High-frequency recombination mediated by polyomavirus large T antigen defective in replication.
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-T-Ag (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 of pmt 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 T-Ag by several orders of magnitude, as well as by mutants defective in the initiation of viral DNA synthesis. Large T-Ag 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 T-Ag has a recombination-promoting activity that can be dissociated from its replicative function.
St-Onge
L
L
Department of Biochemistry, University of Sherbrooke, Quebec, Canada.
Bouchard
L
L
Bastin
M
M
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics*; D002460:Cell Line; D002472:Cell Transformation, Viral; D004261:DNA Replication; D005785:Gene Conversion; D066298:In Vitro Techniques; D009154:Mutation; D051381:Rats; D011995:Recombination, Genetic; D015183:Restriction Mapping; D014779:Virus Replication
D000952:Antigens, Polyomavirus Transforming