Substrate and DNA binding to a 50-residue peptide fragment of DNA polymerase I. Comparison with the enzyme

The fluorescent nucleotide 2',3'-trinitrophenyl-ATP (TNP-ATP) binds at the triphosphate substrate binding site of the large (Klenow) fragment of DNA polymerase I (Pol I) as detected by direct binding studies measuring the increase in fluorescence of this ligand (n = 1.0, KD = 0.07 microM)....

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Veröffentlicht in:The Journal of biological chemistry 1989-11, Vol.264 (33), p.19637-19647
Hauptverfasser: MULLEN, G. P, SHENBAGAMURTHI, P, MILDVAN, A. S
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container_end_page 19647
container_issue 33
container_start_page 19637
container_title The Journal of biological chemistry
container_volume 264
creator MULLEN, G. P
SHENBAGAMURTHI, P
MILDVAN, A. S
description The fluorescent nucleotide 2',3'-trinitrophenyl-ATP (TNP-ATP) binds at the triphosphate substrate binding site of the large (Klenow) fragment of DNA polymerase I (Pol I) as detected by direct binding studies measuring the increase in fluorescence of this ligand (n = 1.0, KD = 0.07 microM). The enzyme-TNP-ATP complex binds Mg2+ and Mn2+ tightly (KD = 0.05 microM) as measured by an increase in fluorescence on titrating with these metals. The substrate dGTP competitively displaces TNP-ATP from the enzyme (KD = 5.7 microM) de-enhancing the fluorescence. The polymerase reaction is half-maximally inhibited by 0.8 microM TNP-ATP in the presence of dATP (10 microM) as substrate. A region of the amino acid sequence of Pol I (peptide I) consisting of residues 728-777 has been synthesized and found to contain significant secondary structure by CD both in water and 50% methanol/water. In water at 3 degrees C, peptide I binds the substrate analog TNP-ATP (KD = 0.03 microM) with a stoichiometry of 0.2. In 50% methanol at 3 degrees C, peptide I binds TNP-ATP with a higher stoichiometry than in water, consistent with a 1:1 complex, but biphasically (16% of the peptide, KD = 0.09 microM; 84% of the peptide, KD = 5.0 microM), and competitively binds the Pol I substrates dATP, TTP, and dGTP (KD = 230-570 microM). Evidence from size exclusion high performance liquid chromatography suggests that these two forms of the peptide are monomer and dimer, respectively. Significantly, the peptide I-TNP-ATP complex binds duplex DNA, tightly (KD = 0.1-0.5 microM) and stoichiometrically, and single stranded DNA more weakly. The peptide I-duplex DNA complex binds both TNP-ATP (KD = 0.5-1.5 microM) and Pol I substrates (KD = 350-2100 microM) stoichiometrically. In a control experiment, a second peptide, peptide II, based on residues 840-888 of the Pol I sequence, retains secondary structure, as detected by CD, but displays no binding of TNP-ATP. The ability of peptide I, which represents only 8% of the large fragment of Pol I, to bind both substrates and duplex DNA indicates that residues 728-777 constitute a major portion of the substrate binding site of this enzyme.
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Comparison with the enzyme</title><source>MEDLINE</source><source>EZB-FREE-00999 freely available EZB journals</source><source>Alma/SFX Local Collection</source><creator>MULLEN, G. P ; SHENBAGAMURTHI, P ; MILDVAN, A. S</creator><creatorcontrib>MULLEN, G. P ; SHENBAGAMURTHI, P ; MILDVAN, A. S</creatorcontrib><description>The fluorescent nucleotide 2',3'-trinitrophenyl-ATP (TNP-ATP) binds at the triphosphate substrate binding site of the large (Klenow) fragment of DNA polymerase I (Pol I) as detected by direct binding studies measuring the increase in fluorescence of this ligand (n = 1.0, KD = 0.07 microM). The enzyme-TNP-ATP complex binds Mg2+ and Mn2+ tightly (KD = 0.05 microM) as measured by an increase in fluorescence on titrating with these metals. The substrate dGTP competitively displaces TNP-ATP from the enzyme (KD = 5.7 microM) de-enhancing the fluorescence. The polymerase reaction is half-maximally inhibited by 0.8 microM TNP-ATP in the presence of dATP (10 microM) as substrate. A region of the amino acid sequence of Pol I (peptide I) consisting of residues 728-777 has been synthesized and found to contain significant secondary structure by CD both in water and 50% methanol/water. In water at 3 degrees C, peptide I binds the substrate analog TNP-ATP (KD = 0.03 microM) with a stoichiometry of 0.2. In 50% methanol at 3 degrees C, peptide I binds TNP-ATP with a higher stoichiometry than in water, consistent with a 1:1 complex, but biphasically (16% of the peptide, KD = 0.09 microM; 84% of the peptide, KD = 5.0 microM), and competitively binds the Pol I substrates dATP, TTP, and dGTP (KD = 230-570 microM). Evidence from size exclusion high performance liquid chromatography suggests that these two forms of the peptide are monomer and dimer, respectively. Significantly, the peptide I-TNP-ATP complex binds duplex DNA, tightly (KD = 0.1-0.5 microM) and stoichiometrically, and single stranded DNA more weakly. The peptide I-duplex DNA complex binds both TNP-ATP (KD = 0.5-1.5 microM) and Pol I substrates (KD = 350-2100 microM) stoichiometrically. In a control experiment, a second peptide, peptide II, based on residues 840-888 of the Pol I sequence, retains secondary structure, as detected by CD, but displays no binding of TNP-ATP. The ability of peptide I, which represents only 8% of the large fragment of Pol I, to bind both substrates and duplex DNA indicates that residues 728-777 constitute a major portion of the substrate binding site of this enzyme.</description><identifier>ISSN: 0021-9258</identifier><identifier>EISSN: 1083-351X</identifier><identifier>PMID: 2684960</identifier><identifier>CODEN: JBCHA3</identifier><language>eng</language><publisher>Bethesda, MD: American Society for Biochemistry and Molecular Biology</publisher><subject>Adenosine Triphosphate - metabolism ; Adenosine Triphosphate - pharmacology ; Amino Acid Sequence ; Analytical, structural and metabolic biochemistry ; Biological and medical sciences ; Circular Dichroism ; DNA - metabolism ; DNA Polymerase I - metabolism ; DNA-Binding Proteins - metabolism ; Enzymes and enzyme inhibitors ; Escherichia coli - enzymology ; Fluorescent Dyes ; Fundamental and applied biological sciences. Psychology ; Kinetics ; Mathematics ; Models, Structural ; Models, Theoretical ; Molecular Sequence Data ; Peptide Fragments - isolation &amp; purification ; Peptide Fragments - metabolism ; Protein Binding ; Protein Conformation ; Spectrometry, Fluorescence ; Transferases</subject><ispartof>The Journal of biological chemistry, 1989-11, Vol.264 (33), p.19637-19647</ispartof><rights>1991 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>315,781,785</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&amp;idt=19466903$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/2684960$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>MULLEN, G. P</creatorcontrib><creatorcontrib>SHENBAGAMURTHI, P</creatorcontrib><creatorcontrib>MILDVAN, A. S</creatorcontrib><title>Substrate and DNA binding to a 50-residue peptide fragment of DNA polymerase I. Comparison with the enzyme</title><title>The Journal of biological chemistry</title><addtitle>J Biol Chem</addtitle><description>The fluorescent nucleotide 2',3'-trinitrophenyl-ATP (TNP-ATP) binds at the triphosphate substrate binding site of the large (Klenow) fragment of DNA polymerase I (Pol I) as detected by direct binding studies measuring the increase in fluorescence of this ligand (n = 1.0, KD = 0.07 microM). The enzyme-TNP-ATP complex binds Mg2+ and Mn2+ tightly (KD = 0.05 microM) as measured by an increase in fluorescence on titrating with these metals. The substrate dGTP competitively displaces TNP-ATP from the enzyme (KD = 5.7 microM) de-enhancing the fluorescence. The polymerase reaction is half-maximally inhibited by 0.8 microM TNP-ATP in the presence of dATP (10 microM) as substrate. A region of the amino acid sequence of Pol I (peptide I) consisting of residues 728-777 has been synthesized and found to contain significant secondary structure by CD both in water and 50% methanol/water. In water at 3 degrees C, peptide I binds the substrate analog TNP-ATP (KD = 0.03 microM) with a stoichiometry of 0.2. In 50% methanol at 3 degrees C, peptide I binds TNP-ATP with a higher stoichiometry than in water, consistent with a 1:1 complex, but biphasically (16% of the peptide, KD = 0.09 microM; 84% of the peptide, KD = 5.0 microM), and competitively binds the Pol I substrates dATP, TTP, and dGTP (KD = 230-570 microM). Evidence from size exclusion high performance liquid chromatography suggests that these two forms of the peptide are monomer and dimer, respectively. Significantly, the peptide I-TNP-ATP complex binds duplex DNA, tightly (KD = 0.1-0.5 microM) and stoichiometrically, and single stranded DNA more weakly. The peptide I-duplex DNA complex binds both TNP-ATP (KD = 0.5-1.5 microM) and Pol I substrates (KD = 350-2100 microM) stoichiometrically. In a control experiment, a second peptide, peptide II, based on residues 840-888 of the Pol I sequence, retains secondary structure, as detected by CD, but displays no binding of TNP-ATP. 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Psychology</subject><subject>Kinetics</subject><subject>Mathematics</subject><subject>Models, Structural</subject><subject>Models, Theoretical</subject><subject>Molecular Sequence Data</subject><subject>Peptide Fragments - isolation &amp; purification</subject><subject>Peptide Fragments - metabolism</subject><subject>Protein Binding</subject><subject>Protein Conformation</subject><subject>Spectrometry, Fluorescence</subject><subject>Transferases</subject><issn>0021-9258</issn><issn>1083-351X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1989</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqN0LtOwzAUBuAIgUopPAKSB2ALsuNL7LEqV6mCAZDYIjs5bVzlhu2oKk-PRStmzhnO8H86w3-UTAmWNKWcfB4nU4wzkqqMy9PkzPsNjsMUmSSTTEimBJ4mm7fR-OB0AKS7Ct29zJGxXWW7NQo90ojj1IG31QhogCHYCtDK6XULXUD96tcPfbNrwWkP6PkWLfp20M76vkNbG2oUakDQfUdxnpysdOPh4nBnycfD_fviKV2-Pj4v5su0zpQIqSwp0yWJi1emZETlwCrCJCE8o6WQhgjIcqwlJ8JgapSWREhCNc8VMRmns-Rm_3dw_dcIPhSt9SU0je6gH32RK0oF_gcknPGcyizCywMcTQtVMTjbarcrDi3G_PqQa1_qJhbUldb_MaKYEArT6K72rrbremsdFMb2ZQ1t_MQKSqMUNKc_q5eFmg</recordid><startdate>19891125</startdate><enddate>19891125</enddate><creator>MULLEN, G. P</creator><creator>SHENBAGAMURTHI, P</creator><creator>MILDVAN, A. S</creator><general>American Society for Biochemistry and Molecular Biology</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>7QL</scope><scope>7TM</scope><scope>C1K</scope><scope>7X8</scope></search><sort><creationdate>19891125</creationdate><title>Substrate and DNA binding to a 50-residue peptide fragment of DNA polymerase I. Comparison with the enzyme</title><author>MULLEN, G. P ; SHENBAGAMURTHI, P ; MILDVAN, A. S</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-h296t-8c34ac1c1c0fbc4197e4d14811523c68b16e270a8516b03b9a816813a5791b253</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1989</creationdate><topic>Adenosine Triphosphate - metabolism</topic><topic>Adenosine Triphosphate - pharmacology</topic><topic>Amino Acid Sequence</topic><topic>Analytical, structural and metabolic biochemistry</topic><topic>Biological and medical sciences</topic><topic>Circular Dichroism</topic><topic>DNA - metabolism</topic><topic>DNA Polymerase I - metabolism</topic><topic>DNA-Binding Proteins - metabolism</topic><topic>Enzymes and enzyme inhibitors</topic><topic>Escherichia coli - enzymology</topic><topic>Fluorescent Dyes</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Kinetics</topic><topic>Mathematics</topic><topic>Models, Structural</topic><topic>Models, Theoretical</topic><topic>Molecular Sequence Data</topic><topic>Peptide Fragments - isolation &amp; purification</topic><topic>Peptide Fragments - metabolism</topic><topic>Protein Binding</topic><topic>Protein Conformation</topic><topic>Spectrometry, Fluorescence</topic><topic>Transferases</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>MULLEN, G. P</creatorcontrib><creatorcontrib>SHENBAGAMURTHI, P</creatorcontrib><creatorcontrib>MILDVAN, A. S</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Nucleic Acids Abstracts</collection><collection>Environmental Sciences and Pollution Management</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>MULLEN, G. P</au><au>SHENBAGAMURTHI, P</au><au>MILDVAN, A. S</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Substrate and DNA binding to a 50-residue peptide fragment of DNA polymerase I. Comparison with the enzyme</atitle><jtitle>The Journal of biological chemistry</jtitle><addtitle>J Biol Chem</addtitle><date>1989-11-25</date><risdate>1989</risdate><volume>264</volume><issue>33</issue><spage>19637</spage><epage>19647</epage><pages>19637-19647</pages><issn>0021-9258</issn><eissn>1083-351X</eissn><coden>JBCHA3</coden><abstract>The fluorescent nucleotide 2',3'-trinitrophenyl-ATP (TNP-ATP) binds at the triphosphate substrate binding site of the large (Klenow) fragment of DNA polymerase I (Pol I) as detected by direct binding studies measuring the increase in fluorescence of this ligand (n = 1.0, KD = 0.07 microM). The enzyme-TNP-ATP complex binds Mg2+ and Mn2+ tightly (KD = 0.05 microM) as measured by an increase in fluorescence on titrating with these metals. The substrate dGTP competitively displaces TNP-ATP from the enzyme (KD = 5.7 microM) de-enhancing the fluorescence. The polymerase reaction is half-maximally inhibited by 0.8 microM TNP-ATP in the presence of dATP (10 microM) as substrate. A region of the amino acid sequence of Pol I (peptide I) consisting of residues 728-777 has been synthesized and found to contain significant secondary structure by CD both in water and 50% methanol/water. In water at 3 degrees C, peptide I binds the substrate analog TNP-ATP (KD = 0.03 microM) with a stoichiometry of 0.2. In 50% methanol at 3 degrees C, peptide I binds TNP-ATP with a higher stoichiometry than in water, consistent with a 1:1 complex, but biphasically (16% of the peptide, KD = 0.09 microM; 84% of the peptide, KD = 5.0 microM), and competitively binds the Pol I substrates dATP, TTP, and dGTP (KD = 230-570 microM). Evidence from size exclusion high performance liquid chromatography suggests that these two forms of the peptide are monomer and dimer, respectively. Significantly, the peptide I-TNP-ATP complex binds duplex DNA, tightly (KD = 0.1-0.5 microM) and stoichiometrically, and single stranded DNA more weakly. The peptide I-duplex DNA complex binds both TNP-ATP (KD = 0.5-1.5 microM) and Pol I substrates (KD = 350-2100 microM) stoichiometrically. In a control experiment, a second peptide, peptide II, based on residues 840-888 of the Pol I sequence, retains secondary structure, as detected by CD, but displays no binding of TNP-ATP. The ability of peptide I, which represents only 8% of the large fragment of Pol I, to bind both substrates and duplex DNA indicates that residues 728-777 constitute a major portion of the substrate binding site of this enzyme.</abstract><cop>Bethesda, MD</cop><pub>American Society for Biochemistry and Molecular Biology</pub><pmid>2684960</pmid><tpages>11</tpages></addata></record>
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identifier ISSN: 0021-9258
ispartof The Journal of biological chemistry, 1989-11, Vol.264 (33), p.19637-19647
issn 0021-9258
1083-351X
language eng
recordid cdi_proquest_miscellaneous_79336025
source MEDLINE; EZB-FREE-00999 freely available EZB journals; Alma/SFX Local Collection
subjects Adenosine Triphosphate - metabolism
Adenosine Triphosphate - pharmacology
Amino Acid Sequence
Analytical, structural and metabolic biochemistry
Biological and medical sciences
Circular Dichroism
DNA - metabolism
DNA Polymerase I - metabolism
DNA-Binding Proteins - metabolism
Enzymes and enzyme inhibitors
Escherichia coli - enzymology
Fluorescent Dyes
Fundamental and applied biological sciences. Psychology
Kinetics
Mathematics
Models, Structural
Models, Theoretical
Molecular Sequence Data
Peptide Fragments - isolation & purification
Peptide Fragments - metabolism
Protein Binding
Protein Conformation
Spectrometry, Fluorescence
Transferases
title Substrate and DNA binding to a 50-residue peptide fragment of DNA polymerase I. Comparison with the enzyme
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