Jan 9, 1992 - SV40 DNA replication, although they did partially replace Py aux-2. Stimulation required the presence of proteins consisting of the GAL4 DNA ...
Vol. 12, No. 6
MOLECULAR AND CELLULAR BIOLOGY, June 1992, p. 2514-2524 0270-7306/92/062514-11$02.00/0 Copyright © 1992, American Society for Microbiology
Specific Transcription Factors Stimulate Simian Virus 40 and Polyomavirus Origins of DNA Replication ZONG-SHENG GUOt AND MELVIN L. DEPAMPHILIS*
Department of Cell and Developmental Biology, Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110 Received 9 January 1992/Accepted 5 March 1992
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. tion and organizational motifs (26, 29). They contain all of the cis-acting genetic information necessary for initiating bidirectional DNA replication in the presence of its cognate T-ag and appropriate permissive cell factors (19, 24, 56, 73). The only viral protein required for DNA replication is T-ag, which binds specifically to its cognate on-core and initiates DNA unwinding (8, 83). Cellular proteins then initiate bidirectional DNA synthesis on the exposed DNA templates and assemble the newly replicated DNA into chromatin (13, 26). on-core is flanked by two auxiliary components, aux-1 and aux-2, that are not interchangeable yet function synergistically to facilitate on-core activity (24, 34, 35, 37, 40, 48, 50). aux-l lies adjacent to the inverted repeat element domain of oni-core, contains a strong binding site for T-ag (SV40 T-ag binding site I [24] and Py T-ag binding sites A and B [93]), and stimulates on-core activity 5- to 10-fold. aux-2 lies on the opposite side of on-core, proximal to the A+T-rich element of oni-core. In SV40, aux-2 is part of the early gene promoter (9, 24, 34, 40, 48, 50, 69). Deletion of SV40 aux-2 reduces replication from 2- to 100-fold, depending on experimental conditions (34, 35). In Py, aux-2 consists of functionally redundant enhancer elements that stimulate Py replication from 200- to 1,000-fold (64, 90). Both aux-1 and aux-2 are auxiliary components, because they are dispensable for replication under some conditions and do not affect the mechanism of replication. For example, deletion of aux-1 does not affect selection of initiation sites for RNA-primed DNA synthesis or the bidirectional movement of replication forks (35). In the presence of T-ag, Py on replicates as efficiently without aux-2 when injected into mouse one-cell embryos as it does with aux-2 when injected into two-cell embryos (56, 57). Both Py and SV40 oni-cores can replicate efficiently under some in vitro conditions (19, 73) but respond to oni-auxiliary sequences under other in vitro conditions (34).
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). Nevertheless, despite the remarkable similarity between their on regions, they do not replicate in the same host cell, and their requirements for auxiliary components appear to differ significantly. SV40 and Py on-cores are strikingly similar in sequence composi*
Corresponding author.
t Present address: Howard Hughes Medical Institute, New York
University Medical Center, New York, NY 10016. 2514
VOL. 12, 1992
TRANSCRIPTION FACIORS IN DNA REPLICATION
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TABLE 1. Transcription factor binding sites used to replace SV40 aux-2 Oligonucleotidea
Sequence
SV40 NcoI site 21core .....................................................
5'-TAGTCAGC CATGGGGCGGAGATfGGGG AACTfGGG G
AGT-3'
[T-ag]3core ..................................................... CATGGAGAQGGATCTAA(AOGATCTAAGA(Q ATCTG [Spl]3core ................................................................. G (
