Laser-induced protein-DNA cross-links via psoralen furanside monoadducts.
Identifieur interne : 004519 ( Main/Exploration ); précédent : 004518; suivant : 004520Laser-induced protein-DNA cross-links via psoralen furanside monoadducts.
Auteurs : S S Sastry [États-Unis] ; H P Spielmann ; Q S Hoang ; A M Phillips ; A. Sancar ; J E HearstSource :
- Biochemistry [ 0006-2960 ] ; 1993.
Descripteurs français
- KwdFr :
- ADN (métabolisme), Bactériophage T7 (enzymologie), Chromatographie en phase liquide à haute performance, Cinétique, DNA-directed RNA polymerases (métabolisme), Données de séquences moléculaires, Escherichia coli (métabolisme), Fragments peptidiques (isolement et purification), Lasers, Lumière, Oligodésoxyribonucléotides (métabolisme), Oligodésoxyribonucléotides (synthèse chimique), Protéines de liaison à l'ADN (métabolisme), Relation dose-effet des radiations, Réactifs réticulants (pharmacologie), Séquence nucléotidique, Trioxysalène (analogues et dérivés), Trioxysalène (métabolisme), Trioxysalène (pharmacologie), Trypsine.
- MESH :
- analogues et dérivés : Trioxysalène.
- enzymologie : Bactériophage T7.
- isolement et purification : Fragments peptidiques.
- métabolisme : ADN, DNA-directed RNA polymerases, Escherichia coli, Oligodésoxyribonucléotides, Protéines de liaison à l'ADN, Trioxysalène.
- pharmacologie : Réactifs réticulants, Trioxysalène.
- synthèse chimique : Oligodésoxyribonucléotides.
- Chromatographie en phase liquide à haute performance, Cinétique, Données de séquences moléculaires, Lasers, Lumière, Relation dose-effet des radiations, Séquence nucléotidique, Trypsine.
English descriptors
- KwdEn :
- Bacteriophage T7 (enzymology), Base Sequence, Chromatography, High Pressure Liquid, Cross-Linking Reagents (pharmacology), DNA (metabolism), DNA-Binding Proteins (metabolism), DNA-Directed RNA Polymerases (metabolism), Dose-Response Relationship, Radiation, Escherichia coli (metabolism), Kinetics, Lasers, Light, Molecular Sequence Data, Oligodeoxyribonucleotides (chemical synthesis), Oligodeoxyribonucleotides (metabolism), Peptide Fragments (isolation & purification), Trioxsalen (analogs & derivatives), Trioxsalen (metabolism), Trioxsalen (pharmacology), Trypsin.
- MESH :
- chemical , analogs & derivatives : Trioxsalen.
- chemical , chemical synthesis : Oligodeoxyribonucleotides.
- chemical , isolation & purification : Peptide Fragments.
- chemical , metabolism : DNA, DNA-Binding Proteins, DNA-Directed RNA Polymerases, Oligodeoxyribonucleotides, Trioxsalen.
- chemical , pharmacology : Cross-Linking Reagents, Trioxsalen.
- enzymology : Bacteriophage T7.
- metabolism : Escherichia coli.
- Base Sequence, Chromatography, High Pressure Liquid, Dose-Response Relationship, Radiation, Kinetics, Lasers, Light, Molecular Sequence Data, Trypsin.
Abstract
We have developed a technique for cross-linking DNA binding proteins to DNA using psoralen furanside monoadducts as photoaffinity probes and a continuous-wave argon ion laser (366 nm) as a light source. Several DNA binding proteins (T7 RNA polymerase, UvrB, single-stranded DNA binding protein of Escherichia coli, T4 gp32, and RecA of E. coli) are shown to cross-link to single-stranded psoralen monoadducted DNA oligos differing in length and sequence. Increasing fluences of laser light on a fixed ratio of DNA/protein resulted in an increase in the yield of cross-links. Titration experiments were carried out to measure the apparent cross-linking constant (KappXL) for T7 RNA polymerase or UvrB to a monoadducted 24 mer DNA. The estimated values for the apparent cross-linking constant were in the range of (2-3) x 10(-7) M for both T7 RNA polymerase and UvrB. The efficiency of cross-linking was investigated as a function of the length of adducted DNA and also as a fraction of the total noncovalent binding of proteins of psoralenated DNAs. The results showed that in the cases of T7 RNA polymerase and UvrB cross-linking was more efficient with short oligos (8 and 19 mers) as compared to longer oligos (50 mer). A tryptic peptide of T7 RNA polymerase that was conjugated to a psoralen furanside monoadducted 12 mer DNA was isolated by high-performance liquid chromatography. Mass spectrometry and amino acid composition of this peptide revealed that it originated from a region between residues 558 and 608 of the primary structure of T7 RNA polymerase. Two other peptides cross-linked to oligos were also purified. Repeated attempts to perform Edman sequencing of the peptide-DNA conjugates failed. Overall evidence indicates that photo-cross-linking of furanside monoadducts occurred at multiple sites on the proteins. We have shown that T7 RNA polymerase molecules in a ternary complex arrested at the furanside monoadduct can be cross-linked to the DNA templates with laser light. Evidence suggests that the arrested polymerase molecules existed in multiple conformations on the DNA template. This method of transcriptional cross-linking offers a new method for preparing highly stable elongation complexes for further studies.
DOI: 10.1021/bi00072a006
PubMed: 8504073
Affiliations:
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Le document en format XML
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<profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Bacteriophage T7 (enzymology)</term>
<term>Base Sequence</term>
<term>Chromatography, High Pressure Liquid</term>
<term>Cross-Linking Reagents (pharmacology)</term>
<term>DNA (metabolism)</term>
<term>DNA-Binding Proteins (metabolism)</term>
<term>DNA-Directed RNA Polymerases (metabolism)</term>
<term>Dose-Response Relationship, Radiation</term>
<term>Escherichia coli (metabolism)</term>
<term>Kinetics</term>
<term>Lasers</term>
<term>Light</term>
<term>Molecular Sequence Data</term>
<term>Oligodeoxyribonucleotides (chemical synthesis)</term>
<term>Oligodeoxyribonucleotides (metabolism)</term>
<term>Peptide Fragments (isolation & purification)</term>
<term>Trioxsalen (analogs & derivatives)</term>
<term>Trioxsalen (metabolism)</term>
<term>Trioxsalen (pharmacology)</term>
<term>Trypsin</term>
</keywords>
<keywords scheme="KwdFr" xml:lang="fr"><term>ADN (métabolisme)</term>
<term>Bactériophage T7 (enzymologie)</term>
<term>Chromatographie en phase liquide à haute performance</term>
<term>Cinétique</term>
<term>DNA-directed RNA polymerases (métabolisme)</term>
<term>Données de séquences moléculaires</term>
<term>Escherichia coli (métabolisme)</term>
<term>Fragments peptidiques (isolement et purification)</term>
<term>Lasers</term>
<term>Lumière</term>
<term>Oligodésoxyribonucléotides (métabolisme)</term>
<term>Oligodésoxyribonucléotides (synthèse chimique)</term>
<term>Protéines de liaison à l'ADN (métabolisme)</term>
<term>Relation dose-effet des radiations</term>
<term>Réactifs réticulants (pharmacologie)</term>
<term>Séquence nucléotidique</term>
<term>Trioxysalène (analogues et dérivés)</term>
<term>Trioxysalène (métabolisme)</term>
<term>Trioxysalène (pharmacologie)</term>
<term>Trypsine</term>
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<keywords scheme="MESH" type="chemical" qualifier="chemical synthesis" xml:lang="en"><term>Oligodeoxyribonucleotides</term>
</keywords>
<keywords scheme="MESH" type="chemical" qualifier="isolation & purification" xml:lang="en"><term>Peptide Fragments</term>
</keywords>
<keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>DNA</term>
<term>DNA-Binding Proteins</term>
<term>DNA-Directed RNA Polymerases</term>
<term>Oligodeoxyribonucleotides</term>
<term>Trioxsalen</term>
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<keywords scheme="MESH" type="chemical" qualifier="pharmacology" xml:lang="en"><term>Cross-Linking Reagents</term>
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<keywords scheme="MESH" qualifier="enzymologie" xml:lang="fr"><term>Bactériophage T7</term>
</keywords>
<keywords scheme="MESH" qualifier="enzymology" xml:lang="en"><term>Bacteriophage T7</term>
</keywords>
<keywords scheme="MESH" qualifier="isolement et purification" xml:lang="fr"><term>Fragments peptidiques</term>
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<keywords scheme="MESH" qualifier="metabolism" xml:lang="en"><term>Escherichia coli</term>
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<keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>ADN</term>
<term>DNA-directed RNA polymerases</term>
<term>Escherichia coli</term>
<term>Oligodésoxyribonucléotides</term>
<term>Protéines de liaison à l'ADN</term>
<term>Trioxysalène</term>
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<keywords scheme="MESH" qualifier="pharmacologie" xml:lang="fr"><term>Réactifs réticulants</term>
<term>Trioxysalène</term>
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<keywords scheme="MESH" qualifier="synthèse chimique" xml:lang="fr"><term>Oligodésoxyribonucléotides</term>
</keywords>
<keywords scheme="MESH" xml:lang="en"><term>Base Sequence</term>
<term>Chromatography, High Pressure Liquid</term>
<term>Dose-Response Relationship, Radiation</term>
<term>Kinetics</term>
<term>Lasers</term>
<term>Light</term>
<term>Molecular Sequence Data</term>
<term>Trypsin</term>
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<term>Cinétique</term>
<term>Données de séquences moléculaires</term>
<term>Lasers</term>
<term>Lumière</term>
<term>Relation dose-effet des radiations</term>
<term>Séquence nucléotidique</term>
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<front><div type="abstract" xml:lang="en">We have developed a technique for cross-linking DNA binding proteins to DNA using psoralen furanside monoadducts as photoaffinity probes and a continuous-wave argon ion laser (366 nm) as a light source. Several DNA binding proteins (T7 RNA polymerase, UvrB, single-stranded DNA binding protein of Escherichia coli, T4 gp32, and RecA of E. coli) are shown to cross-link to single-stranded psoralen monoadducted DNA oligos differing in length and sequence. Increasing fluences of laser light on a fixed ratio of DNA/protein resulted in an increase in the yield of cross-links. Titration experiments were carried out to measure the apparent cross-linking constant (KappXL) for T7 RNA polymerase or UvrB to a monoadducted 24 mer DNA. The estimated values for the apparent cross-linking constant were in the range of (2-3) x 10(-7) M for both T7 RNA polymerase and UvrB. The efficiency of cross-linking was investigated as a function of the length of adducted DNA and also as a fraction of the total noncovalent binding of proteins of psoralenated DNAs. The results showed that in the cases of T7 RNA polymerase and UvrB cross-linking was more efficient with short oligos (8 and 19 mers) as compared to longer oligos (50 mer). A tryptic peptide of T7 RNA polymerase that was conjugated to a psoralen furanside monoadducted 12 mer DNA was isolated by high-performance liquid chromatography. Mass spectrometry and amino acid composition of this peptide revealed that it originated from a region between residues 558 and 608 of the primary structure of T7 RNA polymerase. Two other peptides cross-linked to oligos were also purified. Repeated attempts to perform Edman sequencing of the peptide-DNA conjugates failed. Overall evidence indicates that photo-cross-linking of furanside monoadducts occurred at multiple sites on the proteins. We have shown that T7 RNA polymerase molecules in a ternary complex arrested at the furanside monoadduct can be cross-linked to the DNA templates with laser light. Evidence suggests that the arrested polymerase molecules existed in multiple conformations on the DNA template. This method of transcriptional cross-linking offers a new method for preparing highly stable elongation complexes for further studies.</div>
</front>
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<affiliations><list><country><li>États-Unis</li>
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<region><li>Californie</li>
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<settlement><li>Berkeley (Californie)</li>
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<tree><noCountry><name sortKey="Hearst, J E" sort="Hearst, J E" uniqKey="Hearst J" first="J E" last="Hearst">J E Hearst</name>
<name sortKey="Hoang, Q S" sort="Hoang, Q S" uniqKey="Hoang Q" first="Q S" last="Hoang">Q S Hoang</name>
<name sortKey="Phillips, A M" sort="Phillips, A M" uniqKey="Phillips A" first="A M" last="Phillips">A M Phillips</name>
<name sortKey="Sancar, A" sort="Sancar, A" uniqKey="Sancar A" first="A" last="Sancar">A. Sancar</name>
<name sortKey="Spielmann, H P" sort="Spielmann, H P" uniqKey="Spielmann H" first="H P" last="Spielmann">H P Spielmann</name>
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<country name="États-Unis"><region name="Californie"><name sortKey="Sastry, S S" sort="Sastry, S S" uniqKey="Sastry S" first="S S" last="Sastry">S S Sastry</name>
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