Phage T4 DNA [N 6 -Adenine] Methyltransferase: Kinetic Studies Using Oligonucleotides Containing Native or Modified Recognition Sites
The DNA-[N6-adenine] methyltransferase of T4 phage (T4 Dam MTase) catalyzes methyl group transfer from S-adenosyl-L-methionine (AdoMet) to the N6-position of adenine in the palindromic sequence, GATC. We have investigated the effect of eliminating different structural components of the recognition s...
Gespeichert in:
Veröffentlicht in: | Biological Chemistry 1998-04, Vol.379 (4-5), p.481-488 |
---|---|
Hauptverfasser: | , , , , , |
Format: | Artikel |
Sprache: | eng |
Schlagworte: | |
Online-Zugang: | Volltext |
Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
container_end_page | 488 |
---|---|
container_issue | 4-5 |
container_start_page | 481 |
container_title | Biological Chemistry |
container_volume | 379 |
creator | Zinoviev, Victor V. Evdokimov, Alexei A. Gorbunov, Yuri A. Malygin, Ernst G. Kossykh, Valeri G. Hattman, Stanley |
description | The DNA-[N6-adenine] methyltransferase of T4 phage (T4 Dam MTase) catalyzes methyl group transfer from S-adenosyl-L-methionine (AdoMet) to the N6-position of adenine in the palindromic sequence, GATC. We have investigated the effect of eliminating different structural components of the recognition site on the ability of a substrate to be bound and methylated by T4 Dam. For this purpose, steady state binding (by gel shift assays) and kinetic parameters of methylation (using the methyl donor, [3H-CH3]-AdoMet, at 25 degrees C) were studied using various synthetic duplex oligonucleotides containing some defect in the DNA-target site; e.g., the absence of an internucleotide phosphate or a nucleotide(s) within the recognition site, or a single stranded region. The salient results are summarized as follows: (1) Addition of T4 Dam to a complete reaction mixture (with a 20-mer duplex as substrate) resulted in a 'burst' of 3H-methylated product, followed by a constant rate of product formation that reflected establishment of steady-state conditions. This suggests that the rate-limiting step is release of product methylated DNA from the enzyme [and not the transfer of the methyl group]. (2) A number of the defects in duplex structure had only a weak influence on the binding and Km values, but strongly reduced the kcat. At the same time, several poorly bound duplexes retained good substrate characteristics, especially duplexes having uninterrupted GAT-sequences in both strands. Whereas having only one half of the recognition site element intact was sufficient for stable complex formation, the catalytic turnover process had a strict requirement for an uninterrupted GAT-sequence on both strands. (3) There was no correlation between Km and binding capability; the apparent Kd for some duplexes was 5-70 times higher than Km. This indicates that the T4 Dam methylation reaction can not be explained by a simple Michaelian scheme. |
doi_str_mv | 10.1515/bchm.1998.379.4-5.481 |
format | Article |
fullrecord | <record><control><sourceid>proquest_cross</sourceid><recordid>TN_cdi_proquest_miscellaneous_79937517</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><sourcerecordid>79937517</sourcerecordid><originalsourceid>FETCH-LOGICAL-c342t-98d4479c36a1675931c6321faf5549e4eebcc497a37a8827fac7aafdd60e934e3</originalsourceid><addsrcrecordid>eNo9kc1uEzEQgC0EKlHpI1Tyidsu69her7mFAOUnTUObnhCyHHs2MWzsYnsRfQDeG0eJOpeR5psf2R9Cl6SpCSf8zcbs9jWRsqupkDWreM068gxNCKOiYpTw52jSECEqyiV9iS5S-tmUYJJwLs_QmWynHWVkgv6tdnoLeM3w--UMf1_iFlczC955-IGvIe8ehxy1Tz1EneAt_lpAdgbf5dE6SPg-Ob_FN4PbBj-aAUJ2tpTnwWft_IEtdXZ_AIeIr4N1vQOLb8GErXfZBY_vXIb0Cr3o9ZDg4pTP0f3HD-v5p2pxc_V5PltUhrJprmRnGRPS0FaTVpSXEdPSKel1zzmTwAA2xjApNBW666ai10Zo3VvbNiApA3qOXh_3PsTwe4SU1d4lA8OgPYQxKSElFZyI0siPjSaGlCL06iG6vY6PijTqYEAdDKiDAVUMKKa4KgbK3OXpwLjZg32aOv134dWRu5Th7xPW8ZdqRTmtvq2ZWq0WzdXtF6re0f8rW5LR</addsrcrecordid><sourcetype>Aggregation Database</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>79937517</pqid></control><display><type>article</type><title>Phage T4 DNA [N 6 -Adenine] Methyltransferase: Kinetic Studies Using Oligonucleotides Containing Native or Modified Recognition Sites</title><source>MEDLINE</source><source>De Gruyter journals</source><creator>Zinoviev, Victor V. ; Evdokimov, Alexei A. ; Gorbunov, Yuri A. ; Malygin, Ernst G. ; Kossykh, Valeri G. ; Hattman, Stanley</creator><creatorcontrib>Zinoviev, Victor V. ; Evdokimov, Alexei A. ; Gorbunov, Yuri A. ; Malygin, Ernst G. ; Kossykh, Valeri G. ; Hattman, Stanley</creatorcontrib><description>The DNA-[N6-adenine] methyltransferase of T4 phage (T4 Dam MTase) catalyzes methyl group transfer from S-adenosyl-L-methionine (AdoMet) to the N6-position of adenine in the palindromic sequence, GATC. We have investigated the effect of eliminating different structural components of the recognition site on the ability of a substrate to be bound and methylated by T4 Dam. For this purpose, steady state binding (by gel shift assays) and kinetic parameters of methylation (using the methyl donor, [3H-CH3]-AdoMet, at 25 degrees C) were studied using various synthetic duplex oligonucleotides containing some defect in the DNA-target site; e.g., the absence of an internucleotide phosphate or a nucleotide(s) within the recognition site, or a single stranded region. The salient results are summarized as follows: (1) Addition of T4 Dam to a complete reaction mixture (with a 20-mer duplex as substrate) resulted in a 'burst' of 3H-methylated product, followed by a constant rate of product formation that reflected establishment of steady-state conditions. This suggests that the rate-limiting step is release of product methylated DNA from the enzyme [and not the transfer of the methyl group]. (2) A number of the defects in duplex structure had only a weak influence on the binding and Km values, but strongly reduced the kcat. At the same time, several poorly bound duplexes retained good substrate characteristics, especially duplexes having uninterrupted GAT-sequences in both strands. Whereas having only one half of the recognition site element intact was sufficient for stable complex formation, the catalytic turnover process had a strict requirement for an uninterrupted GAT-sequence on both strands. (3) There was no correlation between Km and binding capability; the apparent Kd for some duplexes was 5-70 times higher than Km. This indicates that the T4 Dam methylation reaction can not be explained by a simple Michaelian scheme.</description><identifier>ISSN: 0177-3593</identifier><identifier>ISSN: 1431-6730</identifier><identifier>EISSN: 1437-4315</identifier><identifier>DOI: 10.1515/bchm.1998.379.4-5.481</identifier><identifier>PMID: 9628341</identifier><language>eng</language><publisher>Berlin, New York: Walter de Gruyter, Berlin / New York</publisher><subject>Bacteriophage T4 - enzymology ; Binding Sites ; DNA Methylation ; Kinetics ; Nucleic Acid Heteroduplexes - metabolism ; Oligodeoxyribonucleotides - metabolism ; Site-Specific DNA-Methyltransferase (Adenine-Specific) - metabolism ; Substrate Specificity ; Temperature ; Viral Proteins</subject><ispartof>Biological Chemistry, 1998-04, Vol.379 (4-5), p.481-488</ispartof><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c342t-98d4479c36a1675931c6321faf5549e4eebcc497a37a8827fac7aafdd60e934e3</citedby><cites>FETCH-LOGICAL-c342t-98d4479c36a1675931c6321faf5549e4eebcc497a37a8827fac7aafdd60e934e3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27924,27925</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/9628341$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Zinoviev, Victor V.</creatorcontrib><creatorcontrib>Evdokimov, Alexei A.</creatorcontrib><creatorcontrib>Gorbunov, Yuri A.</creatorcontrib><creatorcontrib>Malygin, Ernst G.</creatorcontrib><creatorcontrib>Kossykh, Valeri G.</creatorcontrib><creatorcontrib>Hattman, Stanley</creatorcontrib><title>Phage T4 DNA [N 6 -Adenine] Methyltransferase: Kinetic Studies Using Oligonucleotides Containing Native or Modified Recognition Sites</title><title>Biological Chemistry</title><addtitle>Biological Chemistry</addtitle><description>The DNA-[N6-adenine] methyltransferase of T4 phage (T4 Dam MTase) catalyzes methyl group transfer from S-adenosyl-L-methionine (AdoMet) to the N6-position of adenine in the palindromic sequence, GATC. We have investigated the effect of eliminating different structural components of the recognition site on the ability of a substrate to be bound and methylated by T4 Dam. For this purpose, steady state binding (by gel shift assays) and kinetic parameters of methylation (using the methyl donor, [3H-CH3]-AdoMet, at 25 degrees C) were studied using various synthetic duplex oligonucleotides containing some defect in the DNA-target site; e.g., the absence of an internucleotide phosphate or a nucleotide(s) within the recognition site, or a single stranded region. The salient results are summarized as follows: (1) Addition of T4 Dam to a complete reaction mixture (with a 20-mer duplex as substrate) resulted in a 'burst' of 3H-methylated product, followed by a constant rate of product formation that reflected establishment of steady-state conditions. This suggests that the rate-limiting step is release of product methylated DNA from the enzyme [and not the transfer of the methyl group]. (2) A number of the defects in duplex structure had only a weak influence on the binding and Km values, but strongly reduced the kcat. At the same time, several poorly bound duplexes retained good substrate characteristics, especially duplexes having uninterrupted GAT-sequences in both strands. Whereas having only one half of the recognition site element intact was sufficient for stable complex formation, the catalytic turnover process had a strict requirement for an uninterrupted GAT-sequence on both strands. (3) There was no correlation between Km and binding capability; the apparent Kd for some duplexes was 5-70 times higher than Km. This indicates that the T4 Dam methylation reaction can not be explained by a simple Michaelian scheme.</description><subject>Bacteriophage T4 - enzymology</subject><subject>Binding Sites</subject><subject>DNA Methylation</subject><subject>Kinetics</subject><subject>Nucleic Acid Heteroduplexes - metabolism</subject><subject>Oligodeoxyribonucleotides - metabolism</subject><subject>Site-Specific DNA-Methyltransferase (Adenine-Specific) - metabolism</subject><subject>Substrate Specificity</subject><subject>Temperature</subject><subject>Viral Proteins</subject><issn>0177-3593</issn><issn>1431-6730</issn><issn>1437-4315</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1998</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNo9kc1uEzEQgC0EKlHpI1Tyidsu69her7mFAOUnTUObnhCyHHs2MWzsYnsRfQDeG0eJOpeR5psf2R9Cl6SpCSf8zcbs9jWRsqupkDWreM068gxNCKOiYpTw52jSECEqyiV9iS5S-tmUYJJwLs_QmWynHWVkgv6tdnoLeM3w--UMf1_iFlczC955-IGvIe8ehxy1Tz1EneAt_lpAdgbf5dE6SPg-Ob_FN4PbBj-aAUJ2tpTnwWft_IEtdXZ_AIeIr4N1vQOLb8GErXfZBY_vXIb0Cr3o9ZDg4pTP0f3HD-v5p2pxc_V5PltUhrJprmRnGRPS0FaTVpSXEdPSKel1zzmTwAA2xjApNBW666ai10Zo3VvbNiApA3qOXh_3PsTwe4SU1d4lA8OgPYQxKSElFZyI0siPjSaGlCL06iG6vY6PijTqYEAdDKiDAVUMKKa4KgbK3OXpwLjZg32aOv134dWRu5Th7xPW8ZdqRTmtvq2ZWq0WzdXtF6re0f8rW5LR</recordid><startdate>19980401</startdate><enddate>19980401</enddate><creator>Zinoviev, Victor V.</creator><creator>Evdokimov, Alexei A.</creator><creator>Gorbunov, Yuri A.</creator><creator>Malygin, Ernst G.</creator><creator>Kossykh, Valeri G.</creator><creator>Hattman, Stanley</creator><general>Walter de Gruyter, Berlin / New York</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>7X8</scope></search><sort><creationdate>19980401</creationdate><title>Phage T4 DNA [N 6 -Adenine] Methyltransferase: Kinetic Studies Using Oligonucleotides Containing Native or Modified Recognition Sites</title><author>Zinoviev, Victor V. ; Evdokimov, Alexei A. ; Gorbunov, Yuri A. ; Malygin, Ernst G. ; Kossykh, Valeri G. ; Hattman, Stanley</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c342t-98d4479c36a1675931c6321faf5549e4eebcc497a37a8827fac7aafdd60e934e3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1998</creationdate><topic>Bacteriophage T4 - enzymology</topic><topic>Binding Sites</topic><topic>DNA Methylation</topic><topic>Kinetics</topic><topic>Nucleic Acid Heteroduplexes - metabolism</topic><topic>Oligodeoxyribonucleotides - metabolism</topic><topic>Site-Specific DNA-Methyltransferase (Adenine-Specific) - metabolism</topic><topic>Substrate Specificity</topic><topic>Temperature</topic><topic>Viral Proteins</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Zinoviev, Victor V.</creatorcontrib><creatorcontrib>Evdokimov, Alexei A.</creatorcontrib><creatorcontrib>Gorbunov, Yuri A.</creatorcontrib><creatorcontrib>Malygin, Ernst G.</creatorcontrib><creatorcontrib>Kossykh, Valeri G.</creatorcontrib><creatorcontrib>Hattman, Stanley</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>MEDLINE - Academic</collection><jtitle>Biological Chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Zinoviev, Victor V.</au><au>Evdokimov, Alexei A.</au><au>Gorbunov, Yuri A.</au><au>Malygin, Ernst G.</au><au>Kossykh, Valeri G.</au><au>Hattman, Stanley</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Phage T4 DNA [N 6 -Adenine] Methyltransferase: Kinetic Studies Using Oligonucleotides Containing Native or Modified Recognition Sites</atitle><jtitle>Biological Chemistry</jtitle><addtitle>Biological Chemistry</addtitle><date>1998-04-01</date><risdate>1998</risdate><volume>379</volume><issue>4-5</issue><spage>481</spage><epage>488</epage><pages>481-488</pages><issn>0177-3593</issn><issn>1431-6730</issn><eissn>1437-4315</eissn><abstract>The DNA-[N6-adenine] methyltransferase of T4 phage (T4 Dam MTase) catalyzes methyl group transfer from S-adenosyl-L-methionine (AdoMet) to the N6-position of adenine in the palindromic sequence, GATC. We have investigated the effect of eliminating different structural components of the recognition site on the ability of a substrate to be bound and methylated by T4 Dam. For this purpose, steady state binding (by gel shift assays) and kinetic parameters of methylation (using the methyl donor, [3H-CH3]-AdoMet, at 25 degrees C) were studied using various synthetic duplex oligonucleotides containing some defect in the DNA-target site; e.g., the absence of an internucleotide phosphate or a nucleotide(s) within the recognition site, or a single stranded region. The salient results are summarized as follows: (1) Addition of T4 Dam to a complete reaction mixture (with a 20-mer duplex as substrate) resulted in a 'burst' of 3H-methylated product, followed by a constant rate of product formation that reflected establishment of steady-state conditions. This suggests that the rate-limiting step is release of product methylated DNA from the enzyme [and not the transfer of the methyl group]. (2) A number of the defects in duplex structure had only a weak influence on the binding and Km values, but strongly reduced the kcat. At the same time, several poorly bound duplexes retained good substrate characteristics, especially duplexes having uninterrupted GAT-sequences in both strands. Whereas having only one half of the recognition site element intact was sufficient for stable complex formation, the catalytic turnover process had a strict requirement for an uninterrupted GAT-sequence on both strands. (3) There was no correlation between Km and binding capability; the apparent Kd for some duplexes was 5-70 times higher than Km. This indicates that the T4 Dam methylation reaction can not be explained by a simple Michaelian scheme.</abstract><cop>Berlin, New York</cop><pub>Walter de Gruyter, Berlin / New York</pub><pmid>9628341</pmid><doi>10.1515/bchm.1998.379.4-5.481</doi><tpages>8</tpages></addata></record> |
fulltext | fulltext |
identifier | ISSN: 0177-3593 |
ispartof | Biological Chemistry, 1998-04, Vol.379 (4-5), p.481-488 |
issn | 0177-3593 1431-6730 1437-4315 |
language | eng |
recordid | cdi_proquest_miscellaneous_79937517 |
source | MEDLINE; De Gruyter journals |
subjects | Bacteriophage T4 - enzymology Binding Sites DNA Methylation Kinetics Nucleic Acid Heteroduplexes - metabolism Oligodeoxyribonucleotides - metabolism Site-Specific DNA-Methyltransferase (Adenine-Specific) - metabolism Substrate Specificity Temperature Viral Proteins |
title | Phage T4 DNA [N 6 -Adenine] Methyltransferase: Kinetic Studies Using Oligonucleotides Containing Native or Modified Recognition Sites |
url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2024-12-24T06%3A32%3A17IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-proquest_cross&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Phage%20T4%20DNA%20%5BN%206%20-Adenine%5D%20Methyltransferase:%20Kinetic%20Studies%20Using%20Oligonucleotides%20Containing%20Native%20or%20Modified%20Recognition%20Sites&rft.jtitle=Biological%20Chemistry&rft.au=Zinoviev,%20Victor%20V.&rft.date=1998-04-01&rft.volume=379&rft.issue=4-5&rft.spage=481&rft.epage=488&rft.pages=481-488&rft.issn=0177-3593&rft.eissn=1437-4315&rft_id=info:doi/10.1515/bchm.1998.379.4-5.481&rft_dat=%3Cproquest_cross%3E79937517%3C/proquest_cross%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=79937517&rft_id=info:pmid/9628341&rfr_iscdi=true |