Disruption of target DNA binding in Mu DNA transposition by alteration of position 99 in the Mu B protein.
J Mol Biol
; 275(2): 233-43, 1998 Jan 16.
Article
em En
| MEDLINE
| ID: mdl-9466906
ABSTRACT
Target DNA binding by the Mu B protein is an important step in phage Mu transposition; however, the region of Mu B involved in target binding and the mechanism of the interaction are unknown. Previous studies have demonstrated that modification of Mu B with the sulfhydryl-specific reagent N-ethylmaleimide can selectively inhibit target DNA binding. We now show that individual mutation of the three cysteines in Mu B to serine results in proteins which are active in intermolecular strand transfer, but demonstrate variable levels of N-ethylmaleimide resistance. The data indicate that cysteine 99 is the primary site of modification affecting target DNA binding, with a minor contribution resulting from the derivatization of cysteine 129. These findings are confirmed by the construction of Mu B mutants containing a bulky side-chain at the individual cysteine to mimic the N-ethylmaleimide modified protein. The C99Y protein shows a complete loss in target-dependent strand transfer activity under standard reaction conditions and C129Y displays partial activity. The effect of the tyrosine substitutions is specific for target interaction as both mutants show wild-type activity in their ability to stimulate the Mu transposase to perform donor cleavage and intramolecular strand transfer. Finally, a target dissociation assay has shown that the C99Y-DNA complex generated in the presence of ATP-gamma-S has a drastically reduced half-life as previously found for N-ethylmaleimide treated wild-type Mu B. Modification of cysteine 99 is proposed to block target DNA binding by causing steric interference near the DNA binding pocket.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Proteínas Virais
/
Transposases
/
Proteínas de Ligação a DNA
Tipo de estudo:
Prognostic_studies
Idioma:
En
Revista:
J Mol Biol
Ano de publicação:
1998
Tipo de documento:
Article
País de afiliação:
Canadá