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

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...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	VERA,JORGE, VALENZUELA,BEATRIZ, ROTH,MÓNICA J, LEÓN,ÓSCAR
Lenguaje:	English
Publicado:	Sociedad de Biología de Chile 2008
Materias:	integrase retrovirus crosslinking
Acceso en línea:	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0716-97602008000100009
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:scielo:S0716-97602008000100009
record_format	dspace
spelling	oai:scielo:S0716-976020080001000092008-08-21Characterization of the long-terminal repeat single-strand tail-binding site of Moloney-MuLV integrase by crosslinkingVERA,JORGEVALENZUELA,BEATRIZROTH,MÓNICA JLEÓN,ÓSCAR integrase retrovirus crosslinking 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 Cys209info:eu-repo/semantics/openAccessSociedad de Biología de ChileBiological Research v.41 n.1 20082008-01-01text/htmlhttp://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0716-97602008000100009en10.4067/S0716-97602008000100009
institution	Scielo Chile
collection	Scielo Chile
language	English
topic	integrase retrovirus crosslinking
spellingShingle	integrase retrovirus crosslinking VERA,JORGE VALENZUELA,BEATRIZ ROTH,MÓNICA J LEÓN,ÓSCAR Characterization of the long-terminal repeat single-strand tail-binding site of Moloney-MuLV integrase by crosslinking
description	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
author	VERA,JORGE VALENZUELA,BEATRIZ ROTH,MÓNICA J LEÓN,ÓSCAR
author_facet	VERA,JORGE VALENZUELA,BEATRIZ ROTH,MÓNICA J LEÓN,ÓSCAR
author_sort	VERA,JORGE
title	Characterization of the long-terminal repeat single-strand tail-binding site of Moloney-MuLV integrase by crosslinking
title_short	Characterization of the long-terminal repeat single-strand tail-binding site of Moloney-MuLV integrase by crosslinking
title_full	Characterization of the long-terminal repeat single-strand tail-binding site of Moloney-MuLV integrase by crosslinking
title_fullStr	Characterization of the long-terminal repeat single-strand tail-binding site of Moloney-MuLV integrase by crosslinking
title_full_unstemmed	Characterization of the long-terminal repeat single-strand tail-binding site of Moloney-MuLV integrase by crosslinking
title_sort	characterization of the long-terminal repeat single-strand tail-binding site of moloney-mulv integrase by crosslinking
publisher	Sociedad de Biología de Chile
publishDate	2008
url	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0716-97602008000100009
work_keys_str_mv	AT verajorge characterizationofthelongterminalrepeatsinglestrandtailbindingsiteofmoloneymulvintegrasebycrosslinking AT valenzuelabeatriz characterizationofthelongterminalrepeatsinglestrandtailbindingsiteofmoloneymulvintegrasebycrosslinking AT rothmonicaj characterizationofthelongterminalrepeatsinglestrandtailbindingsiteofmoloneymulvintegrasebycrosslinking AT leonoscar characterizationofthelongterminalrepeatsinglestrandtailbindingsiteofmoloneymulvintegrasebycrosslinking
_version_	1718441429319024640

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

Ejemplares similares