Naturally arising variants of simian virus 40 (SV40), generated by serial passage of the virus at high multiplicities of infection, provide important insight into the role of transcription factor-binding sites in enhancing DNA replication. Although the variants that arise from numerous recombination events are the result of selective pressure to replicate more efficiently than the other variants in the infection, there is no transcription pressure. Therefore, it is interesting that a minimum of two viral Sp1 transcription factorbinding sites are retained and that host AP-1 and NF-1 transcription factor-binding sites are incorporated into the 100-bp regulatory region that maximizes DNA replication in these variants. We cotransfected COS-1 cells (that provide viral large T antigen for DNA replication) to examine the effect of transcription factor-binding sites on the replication of plasmid constructs that contain the SV40 origin of replication (ori). The level of relative replication efficiency (RRE) depends on the number and type of transcription factor-binding sites. Replication increases as the number of transcription factor-binding sites increases within the regulatory region of the variants; AP-1 sites are more effective than NF-1 transcription factor-binding sites. Competition between constructs in transfections magnifies the difference in their RREs. The results indicate that transcription factor-binding sites play an important role in enhancing DNA replication.Simian virus 40 (SV40) DNA is replicated by host cell machinery (reviewed in references 5 and 11) and one virally encoded protein, large T antigen (T-ag) (6,45,50,56). SV40 DNA replication is bidirectional (7, 17) from a 64-bp core origin of DNA replication (ori) (4,14,25,45,52). This ori sequence consists of a 15-bp early palindrome, a 27-bp palindrome, and a 17-bp AT-rich region (8). Four pentanucleotides in the central 27-bp palindromic region of ori are binding sites for T-ag (9, 49, 57). Additional regulatory sequences within 180 bp on the late transcription side of ori include three 21-bp repeats (16) and a 72-bp transcriptional enhancer (2,20,23,43). Each 21-bp repeat contains two hexameric GC boxes, which are binding sites for the transcription factor Sp1 (15) and weak binding sites for T-ag (57). The transcriptional enhancer contains a number of protein-binding sites; of these, a binding site for the transcription factor AP-1 is the closest to ori (31,34,63).The involvement of transcription factor-binding sites and the way in which they are arranged within regions that regulate DNA replication are interesting for several reasons. Some arrangements of AP-1 and Sp1 transcription factor-binding sites seem to be favored in evolutionary variants (37). Evolutionary variants arise when SV40 is serially passaged at high multiplicities of infection, whereby recombination leads to new viral species containing deleted and duplicated viral DNA and substituted host DNA. In several SV40 evolutionary variants (ev1101, ev1103, ev1104, and ev1108), the incor...

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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...

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