High-frequency recombination mediated by polyomavirus large T antigen defective in replication

Abstract

We investigated the mechanism by which the large T antigen (T-Ag) of both polyomavirus and simian virus 40 (SV40) promotes homologous recombination in mammalian cells. To this end, we constructed a rat cell line, designated Hy5, that carries two mutated copies of the polyomavirus middle-TAg (pmt) oncogene lying as direct repeats on the same chromosome. The structure of the viral insert was devised so that intrachromosomal recombination between the pmt repeats reconstitutes wild-type pmt and yields cell populations amenable to selection for the transformed phenotype. Correction ofpmt by gene conversion occurred spontaneously at a rate of ca. 1.7 x 10-7 per cell generation and was masked by another recombination event that also led to the transformation of the Hy5 cell line. This event was identified as chromosomal inversion and overexpression of the upstream pmt copy as a result of homologous recombination between adjacent pBR322 sequences. Both events were promoted by the polyomavirus large TAg by several orders of magnitude, as well as by mutants defective in the initiation of viral DNA synthesis. Large TAg also promoted reconstitution of wild-type pmt by unequal exchange between sister chromatids, yielding structures compatible with some of the chromosomal aberrations commonly observed in transformed cells. Our data indicate that large TAg has a recombinationpromoting activity that can be dissociated from its replicative function.

In vivo assay of p53 function in homologous recombination between simian virus 40 chromosomes

“…With this SV40-based method, recombination frequencies in nonmanipulated primary cells were in the order of 10 Ϫ5 . This is clearly below the recombination rates published for SV40 T-Ag or polyomavirus T-Ag by others (25,35,57). The differences can partially be explained by distinct experimental conditions as discussed above.…”

“…Our data allow the interpretation that the stimulatory effect of T-Ag on recombination (25,57) can be explained by alleviating the inhibitory effect of p53 via complex formation. This conclusion is based on our finding that recombination rates are lowered by at least 1 order of magnitude when T-Ag(402H), impaired in its ability to bind p53, is expressed by SV40 instead of wild-type T-Ag in PRK cells (which express wild-type p53).…”

“…In addition, they reflect the confinement to selectively detect double-crossover and gene conversion events in our system. Nevertheless, the recombination frequencies remain at least 10-fold higher than the spontaneous gene conversion rates (5,57) and thus again confirm the recombinogenic character of SV40, though in a quantitatively different manner.…”

“…For the first generation released at 60 hpi, we obtained practically the same rate (10 Ϫ5 ) as for the second generation at 96 hpi. In comparison with the lower rates for spontaneous gene conversion in wild-type p53expressing cells (ranging from 10 Ϫ6 to Ͻ10 Ϫ9 ), these values indicate the stimulatory influence of SV40 with respect to this process (5,57).…”

“…The obvious advantage of applying SV40 virus is the ease in manipulating the recombination target, i.e., the small SV40 genome, in order to measure homologous recombination on DNA with defined indicator sites. Wild-type p53 protein was originally discovered in complex with the viral oncogene product SV40 large tumor antigen (T-Ag) (34,39), which is known to increase the recombination frequencies of cellular and viral DNA by several orders of magnitude (25,57). A recombination assay system based on SV40, therefore, offers the possibility to study a putative role of p53-T-Ag complexes with regard to this process.…”