Relationship among Location of T-Antigen-Induced DNA Distortion, Auxiliary Sequences, and DNA Replication Efficiency

Abstract

T-antigen-induced DNA distortion was studied in a series of simian virus 40 (SV40) plasmid constructs whose relative replication efficiency ranges from 0.2 to 36. Bending was detected in the wild-type SV40 regulatory region consisting of three copies of the GC-rich 21-bp repeat but not in constructs with only one or two copies of the 21-bp repeat. In a construct with enhanced replication efficiency, bending occurred in a 69-bp cellular sequence located upstream of a single copy of the 21-bp repeat. Bending occurred both upstream of ori and in the three 21-bp repeats located downstream of ori in a construct with reduced replication efficiency. In a construct with no 21-bp repeats, DNA distortion occurred downstream of ori. The results indicate that SV40 DNA replication is enhanced when the structure of the regulatory region allows the DNA to form a bent structure upstream of the initial movement of the replication fork.Simian virus 40 (SV40) DNA replication has been widely used as a model to study DNA replication in eukaryotes (reviewed in references 7 and 19); the events before the initiation of replication are particularly interesting. SV40 has a welldefined origin of replication (2,10,22,43), and the initiation of replication depends on a single viral protein, T antigen (T-ag) (3,16,36,59). Except for T-ag, all proteins required for DNA replication are supplied by the host cell, and with the development of an in vitro SV40 replication system, their identity and function have been well characterized (35, 65; reviewed in references 7, 30, and 53). The SV40 core ori is a 64-bp region (20,27,43,54) that consists of three functional domains (13, 36, 45): a 17-bp adenine/thymine-rich (AT) region, a central 27-bp palindrome (PEN), and an early palindrome (EP).Located proximal to the AT tract are the promoter and enhancer elements of the SV40 regulatory region. The bidirectional promoter element for SV40 transcription consists of three 21-bp tandem repeats, each containing two copies of the conserved 5Ј-GGGCGG-3Ј (GC box) sequence. The GC boxes are binding sites for the transcription factor Sp1 (16,24,26). The enhancer contains two 72-bp repeats, each with multiple binding sites for transcription factors, including those belonging to the AP family (31).T-ag binds as a trimer to T-ag binding site I (38) located on the early transcription side of ori. The presence of site I facilitates T-ag-dependent unwinding of the DNA (28) and increases the frequency of initiation of DNA replication (25). T-ag binding site II, located at the PEN region in ori, is essential for the initiation of replication (15,17,20,43,46). T-ag forms double hexameric lobes that surround the PEN region (12, 37), and the helicase activity of the hexamers unwinds the DNA bidirectionally (11, 63). T-ag binding also occurs at site III located within the three 21-bp GC-rich repeats on the late transcription side of ori (23,38,60).T-ag binding to ori and flanking sequences induces structural distortions of the region. Both dimethyl sulfate protection and p...

Production of Papillomavirus‐Based Gene Transfer Vectors

“…The lack of an SV40 ori also precludes amplification of the vector stock (Basic Protocol 2). Addition of a previously reported minimal SV40 ori (construct pC139H; Okuley et al, 2003) to a plasmid of interest effectively enhances conversion of the plasmid into a papillomaviral vector stock.…”