DNA sequence requirements for replication of polyomavirus DNA in vivo and in vitro.

Abstract

Cell extracts of FM3A mouse cells replicate polyomavirus (Py) DNA in the presence of immunoaffinitypurified Py large T antigen, deoxynucleoside triphosphates, ATP, and an ATP-generating system. This system was used to examine the effects of mutations within or adjacent to the Py core origin (on) region in vitro. The analysis of plasmid DNAs containing deletions within the early-gene side of the Py core ori indicated that sequences between nucleotides 41 and 57 define the early boundary of Py DNA replication in vitro. This is consistent with previously published studies on the early-region sequence requirements for Py replication in vivo. Deleting portions of the T-antigen high-affinity binding sites A and B (between nucleotides 57 and 146) on the early-gene side of the core on led to increased levels of replication in vitro and to normal levels of replication in vivo. Point mutations within the core ori region that abolish Py DNA replication in vivo also reduced replication in vitro. A mutant with a reversed orientation of the Py core on region replicated in vitro, but to a lesser extent than wild-type Py DNA. Plasmids with deletions on the late-gene side of the core on, within the enhancer region, that either greatly reduced or virtually abolished Py DNA replication in vivo replicated to levels similar to those of wild-type Py DNA plasmids in vitro. Thus, as has been observed with simian virus 40, DNA sequences needed for Py replication in vivo are different from and more stringent than those required in vitro.Replication of polyomavirus (Py) DNA proceeds bidirectionally from a fixed point within the noncoding region of the viral genome (for a review, see reference 16 and references therein). This replication origin (ori) region (Fig. 1) contains multiple T-antigen (T Ag)-binding sites within a G+C-rich 32-base-pair (bp) sequence of dyad symmetry adjacent to a region in which 13 of 14 residues form A * T pairs. It thus bears structural features similar to the replication ori of simian virus 40 (SV40). However, the relationship of the Py ori to other regulatory signals in the noncoding segment is somewhat different from that of SV40. The number and arrangement of large T Ag high-affinity binding sites on the early side of both viral ori regions differ. Also, the relative affinities of Py and SV40 T Ags for binding sites within their respective ori regions are apparently dissimilar (12,14,28,47). Moreover, while both viruses encode enhancer elements, the Py enhancer is almost directly adjacent to the late boundary of the ori region and consists of a series of discrete elements that bear homologies to other enhancer elements including those of SV40, adenovirus Ela, and mouse immunoglobulin genes (2,22,23,26,31,38,46,51,53). By contrast, the SV40 enhancer, a 72-bp repeated sequence (see reference 22 and references therein), is separated from the core ori by a series of G+C-rich sequences (the 21-bp repeats) that form a distinct transcriptional control element to which the cellular transcription factor, Spl, binds (...

Nuclear organization of DNA replication in primary mammalian cells

“…), suggests that the perinucleolar foci might contain chromosomal regions corresponding to E2F target genes. Physical association between late G 1 transcriptional regulatory proteins and the initiation of DNA synthesis would be consistent with the view of research on a number of organisms that transcriptional activation is linked to the initiation of DNA replication (DeVilliers et al 1984;Prives et al 1987;Guo et al 1989;Mul et al 1990;Cheng et al 1992;Ohba et al 1996;Murakami and Ito 1999). It may also provide a rationale for why origin selection could be different in distinct cell types; that is, the transcriptional profile of a cell in G 1 may influence which origins are ready for firing.…”

The need for enhancers in gene expression first appears during mouse development with formation of the zygotic nucleus.

“…Second, the factors required to initiate replication at a papovavims origin core (DePamphilis 1988) are distinctly different from those needed to activate the tk promoter (McKnight and Kingsbury 1982). Third, PyV origin core can fimction without an enhancer in a cell extract (Prives et al 1987) as well as in one-cell embryos, even though the concen tration of replication factors should be reduced substan tially in the extract. Finally, the sequence requirements for promoter activity corresponded only to the type of nucleus in which the plasmid was placed (Fig.…”

“…This structure must be fundamental to enhancer action because it is common to both pro moters and origins of replication. In the absence of nu clear and chromatin structures, the PyV origin-core fimctions without an enhancer (Prives et al 1987). En hancers can stimulate promoters in soluble in vitro systems, but they have a much smaller stimulatory ef fect in vitro than in vivo (Treisman and Maniatis 1985).…”

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

“…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). TRANSCRIPTION a Each oligonucleotide was designed to contain the minimum number of potential transcription factor binding sites by using a computer program to screen for 40 different known binding sites.…”

“…SV40 and Py on-cores are strikingly similar in sequence composi-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).…”