RESUMEN
The role of transcription in oligonucleotide (ODN)-directed gene modification has been investigated in mammalian cells. The importance of transcription is demonstrated using mammalian cell lines with varying degrees of transcription of the mutant LacZ reporter gene, residing in both episome and chromosome. Gene correction occurs more efficiently when the target gene is actively transcribed and antisense ODN is more active than sense ODN. Using an approach that combines biochemical studies with a cell-based assay to measure the functional activity of intermediates it is shown that a joint molecule, consisting of supercoiled DNA and homologous ODN targeted to correct the mutated base, is a functional intermediate in the gene repair process. Furthermore, this approach showed that a resected joint molecule is a downstream intermediate of the D-loop. These results indicate that the primary reason for efficient gene repair exhibited by the antisense ODN is its increased accessibility to the non-transcribed strand, and as a consequence an increased formation of intermediate during active transcription. Moreover, the processing of intermediates was also affected by transcription, suggesting that ODN-directed gene repair may be linked to transcription-coupled repair. Thus, transcription plays an important role in ODN-directed gene repair by affecting the formation and processing of key intermediates.
Asunto(s)
Reparación del ADN , Oligonucleótidos/genética , Transcripción Genética/genética , Animales , Secuencia de Bases , Células CHO , Cricetinae , ADN/química , ADN/genética , ADN/metabolismo , Operón Lac/genética , Luciferasas/genética , Luciferasas/metabolismo , Datos de Secuencia Molecular , Mutación , Conformación de Ácido Nucleico , Oligonucleótidos/química , Oligonucleótidos/metabolismo , Oligonucleótidos Antisentido/genética , Plásmidos/genética , Rec A Recombinasas/genética , Rec A Recombinasas/metabolismo , Transfección/métodosRESUMEN
To facilitate investigations of replication and host cell interactions in the hepadnavirus system, we have developed cell lines permitting the conditional replication of duck hepatitis B virus (DHBV). With the help of this system, we devised conditions for core particle isolation that preserve replicase activity, which was not found in previous preparations. Investigations of the stability of viral DNA intermediates indicated that both encapsidated DNA and covalently closed circular DNA (cccDNA) were turned over independently of cell division. Moreover, we showed that alpha interferon reduced the accumulation of RNA-containing viral particles. The availability of a synchronized replication system will permit the biochemical analysis of individual steps of the viral replication cycle, including the mechanism and regulation of cccDNA formation.