issn>1362-4962</issn><issn>0305-1048</issn><issn>1362-4962</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2001</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqF0U1v1DAQBuAIgWgpHLkiiwNqD9n6O7HEZVXKh1SKtConhKyJM-mmSuzFdir2xF8nS1dQuHCyLT9jzfgtiueMLhg14tRDPOVmUS2YrOWD4pAJzUtpNH94b39QPEnphlImmZKPiwPGuNZK0cPix0fM6-0AuQ-eNFsCZJwy-EyuOHlzuSRfLo_1SQkt-t7jVzL-0jmCTx1GSEjw-wZ8m0heI5kSXCMJHVmhW5aQUp8ytgR9G_zkBtz549VydX5CdqfbWT8tHnUwJHy2X4-Kz2_Pr87elxef3n04W16UTgqTS-yUMTVzWjEwtaigMbrpGmyEqTTStgXJUSGHDpUyUEPNZStM5xrtnHBaHBWv797dTM2IrUM_DzHYTexHiFsboLd_3_h-ba_DrRWMV2Iuf7Uvj-HbhCnbsU8OhwE8hinZqqKUM8b-C1nNqBRazfDlP_AmTNHPf2A5pUrVlFYzKu-QiyGliN3vhhm1u_jtHL_lxlZ2F__sX9yf8o_e5y1-Atd9rOM</recordid><startdate>20010401</startdate><enddate>20010401</enddate><creator>Minko, I</creator><creator>Hattman, S</creator><creator>Lloyd, R S</creator><creator>Kossykh, V</creator><general>Oxford Publishing Limited (England)</general><general>Oxford University Press</general><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>7QO</scope><scope>7QP</scope><scope>7QR</scope><scope>7SS</scope><scope>7TK</scope><scope>7TM</scope><scope>7U9</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>K9.</scope><scope>M7N</scope><scope>P64</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20010401</creationdate><title>Methylation by a mutant T2 DNA [N(6)-adenine] methyltransferase expands the usage of RecA-assisted endonuclease (RARE) cleavage</title><author>Minko, I ; Hattman, S ; Lloyd, R S ; Kossykh, V</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c439t-ef59981c651a9837ab96bfbeb3976e0dda42e5e2afe559a8a824d39fcb6cc3c63</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2001</creationdate><topic>Bacteriophage lambda - genetics</topic><topic>Base Sequence</topic><topic>Binding Sites</topic><topic>DNA adenine-(N6) methyltransferase</topic><topic>DNA Methylation</topic><topic>DNA Restriction Enzymes - metabolism</topic><topic>DNA, Fungal - genetics</topic><topic>DNA, Fungal - metabolism</topic><topic>DNA, Viral - genetics</topic><topic>DNA, Viral - metabolism</topic><topic>Mutation</topic><topic>Oligonucleotides - genetics</topic><topic>Oligonucleotides - metabolism</topic><topic>Phage ^l</topic><topic>Phage T2</topic><topic>Rec A Recombinases - genetics</topic><topic>Rec A Recombinases - metabolism</topic><topic>Saccharomyces cerevisiae - genetics</topic><topic>Site-Specific DNA-Methyltransferase (Adenine-Specific) - genetics</topic><topic>Site-Specific DNA-Methyltransferase (Adenine-Specific) - metabolism</topic><topic>Viral Proteins</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Minko, I</creatorcontrib><creatorcontrib>Hattman, S</creatorcontrib><creatorcontrib>Lloyd, R S</creatorcontrib><creatorcontrib>Kossykh, V</creatorcontrib><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>Biotechnology Research Abstracts</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Chemoreception Abstracts</collection><collection>Entomology Abstracts (Full archive)</collection><collection>Neurosciences Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</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>Minko, I</au><au>Hattman, S</au><au>Lloyd, R S</au><au>Kossykh, V</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Methylation by a mutant T2 DNA [N(6)-adenine] methyltransferase expands the usage of RecA-assisted endonuclease (RARE) cleavage</atitle><jtitle>Nucleic acids research</jtitle><addtitle>Nucleic Acids Res</addtitle><date>2001-04-01</date><risdate>2001</risdate><volume>29</volume><issue>7</issue><spage>1484</spage><epage>1490</epage><pages>1484-1490</pages><issn>1362-4962</issn><issn>0305-1048</issn><eissn>1362-4962</eissn><coden>NARHAD</coden><abstract>Properties of a mutant bacteriophage T2 DNA [N:(6)-adenine] methyltransferase (T2 Dam MTase) have been investigated for its potential utilization in RecA-assisted restriction endonuclease (RARE) cleavage. Steady-state kinetic analyses with oligonucleotide duplexes revealed that, compared to wild-type T4 Dam, both wild-type T2 Dam and mutant T2 Dam P126S had a 1.5-fold higher k(cat) in methylating canonical GATC sites. Additionally, T2 Dam P126S showed increased efficiencies in methylation of non-canonical GAY sites relative to the wild-type enzymes. In agreement with these steady-state kinetic data, when bacteriophage lambda DNA was used as a substrate, maximal protection from restriction nuclease cleavage in vitro was achieved on the sequences GATC, GATN and GACY, while protection of GACR sequences was less efficient. Collectively, our data suggest that T2 Dam P126S can modify 28 recognition sequences. The feasibility of using the mutant enzyme in RARE cleavage with BCL:I and ECO:RV endonucleases has been shown on phage lambda DNA and with BCL:I and DPN:II endonucleases on yeast chromosomal DNA embedded in agarose.</abstract><cop>England</cop><pub>Oxford Publishing Limited (England)</pub><pmid>11266550</pmid><doi>10.1093/nar/29.7.1484</doi><tpages>7</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Bacteriophage lambda - genetics Base Sequence Binding Sites DNA adenine-(N6) methyltransferase DNA Methylation DNA Restriction Enzymes - metabolism DNA, Fungal - genetics DNA, Fungal - metabolism DNA, Viral - genetics DNA, Viral - metabolism Mutation Oligonucleotides - genetics Oligonucleotides - metabolism Phage ^l Phage T2 Rec A Recombinases - genetics Rec A Recombinases - metabolism Saccharomyces cerevisiae - genetics Site-Specific DNA-Methyltransferase (Adenine-Specific) - genetics Site-Specific DNA-Methyltransferase (Adenine-Specific) - metabolism Viral Proteins |
| title | Methylation by a mutant T2 DNA [N(6)-adenine] methyltransferase expands the usage of RecA-assisted endonuclease (RARE) cleavage |
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