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Nucleic Acids Research, 1992, Vol. 20, No. 11 2785-2794
© 1992

MOLECULAR BIOLOGY

Mechanisms of intermolecular homologous recombination in plants as studied with single- and double-stranded DNA molecules

Marcel J.A. de Groot, Remko Offringa1, Mirjam P. Does1, Paul J.J. Hooykaas1 and Peter J.M. van den Elzen1

MOGEN International nv, Einsteinweg 97, 2333 CB Leiden 1Institute of Molecular Plant Sciences, Clusius Laboratory, Leiden University Wassenaarseweg 64, 2333 AL Leiden, The Netherlands

Received February 14, 1992. Revised May 4, 1992. Accepted May 4, 1992.

To elucidate the mechanism for intermolecular homologous recombination in plants we cotransformed Nicotiana tabacum cv Petit Havana SR1 protoplasts with constructs carrying different defective derivatives of the NPTII gene. The resulting kanamycin resistant clones were screened for possible recombination products by PCR, which proved to be a valuable technique for this analysis. Our results show that the double-stranded circular DNA molecules used in this study recombine predominantly via a pathway consistent with the single-strand annealing (SSA) model as proposed for extrachromosomal recombination in mammalian cells. In the remaining cases recombination occurred via a single reciprocal recombination, gene conversion and possibly double reciprocal recombination. Since single-stranded DNA is considered to be an important intermediate homologous recombination we also established the recombination ability of single-stranded DNA intermolecular recombination. We found that single-stranded DNA enters in recombination processes more efficiently than the corresponding double-stranded DNA. This was also reflected in the recombination mechanisms that generated the functional NPTII gene. Recombination between a single-stranded DNA and the complementing DNA duplex occurred at similar rates via a single reciprocal recombination and the SSA pathway.


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