Circular and linear simian virus 40 DNAs differ in recombination.

Abstract

Linear forms of simian virus 40 (SV40) DNA, when added to transfection mixtures containing circular SV40 and +X174 RFI DNAs, enhanced the frequency of SV40/+X174 recombination, as measured by infectious center in situ plaque hybridization in monkey BSC-1 cells. The sequences required for the enhancement of recombination by linear DNA reside within the SV40 replication origin/regulatory region (nucleotides 5,171 to 5,243/0 to 128). Linearization of +X174 RFI DNA did not increase the recombination frequency. The SV40/+X174 recombinant structures arising from transfections supplemented with linear forms of origin-containing SV40 DNA contained 4X174 DNA sequences interspersed within tandem head-to-tail repeats derived from the recombination-enhancing linear DNA. Evidence is presented that the tandem repeats are not formed by homologous recombination and that linear forms of SV40 DNA must compete with circular SV40 DNA for the available T antigen to enhance recombination. We propose that the enhancement of recombination by linear SV40 DNA results from the entry of that DNA into a rolling circle type of replication pathway which generates highly recombinogenic intermediates.The pathways and underlying mechanisms responsible for recombination in animal cells are poorly understood. Integrative recombination by DNA tumor viruses such as simian virus 40 (SV40), polyoma virus, and adenovirus does not generally involve specific sites in the viral or cellular genome, and extensive base sequence homology is not required (reviewed in reference 69), although a possible role for very short regions of homology has not been excluded (11). Because the molecular analysis of viral integration sites in transformed cells is feasible only many cell generations after the initial cell-virus recombination event, we and others have devised short-term extrachromosomal recombination systems, based either upon the cell-mediated excision of infectious SV40 DNA from transfected plasmid constructs (65,73,74) or upon intermolecular recombination between cotransfected SV40 and foreign DNAs (1,24,31,75,76). Some of these studies have led to the realization that, in contrast to lower eucaryotes, nonhomologous recombination in higher eucaryotes is prevalent.Previously, we have shown that DEAE-dextran-mediated cotransfection of monkey BSC-1 cells with circular SV40 and 4X174 RFI DNAs generates recombinant virus which replicates in the presence of wild-type SV40 (76). The frequency of recombination was measured, in terms of the proportion of SV40-replicating cells which generate SV40/40X174 hybrid virions, by an infectious center in situ plaque hybridization procedure (24). Only recombinants which retain a functional SV40 origin of replication and are of a size which can be encapsidated into SV40 virions are scored by this assay. The number of recombinant-producing cells (10-3 with respect to the total cell population and 10-2 with respect to the successfully transfected subpopulation) is unaffected by the level of gross nucleotide sequence homology bet...

Molecular Biology, Epidemiology, and Pathogenesis of Progressive Multifocal Leukoencephalopathy, the JC Virus-Induced Demyelinating Disease of the Human Brain

“…Replication proceeds bidirectionally, similar to theta replication, and leads to two interlinked DNA circles, which are resolved through the action of topoisomerases I and II (reference 360 and references therein). It has been proposed that linear SV40 genomes can initiate rolling-circle replication, generating concatemers, and that this method of replication may explain some of the recombination seen in viral variants (105,122,459).…”

Intermolecular recombination assay for mammalian cells that produces recombinants carrying both homologous and nonhomologous junctions.

“…We believe that the effect observed when RmI is cut is probably due to another parameter, such as the replicative state of the molecule at the time of recombination. We and others have shown that replication has an influence on the nature and frequency of recombination (9,51). Why did double-strand breaks not increase the ratio of homologous to nonhomologous recombination?…”

Linear simian virus 40 DNA fragments exhibit a propensity for rolling-circle replication.

“…We have documented elsewhere that origin-containing (ori+) linear forms of simian virus 40 (SV40) DNA, when added to transfection mixtures containing circular SV40 and 4X174 replicative form I DNAs, enhance the frequency of SV4O/4X174 recombination as measured by infectiouscenter in situ plaque hybridization in monkey BSC-1 cells (4). A key structural feature of such recombinants is the interspersion of 4X174 DNA within tandem head-to-tail repeats of sequences derived from the ori+ linear SV40 DNA.…”

“…It was also shown that the tandem repeats are generated by a replication-related process rather than by homologous recombination. To account for these findings, we postulated that the recombination-enhancing properties of linear SV40 ori+ DNA are associated with the entry of that DNA into a rolling-circle type of replication which generates recombinogenic intermediates (4). The proposed model envisages that 4X174 replicative form I DNA recombines with the circular template of the intermediate (5) such that continuation of rolling-circle replication generates tandemly repeated segments containing both SV40 ori+ and 4~X174 DNAs.…”