DNA Integration by Ty Integrase in yku70Mutant Saccharomyces cerevisiae Cells

Abstract

In the present work we examined nonhomologous integration of plasmid DNA in a yku70 mutant. Ten of 14 plasmids integrated as composite elements, including Ty sequences probably originating from erroneous strand-switching and/or priming events. Three additional plasmids integrated via Ty integrase without cointegrating Ty sequences, as inferred from 5-bp target site duplication and integration site preferences. Ty integrase-mediated integration of non-Ty DNA has never been observed in wild-type cells, although purified integrase is capable of using non-Ty DNA as a substrate in vitro. Hence our data implicate yKu70 as the cellular function preventing integrase from accepting non-Ty DNA as a substrate.

ACKNOWLEDGMENTS

We thank David Garfinkel for useful discussions and comments on the manuscript. We also thank Dan Gietz and Wolfram Siede for plasmids. Sequencing was generously provided by Genome Therapeutics.

This research was supported by grant CN-83 from the American Cancer Society and Research Career Development Award ES00299 from the National Institute of Environmental Health Sciences to R.H.S., by grant FI4P-CT95-0010 from the European Commission to F.E.-S., and by travel grants from the Neuherberger Forschungsfoerderung to M.K.

M.K. and A.A.F. contributed equally to this work.