Characterization of the long-terminal repeat single-strand tail-binding site of Moloney-MuLV integrase by crosslinking
Biol. Res
; 41(1): 69-80, 2008. ilus, graf
Article
em En
| LILACS
| ID: lil-490634
Biblioteca responsável:
BR1.1
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 A 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 8A 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.
Palavras-chave
Texto completo:
1
Bases de dados:
LILACS
Assunto principal:
DNA Viral
/
Integração Viral
/
Integrases
/
Sequências Repetidas Terminais
/
Vírus da Leucemia Murina de Moloney
Limite:
Animals
Idioma:
En
Revista:
Biol. Res
Assunto da revista:
BIOLOGIA
Ano de publicação:
2008
Tipo de documento:
Article
/
Project document
País de afiliação:
Chile
/
Estados Unidos