Simian virus 40 DNA replication: functional organization of regulatory elements.

Abstract

The efficiency of simian virus 40 (SV40) DNA replication is dependent on the structural organization of the regulatory region. The enhancing effect of the G+C-rich 21-base-pair (bp) repeats on SV40 DNA replication is position and dose dependent and to some extent orientation dependent. The inverted orientation is about 50% as effective as the normal orientation of the 21-bp repeat region. Movement of the 21-bp The double-stranded circular genome of simian virus 40 (SV40) represents a simple model system for studying mechanisms involved in eucaryotic DNA replication (for a review, see reference 8). Replication of the 5,243-base-pair (bp) genome of SV40 begins at a unique site, and the elongation of nascent DNA chains proceeds bidirectionally (9, 14). Viral DNA replication depends mainly on cellular mechanisms. The analysis of naturally arising evolutionary variants and deletion mutants has narrowed the limits of the origin signal for SV40 DNA replication (core ori) to a segment of approximately 65 bp from 0.653 to 0.666 map units including a 27-bp palindrome and an A+T-rich sequence (5,11,12,22,32,42,48). Single nucleotide changes or small deletions within this segment abolish or alter replication (20,42,46). The in vivo initiation sites for DNA synthesis have been mapped within the ori segment in the 27-bp palindrome as well as within the three 21-bp, G+C-rich tandem repeats (25). The 21-bp repeat has an enhancing effect on SV40 DNA replication (5,15,28) and is a bidirectional promoter element for SV40 transcription (3,13,18,40,54). The 72-bp repeat is a position-and orientationindependent enhancer for transcription (2,4,15,16,21,41,55).We examined the effects of the orientation, location, and number of copies of the 21-bp repeat on SV40 DNA replication. The role(s) of the 72-bp repeat in SV40 DNA replication was also investigated. Fragments containing the SV40 ori sequence plus various portions of the normal or relocated regulatory sequences were cloned into bacterial plasmids. By mixed transfection of monkey cells and Southern blot analysis of newly replicated DNAs, we compared the effects of various structural rearrangements of the 21-or 72-bp repeat or both on the replication efficiency of the SV40 ori region. MATERIALS AND METHODSCells and plasmid vector. The BSC-1 line of African green monkey kidney cells was grown in 8% calf serum plus 2% * Corresponding author.fetal calf serum and Eagle minimal essential medium (MEM-10). The COS-1 line of African green monkey kidney cells (19) was grown in 5% fetal calf serum (MEM-5).The plasmid vector pKP45 (kindly provided by K. Peden, The Johns Hopkins University, School of Medicine) is derived from pBR322 by deletion of nucleotides 676 to 2364 which removes the sequence that is "poisonous" for plasmid replication in mammalian cells (23,36,43).Restriction endonuclease digestion and DNA fragment purification. Restriction endonucleases were purchased from New England BioLabs, Inc. (Beverly, Mass.) or Boehringer Mannheim Biochemicals (Indianapolis, Ind.) and used as su...

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

“…On the other hand, a highly replication-efficient, enhancerless plasmid such as p2x210ri given in trans at the same level as the test plasmid effectively inhibits the enhancer-containing test plasmids. Such a competition for replication between two plasmids which is decided in favor of the more efficiently replicating plasmid has been observed previously by other workers (46,47).…”

“…However, even from a distance of 5 kb from the promoter, the 72-bp enhancer can activate transcription by at least 10-fold over that obtained without the enhancer (71). On the other hand, the replication activation property of the SV40 72-bp repeat (which is exhibited only in the absence of the intervening 21-bp repeat [14,36,46,47]) is highly position dependent; replication is activated at 8 to 9 bp from the SV40 core origin, but not at 99 bp or more (11). A similar finding has also been made for the polyomavirus system (33).…”

“…The 72-bp repeat is also capable of a similar enhancement in replication if it is juxtaposed to the core origin by the deletion of the intervening 21-bp repeat (14,36,46,47). However, in the presence of the 21-bp repeat, the 72-bp repeat does not enhance replication any further (4,36,46,47). Unlike SV40, in which the core origin alone can initiate replication at a basal level, the polyomavirus core origin is incapable of initiating replication in the absence of the adjacent transcriptional enhancer region (15,33,48,54,69,70).…”

“…In SV40, the presence of the 21-bp repeat adjacent to the core origin increases the efficiency of replication by about 10-to 20-fold (4,21,36,46,47). The 72-bp repeat is also capable of a similar enhancement in replication if it is juxtaposed to the core origin by the deletion of the intervening 21-bp repeat (14,36,46,47). However, in the presence of the 21-bp repeat, the 72-bp repeat does not enhance replication any further (4,36,46,47).…”

Simian virus 40 enhancer does not affect the tumor specificity of human heparanase gene promoter

“…The SV40 enhancer was one of the first enhancers to exhibit strong activity. It is composed of a 72 bp repeated sequence without obvious partiality in race and can exert a 2-to 20-fold enhancement of gene transcription in different host cells (18,19). Previous results have shown that the transcription activity of human telomerase reverse transcriptase (hTERT) promoter was significantly enhanced after it was modified using SV40 enhancer (20).…”