Lysine directed cross-linking of viral DNA-RNA:DNA hybrid substrate to the isolated RNase H domain of HIV-1 reverse transcriptase.
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 Mn(2+). The Mn(2+) titration of cross-linking paralleled the Mn(2+) 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.
Guaitiao
Juan P
JP
Programa de Virología, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Independencia 1027, Santiago, Chile.
Zúñiga
Roberto A
RA
Roth
Monica J
MJ
Leon
Oscar
O
D001667:Binding, Competitive; D002384:Catalysis; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004279:DNA, Viral / Q000138:chemical synthesis / Q000737:chemistry*; D003857:Deoxyuridine / Q000031:analogs & derivatives* / Q000737:chemistry; D054303:HIV Reverse Transcriptase / Q000737:chemistry*; D006868:Hydrolysis; D007700:Kinetics; D008239:Lysine / Q000737:chemistry*; D008345:Manganese / Q000737:chemistry; D009943:Organophosphorus Compounds / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D017434:Protein Structure, Tertiary; D012367:RNA, Viral / Q000138:chemical synthesis / Q000737:chemistry*; D016914:Ribonuclease H / Q000737:chemistry* / Q000302:isolation & purification; D013379:Substrate Specificity; D013388:Succinimides / Q000737:chemistry
C103219:5'-O-(4,4'-dimethoxytrityl)-5-(S-(2,4-dinitrophenyl)thio)-2'-deoxyuridine 3'-O-(2-cyanoethyl N,N'-diisopropylphosphoramidite); D003432:Cross-Linking Reagents; D004279:DNA, Viral; D009943:Organophosphorus Compounds; D010446:Peptide Fragments; D012367:RNA, Viral; D013388:Succinimides; C096464:disuccinimidyl glutarate; D008345:Manganese; D054303:HIV Reverse Transcriptase; D016914:Ribonuclease H; D008239:Lysine; D003857:Deoxyuridine