Pausing in simian virus 40 DNA replication by a sequence containing (dG-dA)₂₇·(dT-dC)₂₇

Abstract

A 200 bp sequence including a stretch of 54 base pairs of alternating guanosine and adenosine nucleotide residues [(dG-dA)27.(dT-dC)27] was cloned in the simian virus 40 (SV40) genome between the KpnI and HpaII sites. This sequence was discovered earlier as part of a region limiting the amplification of sequences adjacent to an integrated polyoma virus in a transformed rat cell line. The newly constructed DNA was transfected into African Green monkey kidney CV1 cells and the variant virus was isolated by plaque-purification. The insertion was stably maintained and the variant virus grew more slowly than the wild type, had lower titers and gave smaller plaques. In mixed infection experiments, the variant was found to be stable, though the wild type replicated more rapidly. Pulse labeling experiments indicated that the unusual inserted sequence acts as a pause site for fork progression during DNA replication, as evidenced by the rate of incorporation of radioactively labeled nucleotides into various regions of the SV40 genome. Statistical fit of the experimental curves with theoretically generated curves suggested the pause of fork progression to be about one minute.

DNA triple helices: Biological consequences and therapeutic potential

“…Long GAA repeats (> 40 repeats), which form H-DNA, stalled replication fork progression on plasmids in S. cerevisiae [132]. An H-DNA-forming sequence GA (20) in the simian virus 40 (SV40) genome slowed growth in monkey CV1 cells, resulting in lower titers and smaller plaques [133]. GA repeats reduced the rate of nucleotide incorporation into the SV40 genome, resulting in stalled replication forks [133,134].…”

“…An H-DNA-forming sequence GA (20) in the simian virus 40 (SV40) genome slowed growth in monkey CV1 cells, resulting in lower titers and smaller plaques [133]. GA repeats reduced the rate of nucleotide incorporation into the SV40 genome, resulting in stalled replication forks [133,134]. E. coli strains transformed with plasmids containing the 2.5-kbp polypyrimidine sequence from the human PKD1 gene grew slower than those had shorter inserts, and this effect correlated with the level of negative supercoiling of the plasmid DNA in vivo, suggesting that the H-DNA structures formed were responsible for the cell growth retardation [135].…”

Molecular characterization of a common fragile site ( FRA7H ) on human chromosome 7 by the cloning of a simian virus 40 integration site

“…Hence, we searched the FRA7H region for two types of potential non-B-DNA sequences under negative superhelical strain: triple helixes and MARs. Triple-helix-forming sequences play an important role in triggering gene expression and homologous recombination (43), block DNA replication in vitro (44), and decrease the efficiency of replication in vivo (45). The MARs are prone to unwinding by continuous base unpairing (46).…”

Discrete functional elements required for initiation activity of the Chinese hamster dihydrofolate reductase origin beta at ectopic chromosomal sites

“…These final 16 bp, however, were not sufficient for full initiation activity of an ori-beta mutant lacking the preceding repetitive sequences ( Figure 2D, 0.5DNR2 mutant). GA repeats have been shown to impede replication forks [37,47,48] and affect the assembly of DNA into nucleosomes [49], so both of these potential roles were investigated separately ( Figure 3). Importantly, a replication fork barrier (SB2) and an element that contains a nucleosomepositioning element (5S rDNA) were each able to independently replace the function of DNR ( Figure 3C).…”