DNA binding site for a factor(s) required to initiate simian virus 40 DNA replication.

Abstract

Efficient initiation of DNA replication in the absence of nonspecific DNA repair synthesis was obtained by using a modification of the system developed by J. J. Li and T. J. Kelly [(1984) Proc. Natl. Acad. Sci. USA 81, [6973][6974][6975][6976][6977].Circular double-stranded DNA plasmids replicated in extracts of CV-1 cells only when the plasmids contained the cis-acting origin sequence for simian virus 40 DNA replication (on) and the extract contained simian virus 40 large tumor antigen. Competition between plasmids containing on and plasmids carrying deletions in and about on served to identify a sequence that binds the rate-limiting factor(s) required to initiate DNA replication. The minimum binding site (nucleotides 72-5243) encompassed one-half of the simian virus 40 on sequence that is required for initiation of replication (oni-core) plus the contiguous sequence on the late gene side of on-core containing G+C-rich repeats that facilitates initiation (oni-auxiliary). This initiation factor binding site was specific for the simian virus 40 on region, even though it excluded the high-affmlity large tumor antigen DNA binding sites.With the exception of initiation of DNA replication, all subsequent steps in the replication and assembly of simian virus 40 (SV40) chromosomes utilize host-cell components exclusively (1, 2). Initiation ofviral DNA replication requires the cis-acting 63-base-pair (bp) ori-core sequence, SV40 large tumor antigen (T-Ag), and permissive cell factors from monkey or human cells. Bidirectional replication then originates at the junction between the strongest DNA binding site for T-Ag and ori-core (Fig. 1). The 40-45 bp flanking either side of ori-core (ori-auxiliary; Fig. 1) are not required for replication, although they do stimulate replication 3-to 5-fold.Purified SV40 T-Ag binds most strongly to the early gene-side auxiliary sequence (site I), less strongly to the ori-core sequence (site II), and marginally to the late geneside auxiliary sequence and beyond (site III; Fig. 1). Mutations within sites I and II alter the affinity of T-Ag for DNA (6)(7)(8)19). These sites are biologically significant because mutations within ori that reduce or prevent replication can be suppressed by mutations in T-Ag (20), and mutations within site I that reduce the ability of T-Ag to regulate its own synthesis are overcome by mutations in T-Ag (7). Since most of binding sites I and III can be deleted with only a marginal effect on replication, initiation of DNA replication must involve binding of T-Ag to site II (ori-core), perhaps aided by the strong affinity of T-Ag to site I. Thus, one might expect ori-core and perhaps T-Ag binding site I to be the primary binding site for the protein complex responsible for initiating SV40 DNA replication.With the advent of soluble systems capable of initiating SV40 DNA replication in vitro (21-24), it was possible to determine which DNA sequence exhibited the strongest affinity for replication initiation factors by its ability to inhibit replication of a s...

Homologous recombination enhancement conferred by the Z-DNA motif d(TG)30 is abrogated by simian virus 40 T antigen binding to adjacent DNA sequences.

“…An established replication assay was used to determine the relative replication efficiencies of the recombination substrates (3,24,47,52). COS-1 cells were cotransfected with substrate DNA and p2X21ori and incubated for 56 h to allow replication to occur, and low-Mw DNA was isolated.…”

“…The replication assay was essentially as previously described (3,24,47,52). A 100-ng portion of experimental plasmid and 50 ng of internal replication control plasmid p2X21ori were introduced into 2 x 106 COS-1 cells per 100-mm tissue culture dish by the DEAE-dextran transfection procedure detailed above.…”

Initiation of simian virus 40 DNA replication in vitro: aphidicolin causes accumulation of early-replicating intermediates and allows determination of the initial direction of DNA synthesis.

“…HSNE, High-salt nuclear extract; Aph, aphidicolin. polyoma virus ori did not replicate (40). Finally, as described below, partial inhibition of SV40 DNA synthesis by aphidicolin, a specific inhibitor of DNA polymerase a (25), resulted in accumulation of SV40 replicating chromosomes with only their ori regions replicated.…”

“…Alternatively, in the absence of added T-ag or extracts from cells infected with a viral mutant that overproduces T-ag, initiation of SV40 DNA replication was achieved both in SV40 chromo-3816 DECKER ET AL. somes (present communication; R. S. Decker, M. Yamaguchi, M. K. Bradley, and M. L. DePamphilis, submitted for publication) and in plasmids containing SV40 ori (40) by addition of a hydrophilic polymer to extracts of wild-type SV40-infected permissive monkey cells. Using this system, we observed that initiation of DNA synthesis in the ori region of SV40 was much less sensitive to inhibition by aphidicolin than was DNA synthesis in replicative intermediates that had already initiated replication.…”

“…To determine whether the relative insensitivity of initiation of SV40 DNA replication to aphidicolin was due to the nucleoprotein structure of the chromosome substrate, a purified plasmid DNA (pSVori) containing the 206-bp HindIII-SphI fragment of SV40 wt800, which includes the ori sequence, was incubated under in vitro conditions that allowed ori-dependent, T-ag-dependent initiation of DNA replication (40). These conditions were similar to the ones used to initiate replication in SV40 chromosomes and resulted in bidirectional replication from ori.…”