Reconstitution of a functional bovine papillomavirus type 1 origin of replication reveals a modular tripartite replicon with an essential AT-rich element.
A functional replication origin was reconstituted using oligonucleotide cassettes corresponding to three sequence subelements within the Bovine Papillomavirus Type 1 (BPV-1) replication origin: the 23-bp AT-rich region (ATR), the 18-bp binding site for the viral replication initiator protein E1 (E1BS), and a binding site for the viral transcriptional transactivator and replication enhancer protein E2 (E2BS). Replication of the reconstituted origin depended on heterologous expression of both the E1 and E2 proteins and on the presence of both the E1BS and E2BS, indicating that it is functionally analogous to the authentic BPV-1 origin. In addition, pairwise testing of subelement combinations revealed that the ATR was also essential and that a functional origin required at least one copy of all three subelements. While the E1BS and E2BS are sequence-specific elements, the function of the BPV-1 ATR could be at least partially substituted with heterologous AT-rich sequences, suggesting that the role of this element is primarily AT content-dependent rather than sequence-dependent. A stringent requirement for the ATR was also observed in the context of an authentic minimal origin sequence confirming that it is an intrinsic property of the BPV-1 origin and not simply an artifact of the reconstitution system. This study indicates that the minimal functional BPV-1 origin shares the tripartite modular organization characteristic of other simple eukaryotic replication origins. The reconstitution system described now provides a convenient approach to define the physical and functional interrelationships between the three subelements in a systematic fashion.
McShan
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Department of Medical Microbiology and Immunology, Texas A&M University Health Science Center, Room 473, College Station, Texas 77843-1114, USA.
Wilson
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VG
D017502:Adenovirus E2 Proteins / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D052688:Bovine papillomavirus 1 / Q000502:physiology*; D002417:Cattle; D008969:Molecular Sequence Data; D012093:Replicon / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics; D014779:Virus Replication
D017502:Adenovirus E2 Proteins; D014764:Viral Proteins