Figure 2.

DNA sequences that recruit RNA pol III transcription factors can also stimulate replication from a chromosomal ARS1 site. (A) Two-dimensional gel analysis of chromosomal ARS1 replication intermediates generated in the following yeast strains: (1) RL1 expressing Gal4(1–94); (2) YKE1, a derivative of RL1 in which the Gal4-binding sites near ARS1 have been replaced by the pol III B-SNR6 gene cassette; (3) YKE2, which contains the UAS_G-SNR6 cassette. The ARS1 regions of these strains (1–3) are schematically drawn to scale. The bubble arc is indicated by an arrow and is formed by replication intermediates that initiated from ARS1. (B) Primer extension analysis of the B-SNR6 and UAS_G-SNR6 gene products reveals that stimulation of replication by these pol III gene cassettes is not dependent on the transcription process. The endogenous wild-type SNR6 gene of both the YKE1 and YKE2 strains was replaced by a 6 bp shorter SNR6 sequence (SNR6-6) to allow the detection of transcripts specifically synthesized from the pol III gene cassettes near the ARS1 locus. Primer extension analysis of the RL1 endogenous SNR6 gene products served as a positive control for expression and size of the full-length U6 RNA.