Replication from a proximal simian virus 40 origin is severely inhibited by multiple reiterations of the 72-base-pair repeat enhancer sequence.

Abstract

In a previous study in our laboratory, the effect of the reiteration frequency of the simian virus 40 (SV40) 72-base-pair (bp) repeat enhancer on transcription from the proximal SV40 early promoter was investigated (R. Kumar, T. A. Firak, C. T. Schroll, and K. N. Subramanian, Proc. Natl. Acad. Sci. USA 83:3199-3203, 1986). Increasing the enhancer copy number to four increased transcription proportionately; further increments in enhancer copy number reversed this effect, resulting in a decrease in the transcriptional activation. In the present study, the effect of enhancer reiteration on the replication efficiency of plasmids containing the SV40 origin of replication was investigated in transient replication assays in vivo in COS-1 monkey kidney cells producing the SV40 large tumor antigen required for replication. A plasmid containing the SV40 core origin and three copies of the replication-activating, G+C-rich 21-bp repeat promoter element replicated efficiently. Plasmids containing multiple copies of the 72-bp repeat enhancer cloned in head-to-tail linkage adjacent to the 21-bp repeat and the core origin replicated less efficiently; the decrease in replication efficiency could be correlated with the number of copies of the 72-bp repeat; replication was severely curtailed when 10 or more copies of the 72-bp repeat were present. Replication was not significantly inhibited by an increase in the number of copies of the 21-bp repeat to 15 or by the presence of three copies of a 360-bp pBR322 sequence in the immediate vicinity. Multiple copies of the 72-bp enhancer in cis were unable to inhibit replication from a second SV40 origin of replication situated 2 kilobase pairs away from the enhancer reiteration. Replication of four different test plasmids was not inhibited in trans by cotransfection of an excess of a potential competitor plasmid containing a 24-copy reiteration of the 72-bp enhancer. These results indicate that multiple tandem reiterations of the 72-bp enhancer inhibit replication only when they are present in cis adjacent to the origin of replication. Possible explanations for this inhibitory effect, such as an unfavorable local chromatin structure induced by the multimeric enhancer region or reduced or improper communications between factors bound to the multimeric enhancer region and the adjacent replication origin, are discussed.

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.

“…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. p2X21ori is a small plasmid, containing the SV40 origin of replication and two copies of the 21-bp repeats, that replicates efficiently in COS-1 cells (24). The low-Mw DNA was extensively purified and linearized by restriction endonuclease digestion with Sall, which cuts only once within p2X21ori and the substrates.…”

“…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.…”

“…Cells (106) were placed on 100-mm tissue culture plates 18 h prior to transfection. The medium was removed, and 1 ml of serum-free Dulbecco modified Eagle medium containing 400 ,ug of DEAE-dextran per ml (Mw, 500,000) and DNA was layered on the cells and incubated with occasional agitation for 3 h. The medium was removed, and the cells were treated for 2 min with 10% dimethyl sulfoxide in 138 mM NaCl-5 mM KCI-0.7 mM Na2HPO4-6 mM dextrose-20 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (pH 6.92) ( 24 h in the absence of selection and then transferred to 100-mm plates for selection in Dulbecco modified Eagle medium containing G418 at 400 ,ug/ml. G418F colonies were visible 7 to 10 days after transfection.…”

“…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.…”

Origin auxiliary sequences can facilitate initiation of simian virus 40 DNA replication in vitro as they do in vivo.

“…Deletion of these sequences (pSVori) increased replication by as much as fourfold, depending on the T-Ag concentration. In fact, these sequences have been shown to inhibit ori activity in proportion to their copy numbers (26). pSVori consistently replicated the fastest under all conditions examined.…”