Identification of minimal in vitro targeted strand transfer components.
A, in vitro strand-transfer assay. Reactions contained VLPs as a source of Ty3 cDNA and IN and TFIIIB or TFP (Brf11–382-TBP61–240-Brf1439–596). A functionally symmetric TATA box (ATAT) binds TBP in either orientation allowing bidirectional transcription initiation (L and R arrows). Strand-transfer products were detected in PCR primed from within Ty3 and SNR6 (primers 411 and 242, respectively). DNA recovery was monitored with PCR using primers annealed to the backbone of the target plasmid (primers 679 and 680). B, TFP substitutes for TFIIIB in targeting Ty3 strand transfer. Strand-transfer reactions with test reactants (lanes 1–3), positive control (P) plasmids with a Ty3 LTR at leftward (pLY1842) and rightward TSS (pDLC370) (lane 4), or negative control (N) containing water alone (lane 5) were used to template PCR using primers 411 and 242. PCR products were separated on a nondenaturing 8% polyacrylamide gel. Products amplified from leftward (L) and rightward (R) TSS are indicated.