FIG. 7.

PCR analysis of the autointegration hot spot mediated by the cPPT. (A) The autointegration hot spot region adjacent to the cPPT (nucleotide 3782) is shown along with primers 4203c, 52130c, 5483c, and TyBout (5486) and one deletion circle (dotted line). DNA from strains expressing wild-type Ty1, several defective elements (in-2600, cppt-74, in-K596,597G, and the FS mutant), and an empty GAL1 vector was used. If the hot spot were adjacent to the cPPT and begins at Ty1 nucleotide ∼3808 (inferred from analyzing Ty1Zeo circles), PCR products of ∼827 bp (TyBout plus 4203c), ∼1,754 bp (TyBout plus 5130c), ∼2,107 bp (TyBout plus 5483c) should be amplified. (B) Genomic DNA from cells expressing wild-type, in-2600, cppt-74, FS, nuclear-localization-defective (in-K596,597G) Ty1 elements or an empty vector were used in PCRs with the primer pairs shown below, and the amplified fragments were separated by agarose gel electrophoresis. PCR products resulting from autointegration events associated with the cPPT are noted. The asterisks indicate PCR products in the expected size range. The positions of molecular size standards (in base pairs) are noted to the left of the leftmost gel. (C) The ∼827-bp PCR product resulting from amplification with primers TyBout plus 4203c was sequenced, and the autointegration insertion sites were compared with those obtained from sequencing Ty1Zeo circles (Fig. 5). The resolution is about 5 bp (see file S2 in the supplemental material for insertion sites). Also shown are the central Ty1 DNA flaps resulting from cPPT-initiated DNA synthesis and variable termination of the incoming plus strand at the CTS (rectangle), with 30% of strands terminating 30 to 50 nucleotides downstream of the cPPT (filled rectangle) (30).