DNA integration by Ty integrase in yku70 mutant 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.

FIG. 1

Sequence elements identified adjacent to pM151 sequences after analysis of rescued plasmids. Indicated are the areas sequenced by using primer P1 (on the left side of the plasmid) and primer P2 (on the right side). For better comparison, different sequence element types (unique, rDNA, LTR, tRNA pbs, and Ty ORF) are differentiated by shading types (see the key); elements were not drawn to scale. Numbers below the elements refer to SGD genomic coordinates, while coordinates given above the Ty elements refer to those Ty1 element sequences which gave the best fit in BLASTN analysis (see Table 1). The numbers of nucleotides missing from the 5′ protruding single-stranded plasmid ends are given just above the plasmid junctions. Asterisks indicate samples obtained after irradiation with 50 Gy. (A) Integration event probably mediated by topoisomerase I. (B) Three plasmid integrations without cointegrating Ty sequences. These events were most likely mediated by Ty integrase, since they created a 5-bp target site duplication. (C) Apparent cointegration events of pM151 and Ty sequences. For MK26 the junction between chromosomal DNA and cointegrating Ty sequences is also shown. RDN, rDNA.

FIG. 2

Nucleotide sequences of junctions between plasmid and chromosomal or Ty1-derived DNA. Sequences determined after plasmid rescue (middle row) are depicted in 5′-to-3′ orientation and aligned with the plasmid sequence (top row) and chromosomal or Ty1-derived sequences (bottom row). Sequence coordinates are given as described for Fig. 1. For MK26, the junction between chromosome VII DNA and Ty1-derived sequences is also given. Underlined sequences indicate confirmed 5-bp duplications at chromosomal target sites. Shading in the MK23 sequence indicates a putative topoisomerase I target site.

FIG. 3

Locations of junctions between pM151 and those Ty1 sequences that could not be demonstrated to have preexisted at the integration site, relative to a standard Ty1 element.