Lysine Directed Cross-Linking of Viral DNA−RNA:DNA Hybrid Substrate to the Isolated RNase H Domain of HIV-1 Reverse Transcriptase†
Identifieur interne : 003088 ( Main/Exploration ); précédent : 003087; suivant : 003089Lysine Directed Cross-Linking of Viral DNA−RNA:DNA Hybrid Substrate to the Isolated RNase H Domain of HIV-1 Reverse Transcriptase†
Auteurs : Juan P. Guaitiao [États-Unis] ; Roberto A. Zú Iga [États-Unis] ; Monica J. Roth [États-Unis] ; Oscar Leon [États-Unis, Chili]Source :
- Biochemistry [ 0006-2960 ] ; 2004.
Abstract
An isolated ribonuclease H domain of HIV-1 reverse transcriptase is capable of specifically removing the tRNA primer within an oligonucleotide mimic. The determinants for substrate specificity are located in a region within the terminal octanucleotide of the acceptor stem of the tRNA. Recognition of the substrate by HIV-1 RNase H was analyzed by the introduction of a cross-linking reagent directed toward lysines on the thymine residue complementary to the scissile bond, facing the major groove of the DNA−RNA:DNA substrate. Cross-linking of the modified substrate to RNase H required the presence of Mn2+. The Mn2+ titration of cross-linking paralleled the Mn2+ requirement for activity. Modified substrate quenched with glycine prior to binding of substrate was efficiently cleaved, whereas the RNA within the cross-linked product was intact. Tryptic digestion of the isolated RNase H−nucleic acid covalent complex revealed a main cross-linked peptide whose N-terminal peptide sequence is VVTLTDTTNQ, indicating that the cross-linked lysine corresponds to Lys476. Cross-linking to K476 was confirmed by analysis of K476C RNase H. Mutation of K476C disrupted the chemical cross-linking while maintaining activity. On the basis of the size of the cross-linker arm, the results indicate that K476 is in closer proximity to the tRNA mimic substrate within the isolated RNase H domain than observed for the RNase H-resistant polypurine tract (PPT) substrate within the HIV-1 RT.
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DOI: 10.1021/bi035454y
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The determinants for substrate specificity are located in a region within the terminal octanucleotide of the acceptor stem of the tRNA. Recognition of the substrate by HIV-1 RNase H was analyzed by the introduction of a cross-linking reagent directed toward lysines on the thymine residue complementary to the scissile bond, facing the major groove of the DNA−RNA:DNA substrate. Cross-linking of the modified substrate to RNase H required the presence of Mn2+. The Mn2+ titration of cross-linking paralleled the Mn2+ requirement for activity. Modified substrate quenched with glycine prior to binding of substrate was efficiently cleaved, whereas the RNA within the cross-linked product was intact. Tryptic digestion of the isolated RNase H−nucleic acid covalent complex revealed a main cross-linked peptide whose N-terminal peptide sequence is VVTLTDTTNQ, indicating that the cross-linked lysine corresponds to Lys476. Cross-linking to K476 was confirmed by analysis of K476C RNase H. Mutation of K476C disrupted the chemical cross-linking while maintaining activity. On the basis of the size of the cross-linker arm, the results indicate that K476 is in closer proximity to the tRNA mimic substrate within the isolated RNase H domain than observed for the RNase H-resistant polypurine tract (PPT) substrate within the HIV-1 RT.</div></front></TEI><affiliations><list><country><li>Chili</li><li>États-Unis</li></country><region><li>New Jersey</li></region></list><tree><country name="États-Unis"><region name="New Jersey"><name sortKey="Guaitiao, Juan P" sort="Guaitiao, Juan P" uniqKey="Guaitiao J" first="Juan P." last="Guaitiao">Juan P. Guaitiao</name></region><name sortKey="Leon, Oscar" sort="Leon, Oscar" uniqKey="Leon O" first="Oscar" last="Leon">Oscar Leon</name><name sortKey="Roth, Monica J" sort="Roth, Monica J" uniqKey="Roth M" first="Monica J." last="Roth">Monica J. Roth</name><name sortKey="Zu Iga, Roberto A" sort="Zu Iga, Roberto A" uniqKey="Zu Iga R" first="Roberto A." last="Zú Iga">Roberto A. Zú Iga</name></country><country name="Chili"><noRegion><name sortKey="Leon, Oscar" sort="Leon, Oscar" uniqKey="Leon O" first="Oscar" last="Leon">Oscar Leon</name></noRegion></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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