Extrachromosomal recombination is deranged in the rec2 mutant of Ustilago maydis

Abstract

Transformation of a leu1 auxotroph of Ustilago maydis to prototrophy with an autonomously replicating plasmid containing the selectable LEU1 gene was found to be efficient regardless of whether the transforming DNA was circular or linear. When pairs of autonomously replicating plasmids bearing noncomplementing leu1 alleles were used to cotransform strains deleted entirely for the genomic copy of the LEU1 gene, Leu+ transformants were observed to arise by extrachromosomal recombination. The frequency of recombination increased severalfold when one plasmid of the pair was made linear by cleavage at one end of the leu1 gene, but increased 10-100-fold when both plasmids were first made linear. The increase in recombination noted in wild-type and rec1 strains was not apparent in the rec2 mutant unless the members of the pair of plasmids were cut at opposite ends of the leu1 gene to yield linear molecules offset in only one of the two possible configurations. Use of a pair of plasmid substrates designed to measure nonreciprocal and multiple exchange events revealed only a minor fraction of the total events arise through these modes, and further that no stimulation occurred when the plasmid DNA was linear. It is unlikely that the defect in rec2 lies in a mismatch correction step since a high yield of Leu+ recombinants was obtained from the rec2 mutant when it was transformed with heteroduplex DNA constructed from plasmids with the two different leu1 alleles.