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low molecular weight DNA polymerase from wheat embryos
The study of plant DNA polymerases lags far behind that concerning their animal or yeast counterpart. In this work we describe the first extensive purification to apparent homogeneity, as well as a detailed biochemical and immunological characterization, of a low molecular weight DNA polymerase (DNA...
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Published in: | Plant molecular biology 1990-09, Vol.15 (3), p.383-397 |
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description | The study of plant DNA polymerases lags far behind that concerning their animal or yeast counterpart. In this work we describe the first extensive purification to apparent homogeneity, as well as a detailed biochemical and immunological characterization, of a low molecular weight DNA polymerase (DNA polymerase CI) purified from wheat embryos. The monomeric enzyme is a basic protein having a molecular weight of 52 kDa. Polyclonal antibodies raised in rabbits against DNA polymerase CI did not inhibit animal DNA polymerases alpha and beta or wheat DNA polymerase A, whereas wheat DNA polymerases CII and B were much less affected than the CI enzyme. Several properties of enzyme CI were studied. Some known inhibitors of DNA polymerase activity including aphidicolin, phosphonoacetic acid and heparin, did not affect DNA polymerase CI while the activity of this enzyme was strongly inhibited by ddTTP and N-ethylmaleimide. The polyamine spermine decreased markedly the enzyme activity, while spermidine produced a strong stimulation at the same concentrations that spermine inhibited the enzyme. The best template for this enzyme is poly dA-oligo dT, although polymerase CI can recognize significantly some synthetic polyribonucleotide templates (poly rC-oligo dG, poly rA-oligo dT) but only at a given protein/template primer ratio. The enzyme is blocked at the amino terminus, thus preventing the automatic sequencing of the protein. The amino acid analysis showed a striking similarity with the animal low molecular weight DNA polymerase P. The latter observation, as well as the effect of inhibitors (except N-ethylmaleimide which does not inhibit the animal polymerase) indicate that the DNA polymerase described in this work is a plant DNA polymerase very similar to the low molecular weight animal DNA polymerase beta, an enzyme believed to be involved in nuclear DNA repair. |
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In this work we describe the first extensive purification to apparent homogeneity, as well as a detailed biochemical and immunological characterization, of a low molecular weight DNA polymerase (DNA polymerase CI) purified from wheat embryos. The monomeric enzyme is a basic protein having a molecular weight of 52 kDa. Polyclonal antibodies raised in rabbits against DNA polymerase CI did not inhibit animal DNA polymerases alpha and beta or wheat DNA polymerase A, whereas wheat DNA polymerases CII and B were much less affected than the CI enzyme. Several properties of enzyme CI were studied. Some known inhibitors of DNA polymerase activity including aphidicolin, phosphonoacetic acid and heparin, did not affect DNA polymerase CI while the activity of this enzyme was strongly inhibited by ddTTP and N-ethylmaleimide. The polyamine spermine decreased markedly the enzyme activity, while spermidine produced a strong stimulation at the same concentrations that spermine inhibited the enzyme. The best template for this enzyme is poly dA-oligo dT, although polymerase CI can recognize significantly some synthetic polyribonucleotide templates (poly rC-oligo dG, poly rA-oligo dT) but only at a given protein/template primer ratio. The enzyme is blocked at the amino terminus, thus preventing the automatic sequencing of the protein. The amino acid analysis showed a striking similarity with the animal low molecular weight DNA polymerase P. The latter observation, as well as the effect of inhibitors (except N-ethylmaleimide which does not inhibit the animal polymerase) indicate that the DNA polymerase described in this work is a plant DNA polymerase very similar to the low molecular weight animal DNA polymerase beta, an enzyme believed to be involved in nuclear DNA repair.</description><identifier>ISSN: 0167-4412</identifier><identifier>EISSN: 1573-5028</identifier><identifier>DOI: 10.1007/BF00019156</identifier><identifier>PMID: 2103459</identifier><identifier>CODEN: PMBIDB</identifier><language>eng</language><publisher>Dordrecht: Springer</publisher><subject>amino acid composition ; amino acids ; Amino Acids - analysis ; Analytical, structural and metabolic biochemistry ; Animals ; Biological and medical sciences ; DNA Polymerase I - analysis ; DNA replication ; DNA-directed DNA polymerase ; DNA-Directed DNA Polymerase - immunology ; DNA-Directed DNA Polymerase - isolation & purification ; DNA-Directed DNA Polymerase - metabolism ; embryo (plant) ; enzyme activity ; enzyme inhibitors ; Enzymes and enzyme inhibitors ; Fundamental and applied biological sciences. Psychology ; Molecular Weight ; Nucleic Acid Synthesis Inhibitors ; physicochemical properties ; Plant Proteins - antagonists & inhibitors ; Plant Proteins - immunology ; Plant Proteins - isolation & purification ; Plant Proteins - metabolism ; purification ; Rats ; Species Specificity ; Substrate Specificity ; Templates, Genetic ; Transferases ; Triticum - embryology ; Triticum - enzymology ; Triticum - immunology ; Triticum aestivum</subject><ispartof>Plant molecular biology, 1990-09, Vol.15 (3), p.383-397</ispartof><rights>1991 INIST-CNRS</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c367t-a6a00a91cd4c47372bd7edf6be4fb7b973f7c9ba553f2f7da6605db1a81289103</citedby><cites>FETCH-LOGICAL-c367t-a6a00a91cd4c47372bd7edf6be4fb7b973f7c9ba553f2f7da6605db1a81289103</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27924,27925</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=19269110$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/2103459$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Castroviejo, M</creatorcontrib><creatorcontrib>Gatius, M.T</creatorcontrib><creatorcontrib>Litvak, S</creatorcontrib><title>low molecular weight DNA polymerase from wheat embryos</title><title>Plant molecular biology</title><addtitle>Plant Mol Biol</addtitle><description>The study of plant DNA polymerases lags far behind that concerning their animal or yeast counterpart. In this work we describe the first extensive purification to apparent homogeneity, as well as a detailed biochemical and immunological characterization, of a low molecular weight DNA polymerase (DNA polymerase CI) purified from wheat embryos. The monomeric enzyme is a basic protein having a molecular weight of 52 kDa. Polyclonal antibodies raised in rabbits against DNA polymerase CI did not inhibit animal DNA polymerases alpha and beta or wheat DNA polymerase A, whereas wheat DNA polymerases CII and B were much less affected than the CI enzyme. Several properties of enzyme CI were studied. Some known inhibitors of DNA polymerase activity including aphidicolin, phosphonoacetic acid and heparin, did not affect DNA polymerase CI while the activity of this enzyme was strongly inhibited by ddTTP and N-ethylmaleimide. The polyamine spermine decreased markedly the enzyme activity, while spermidine produced a strong stimulation at the same concentrations that spermine inhibited the enzyme. The best template for this enzyme is poly dA-oligo dT, although polymerase CI can recognize significantly some synthetic polyribonucleotide templates (poly rC-oligo dG, poly rA-oligo dT) but only at a given protein/template primer ratio. The enzyme is blocked at the amino terminus, thus preventing the automatic sequencing of the protein. The amino acid analysis showed a striking similarity with the animal low molecular weight DNA polymerase P. The latter observation, as well as the effect of inhibitors (except N-ethylmaleimide which does not inhibit the animal polymerase) indicate that the DNA polymerase described in this work is a plant DNA polymerase very similar to the low molecular weight animal DNA polymerase beta, an enzyme believed to be involved in nuclear DNA repair.</description><subject>amino acid composition</subject><subject>amino acids</subject><subject>Amino Acids - analysis</subject><subject>Analytical, structural and metabolic biochemistry</subject><subject>Animals</subject><subject>Biological and medical sciences</subject><subject>DNA Polymerase I - analysis</subject><subject>DNA replication</subject><subject>DNA-directed DNA polymerase</subject><subject>DNA-Directed DNA Polymerase - immunology</subject><subject>DNA-Directed DNA Polymerase - isolation & purification</subject><subject>DNA-Directed DNA Polymerase - metabolism</subject><subject>embryo (plant)</subject><subject>enzyme activity</subject><subject>enzyme inhibitors</subject><subject>Enzymes and enzyme inhibitors</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Molecular Weight</subject><subject>Nucleic Acid Synthesis Inhibitors</subject><subject>physicochemical properties</subject><subject>Plant Proteins - antagonists & inhibitors</subject><subject>Plant Proteins - immunology</subject><subject>Plant Proteins - isolation & purification</subject><subject>Plant Proteins - metabolism</subject><subject>purification</subject><subject>Rats</subject><subject>Species Specificity</subject><subject>Substrate Specificity</subject><subject>Templates, Genetic</subject><subject>Transferases</subject><subject>Triticum - embryology</subject><subject>Triticum - enzymology</subject><subject>Triticum - immunology</subject><subject>Triticum aestivum</subject><issn>0167-4412</issn><issn>1573-5028</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1990</creationdate><recordtype>article</recordtype><recordid>eNqF0DFPwzAQBWALgaAUFnZEFhiQAnd2YsdjKRSQKhiAObo4Ni1KcLFbVf33BFrRkemG-_R09xg7QbhCAHV9MwIA1JjLHdbDXIk0B17ssh6gVGmWIT9ghzF-dAhAyH22zxFElusek41fJq1vrFk0FJKlnb5P5snt0yCZ-WbV2kDRJi74NllOLM0T21Zh5eMR23PURHu8mX32Nrp7HT6k4-f7x-FgnBoh1TwlSQCk0dSZyZRQvKqVrZ2sbOYqVWklnDK6ojwXjjtVk5SQ1xVSgbzQ3Y19drHOnQX_tbBxXrbTaGzT0Kf1i1gWIApZaP4vxLwQWKDu4OUamuBjDNaVszBtKaxKhPKnzXLbZodPN6mLqrX1H93U1-3PN3uKhhoX6NNM4zZRc6nx942ztXPkS3oPnXl74YACUIlf8Q2ZaYOe</recordid><startdate>19900901</startdate><enddate>19900901</enddate><creator>Castroviejo, M</creator><creator>Gatius, M.T</creator><creator>Litvak, S</creator><general>Springer</general><scope>FBQ</scope><scope>IQODW</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>19900901</creationdate><title>low molecular weight DNA polymerase from wheat embryos</title><author>Castroviejo, M ; Gatius, M.T ; Litvak, S</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c367t-a6a00a91cd4c47372bd7edf6be4fb7b973f7c9ba553f2f7da6605db1a81289103</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1990</creationdate><topic>amino acid composition</topic><topic>amino acids</topic><topic>Amino Acids - analysis</topic><topic>Analytical, structural and metabolic biochemistry</topic><topic>Animals</topic><topic>Biological and medical sciences</topic><topic>DNA Polymerase I - analysis</topic><topic>DNA replication</topic><topic>DNA-directed DNA polymerase</topic><topic>DNA-Directed DNA Polymerase - immunology</topic><topic>DNA-Directed DNA Polymerase - isolation & purification</topic><topic>DNA-Directed DNA Polymerase - metabolism</topic><topic>embryo (plant)</topic><topic>enzyme activity</topic><topic>enzyme inhibitors</topic><topic>Enzymes and enzyme inhibitors</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Molecular Weight</topic><topic>Nucleic Acid Synthesis Inhibitors</topic><topic>physicochemical properties</topic><topic>Plant Proteins - antagonists & inhibitors</topic><topic>Plant Proteins - immunology</topic><topic>Plant Proteins - isolation & purification</topic><topic>Plant Proteins - metabolism</topic><topic>purification</topic><topic>Rats</topic><topic>Species Specificity</topic><topic>Substrate Specificity</topic><topic>Templates, Genetic</topic><topic>Transferases</topic><topic>Triticum - embryology</topic><topic>Triticum - enzymology</topic><topic>Triticum - immunology</topic><topic>Triticum aestivum</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Castroviejo, M</creatorcontrib><creatorcontrib>Gatius, M.T</creatorcontrib><creatorcontrib>Litvak, S</creatorcontrib><collection>AGRIS</collection><collection>Pascal-Francis</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>Plant molecular biology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Castroviejo, M</au><au>Gatius, M.T</au><au>Litvak, S</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>low molecular weight DNA polymerase from wheat embryos</atitle><jtitle>Plant molecular biology</jtitle><addtitle>Plant Mol Biol</addtitle><date>1990-09-01</date><risdate>1990</risdate><volume>15</volume><issue>3</issue><spage>383</spage><epage>397</epage><pages>383-397</pages><issn>0167-4412</issn><eissn>1573-5028</eissn><coden>PMBIDB</coden><abstract>The study of plant DNA polymerases lags far behind that concerning their animal or yeast counterpart. In this work we describe the first extensive purification to apparent homogeneity, as well as a detailed biochemical and immunological characterization, of a low molecular weight DNA polymerase (DNA polymerase CI) purified from wheat embryos. The monomeric enzyme is a basic protein having a molecular weight of 52 kDa. Polyclonal antibodies raised in rabbits against DNA polymerase CI did not inhibit animal DNA polymerases alpha and beta or wheat DNA polymerase A, whereas wheat DNA polymerases CII and B were much less affected than the CI enzyme. Several properties of enzyme CI were studied. Some known inhibitors of DNA polymerase activity including aphidicolin, phosphonoacetic acid and heparin, did not affect DNA polymerase CI while the activity of this enzyme was strongly inhibited by ddTTP and N-ethylmaleimide. The polyamine spermine decreased markedly the enzyme activity, while spermidine produced a strong stimulation at the same concentrations that spermine inhibited the enzyme. The best template for this enzyme is poly dA-oligo dT, although polymerase CI can recognize significantly some synthetic polyribonucleotide templates (poly rC-oligo dG, poly rA-oligo dT) but only at a given protein/template primer ratio. The enzyme is blocked at the amino terminus, thus preventing the automatic sequencing of the protein. The amino acid analysis showed a striking similarity with the animal low molecular weight DNA polymerase P. The latter observation, as well as the effect of inhibitors (except N-ethylmaleimide which does not inhibit the animal polymerase) indicate that the DNA polymerase described in this work is a plant DNA polymerase very similar to the low molecular weight animal DNA polymerase beta, an enzyme believed to be involved in nuclear DNA repair.</abstract><cop>Dordrecht</cop><pub>Springer</pub><pmid>2103459</pmid><doi>10.1007/BF00019156</doi><tpages>15</tpages><oa>free_for_read</oa></addata></record> |
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subjects | amino acid composition amino acids Amino Acids - analysis Analytical, structural and metabolic biochemistry Animals Biological and medical sciences DNA Polymerase I - analysis DNA replication DNA-directed DNA polymerase DNA-Directed DNA Polymerase - immunology DNA-Directed DNA Polymerase - isolation & purification DNA-Directed DNA Polymerase - metabolism embryo (plant) enzyme activity enzyme inhibitors Enzymes and enzyme inhibitors Fundamental and applied biological sciences. Psychology Molecular Weight Nucleic Acid Synthesis Inhibitors physicochemical properties Plant Proteins - antagonists & inhibitors Plant Proteins - immunology Plant Proteins - isolation & purification Plant Proteins - metabolism purification Rats Species Specificity Substrate Specificity Templates, Genetic Transferases Triticum - embryology Triticum - enzymology Triticum - immunology Triticum aestivum |
title | low molecular weight DNA polymerase from wheat embryos |
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