Herpes simplex virus type 1 helicase-primase: DNA binding and consequent protein oligomerization and primase activation.
J Virol
; 85(2): 968-78, 2011 Jan.
Article
em En
| MEDLINE
| ID: mdl-21068246
ABSTRACT
The heterotrimeric helicase-primase complex of herpes simplex virus type I (HSV-1), consisting of UL5, UL8, and UL52, possesses 5' to 3' helicase, single-stranded DNA (ssDNA)-dependent ATPase, primase, and DNA binding activities. In this study we confirm that the UL5-UL8-UL52 complex has higher affinity for forked DNA than for ssDNA and fails to bind to fully annealed double-stranded DNA substrates. In addition, we show that a single-stranded overhang of greater than 6 nucleotides is required for efficient enzyme loading and unwinding. Electrophoretic mobility shift assays and surface plasmon resonance analysis provide additional quantitative information about how the UL5-UL8-UL52 complex associates with the replication fork. Although it has previously been reported that in the absence of DNA and nucleoside triphosphates the UL5-UL8-UL52 complex exists as a monomer in solution, we now present evidence that in the presence of forked DNA and AMP-PNP, higher-order complexes can form. Electrophoretic mobility shift assays reveal two discrete complexes with different mobilities only when helicase-primase is bound to DNA containing a single-stranded region, and surface plasmon resonance analysis confirms larger amounts of the complex bound to forked substrates than to single-overhang substrates. Furthermore, we show that primase activity exhibits a cooperative dependence on protein concentration while ATPase and helicase activities do not. Taken together, these data suggest that the primase activity of the helicase-primase requires formation of a dimer or higher-order structure while ATPase activity does not. Importantly, this provides a simple mechanism for generating a two-polymerase replisome at the replication fork.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Proteínas Virais
/
DNA
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DNA de Cadeia Simples
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Herpesvirus Humano 1
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DNA Helicases
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DNA Primase
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Multimerização Proteica
Limite:
Animals
Idioma:
En
Revista:
J Virol
Ano de publicação:
2011
Tipo de documento:
Article
País de afiliação:
Estados Unidos