RESUMEN
Previous studies have shown that the sequence-independent adenovirus DNA binding protein (DBP) increases transcription from several promoters, notably from the adenovirus major late promoter (MLP) and the adeno-associated virus P5 promoter, both of which contain a USF/MLTF binding site. In order to study this mechanism, we have investigated the effects of DBP on the binding of USF/MLTF to MLP and on transcription from MLP by a reconstituted in vitro system. As shown by gel retardation and DNase I footprinting, upon saturation of DNA, DBP enhances the binding affinity of USF43 to the promoter three- to fourfold without changing the footprint pattern. In contrast, the binding of the TATA box binding protein to the promoter is not influenced by DBP. No protein-protein interactions between DBP and USF43 could be observed in the absence of DNA, suggesting that enhanced binding is caused by a change in DNA structure induced by the DBP-DNA complex. Employing a transcription system reconstituted with purified general transcription factors, we show that USF43 enhances basal transcription and that USF43-dependent transcription is further increased by DBP, while DBP alone does not have an effect on basal transcription. Our results suggest that transcription enhancement by DBP is based on a specific increase in the binding of a transcription factor to a promoter through subtle changes in DNA structure, similar to the mechanism by which DBP stimulates the initiation of DNA replication.
Asunto(s)
Adenoviridae/metabolismo , Proteínas de Unión al ADN/metabolismo , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Transcripción Genética , Adenoviridae/genética , Sitios de Unión , Cromatografía de Afinidad , Clonación Molecular , Proteínas de Unión al ADN/aislamiento & purificación , Desoxirribonucleasa I , Dependovirus/genética , Dependovirus/metabolismo , Cinética , Factores de Transcripción NFI , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , TATA Box , Proteína de Unión a TATA-Box , Factores de Transcripción/aislamiento & purificación , Factores Estimuladores hacia 5'RESUMEN
The adenovirus DNA-binding protein (DBP) is a multifunctional protein that is essential for viral DNA replication. DBP binds both single-stranded and double-stranded DNA as well as RNA in a sequence-independent manner. Previous studies showed that DBP does not promote melting of duplex poly(dA-dT) in contrast to prokaryotic single-strand-binding proteins. However, here we show that DBP can displace oligonucleotides annealed to single-stranded M13 DNA. Depending upon the DBP concentration, strands of at least 200 nucleotides can be unwound. Although unwinding of short (17-bp), fully duplex DNA is facilitated by DBP, unwinding of larger (28-bp) duplexes is only possible if single-stranded protruding ends are present. These protruding ends must be at least 4 nucleotides long for optimal unwinding, and both 5' and 3' single-stranded overhangs suffice. DBP-promoted strand displacement is sensitive to MgCl2 and NaCl and not dependent upon ATP. Our results suggest that DBP, through formation of a protein chain on the displaced strand, may destabilize duplex DNA ahead of the replication fork, thereby assisting in strand displacement during replication.
Asunto(s)
Adenoviridae/enzimología , Proteínas E2 de Adenovirus/metabolismo , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Conformación de Ácido Nucleico , Cloruro de Magnesio/farmacología , Modelos Biológicos , Conformación de Ácido Nucleico/efectos de los fármacos , Oligonucleótidos/metabolismo , Cloruro de Sodio/farmacologíaRESUMEN
The Adenovirus DNA binding protein (DBP) imposes a regular, rigid and extended conformation on single stranded DNA (ssDNA) and removes secondary structure. Here we show that DBP promotes renaturation of complementary single DNA strands. Enhancement of intermolecular renaturation is sequence independent, can be observed over a broad range of ionic conditions and occurs only when the DNA strands are completely covered with DBP. When one strand of DNA is covered with DBP and its complementary strand with T4 gene 32 protein, renaturation is still enhanced compared to protein-free DNA, indicating that the structures of both protein-DNA complexes are compatible for renaturation. In contrast to promoting intermolecular renaturation, DBP strongly inhibits intramolecular renaturation required for the formation of a panhandle from an ssDNA molecule with an inverted terminal repeat. We explain this by the rigidity of an ssDNA-DBP complex. These results will be discussed in view of the crystal structure of DBP that has recently been determined.
