Characterization of the long-term-terminal repeat single-strand tail-binding site of Moloney-MuLV integrase by crosslinking

Vera, J; Valenzuela,B; Leon, O.; Roth M.J.

Keywords: sequence, chemistry, enzymology, acid, dna, animals, binding, protein, cell, long, structure, integration, single, sequences, metabolism, ultraviolet, virus, site, radiation, cross-linking, murine, leukemia, genetics, cysteine, sites, domain, interaction, article, oligonucleotide, base, reagent, cross, reagents, viral, oligonucleotides, integrase, strand, maleimide, controlled, animal, repeat, study, nucleotide, retrovirus, linking, amino, nonhuman, DNA,, stranded, Ethylmaleimide, n, terminal, uridine, moloney, oncovirus, Integrases

Abstract

Processing of viral DNA by retroviral integrase leaves a dinucleotide single-strand overhang in the unprocessed strand. Previous studies have stressed the importance of the 5′ single-stranded (ss) tail in the integration process. To characterize the ss-tail binding site on M-MuLV integrase, we carried out crosslinking studies utilizing a disintegration substrate that mimics the covalent intermediate formed during integration. This substrate carried reactive groups at the 5′ ss tail. A bromoacetyl derivative with a side chain of 6 Å was crosslinked to the mutant IN 106-404, which lacks the N-terminal domain, yielding a crosslinked complex of 50 kDa. Treatment of IN 106-404 with N-ethylmaleimide (NEM) prevented crosslinking, suggesting that Cys209 was involved in the reaction. The reactivity of Cys209 was confirmed by crosslinking of a more specific derivative carrying maleimide groups that spans 8Å approximately. In contrast, WT IN was not reactive, suggesting that the N-terminal domain modifies the reactivity of the Cys209 or the positioning of the crosslinker side chain. A similar oligonucleotide-carrying iodouridine at the 5′ ss tail reacted with both IN 106-404 and WT IN upon UV irradiation. This reaction was also prevented by NEM, suggesting that the ss-tail positions near a peptide region that includes Cys209.

Más información

Título según SCOPUS: Characterization of the long-term-terminal repeat single-strand tail-binding site of Moloney-MuLV integrase by crosslinking
Título de la Revista: BIOLOGICAL RESEARCH
Volumen: 41
Número: 1
Editorial: SOC BIOLGIA CHILE
Fecha de publicación: 2008
Página de inicio: 69
Página final: 80
Idioma: eng
URL: http://www.scopus.com/inward/record.url?eid=2-s2.0-51449089471&partnerID=q2rCbXpz
Notas: SCOPUS