Intrachromosomal recombination mediated by papovavirus large T antigens

Abstract

To investigate the mechanism by which the large T antigen (T-Ag) of polyomavirus and simian virus 40 can promote recombination in mammalian cells, we analyzed homologous recombination events occurring between two defective copies of the polyomavirus middle T (pmt) oncogene lying in close proximity on the same chromosome in a rat cell line. Reconstitution of a functional pmt gene by spontaneous recombination occurred at a rate of about 2 x 10-7 per cell generation. Introduction of the polyomavirus large T (plt) oncogene into the cell line by DNA transfection promoted recombination very efficiently, with rates in the range of 10-1 to 10-2 per cell generation. Recombination was independent of any amplification of viral sequences and could even be promoted by the large TAg from simian virus 40, which cannot activate polyomavirus DNA replication. To explain the role of large TAg , we propose a novel mechanism of nonconservative recombination involving slipped-strand mispairing between the two viral repeats followed by gap repair synthesis.

Polyomavirus large and small T antigens cooperate in induction of the S phase in serum-starved 3T3 mouse fibroblasts

“…It should also be remembered that immortal cells arising by transfection with viral genetic information usually exhibit a variety of chromosomal aberrations which may be intimately related to their capability to escape senescence. In fact, a mutagenic and/or recombinogenic activity of papovavirus LT antigens might be important for the immortalizing function of these proteins (48,52,53).…”

p53 is linked directly to homologous recombination processes via RAD51/RecA protein interaction.

“…The large tumour antigens of polyomavirus and SV40 are known to promote recombination events very efficiently (St-Onge et al, 1990;St-Onge and Bastin, 1993). T antigen has been proposed to facilitate recombinatorial processes by melting and unwinding of DNA at the viral origin of replication, so as to create a favourable substrate for homologous recombination (St-Onge et al, 1990;St-Onge and Bastin, 1993). To test whether, in addition to these mechanisms, SV40 T antigen would cause a direct effect on p53-controlled recombinatorial activities, increasing amounts of purified T antigen were added to the RecA ATPase reaction under p53 block.…”

Amplification of polyomavirus DNA sequences stably integrated in rat cells

“…At 39°C, Hy2-ts5 cells produced the lOOK large T-Ag as well as another polypeptide of about 53K (Figure 4). This polypeptide which was also present in the parental Hy2 cell line was identified as a truncated middle T-Ag expressed from the pmt locus (26). The 56K middle T-Ag was clearly produced in four foci picked at random following the temperature shift, indicating that exposure to functional large T-Ag triggered recombination in the pmt locus so as to reconstitute a complete and fully active pmt oncogene.…”

“…Details on the construction of the Hy2 cell line have been reported (26). The arrangement of the viral insert is represented in Figure IA.…”

“…Previously we described a system to analyze recombination between two repeats of polyomavirus DNA sequences lying in close proximity in the genome of a rat cell line (26). Both polyomavirus and SV40 large T-Ag's promoted homologous recombination in the viral insert.…”