Specific transcription factors stimulate simian virus 40 and polyomavirus origins of DNA replication.

Abstract

The origins of DNA replication (ori) in simian virus 40 (SV40) and polyomavirus (Py) contain an auxiliary component (aux-2) composed of multiple transcription factor binding sites. To determine whether this component stimulated replication by binding specific transcription factors, aux-2 was replaced by synthetic oligonucleotides that bound a single transcription factor. Spl and T-antigen (T-ag) sites, which exist in the natural SV40 aux-2 sequence, provided -75 and -20%, respectively, of aux-2 activity when transfected into monkey cells. In cell extracts, only T-ag sites were active. AP1 binding sites could replace completely either SV40 or Py aux-2. Mutations that eliminated API binding also eliminated AP1 stimulation of replication. Yeast GAL4 binding sites that strongly stimulated transcription in the presence of GAL4 proteins failed to stimulate SV40 DNA replication, although they did partially replace Py aux-2. Stimulation required the presence of proteins consisting of the GAL4 DNA binding domain fused to specific activation domains such as VP16 or c-Jun. These data demonstrate a clear role for transcription factors with specific activation domains in activating both SV40 and Py ori. However, no correlation was observed between the ability of specific proteins to stimulate promoter activity and their ability to stimulate origin activity. We propose that only transcription factors whose specific activation domains can interact with the T-ag initiation complex can stimulate SV40 and Py ori-core activity.Most, if not all, of the eukaryotic origins of DNA replication (on) characterized so far consist of two principal components: the core component that determines where replication begins in the chromosome and in which animal species replication occurs, and one or more auxiliary components that stimulate replication in certain cell types (28,30,96). on-core is the minimal cis-acting sequence required to initiate DNA replication under all conditions; it is analogous to a transcription promoter. on-auxiliary sequences generally consist of transcription factor binding sites that are dispensable under some conditions; they are analogous to transcription enhancers. The purpose of auxiliary components may be to regulate DNA replication by determining when initiation occurs. For example, the polyomavirus (Py) on functions only in those mouse cell types that can activate its enhancer (6,12,58,74). In mammalian chromosomes, active genes are replicated early during S phase while quiescent genes are replicated late (82), and initiation of replication may require specific transcription factors (76) that bind DNA sites where replication begins (11).To elucidate the function of on-auxiliary components in mammals, we turned our attention to the origins of DNA replication in simian virus 40 (SV40) and Py. These two closely related viral chromosomes replicate in the nuclei of mammalian cells and, with the exception of a single viral protein (large tumor antigen [T-ag]), rely entirely on the host to replicate their DNA (13,29)....

Transcription factors in DNA replication

“…Aux2, which is located on the late side of the core origin, contains binding sites for various transcription factors including SP1, AP1, AP2, AP4 and p53. The Aux2 sequence enhances SV40 DNA replication between 10 and 100 fold in vivo, depending on the assay conditions (31). Aux2 can be replaced with tandem multimers of single transcription factor binding sites such as those of SP1, AP1, ATF and NFI (16,31,36,51).…”

“…However, Py is able to replicate in hematopoietic cells when the native enhancer is replaced with the enhancer of the immunoglobulin gene. Like SV40, the enhancer of polyomavirus can be replaced by a single kind of transcription factor binding site and many kinds of transcription factors including AP1, Ets, c-Rel, p53, Gal4 , BPV E2 and Gal4-VP16 have been shown to stimulate Py DNA replication (6,8,31,41,46,66,69,70,102). Except for c-Rel (41), these transcription factors stimulate DNA replication through their transcription activation domains which are distinct from their DNA binding domains.…”

“…However, there are some arguments against the model when anti-repression is proposed to be the primary cause of the enhancement of DNA replication. For example, Gal4-VP16, which can alleviate repression by nucleosomes in vitro, did not stimulate SV40 DNA replication in vivo (31) and prebinding of T antigen to origin DNA is sufficient to overcome chromatin repression (42). Thus, more detailed in vivo studies are required to establish the anti-repression effect as one of the prime roles of transcription factors in viral DNA replication.…”

“…Aux2 can be replaced with tandem multimers of single transcription factor binding sites such as those of SP1, AP1, ATF and NFI (16,31,36,51). However, binding sites for the transcription factor Gal4 cannot substitute for the native binding sites, even when fusion proteins containing the Gal4 DNA binding domain fused to strong transcription activation domains such as those of VP16 or c-Jun are exogeneously expressed (31).…”

“…Like SV40, a 68 bp-long core origin, which provides the binding sites for T antigen, is associated with two auxiliary sequences (figure 1). Aux1 contains the T antigen binding sites and Aux2 comprises the transcriptional enhancer which is made up of various transcription factor binding sites (31,79). The requirement for Aux2 in Py DNA replication is more strict than in SV40.…”