Functional Centromeres Determine the Activation Time of Pericentric Origins of DNA Replication in Saccharomyces cerevisiae
DNA fragments containing a functional origin are detected as a bubble arc (depicted by the bubble fragment) while fragments that are passively replicated are detected as a Y-arc (depicted as a Y shaped fragment). (A) ARS1410 and ARS1426 are functional origins in the WT and rearranged strains. (B) 2D gel analysis of the MET2 or met2 locus in the WT and the rearranged strains. WT DNA was digested with the restriction enzyme StuI giving a fragment of 3.96 kb centered on MET2 (grey arrow). Rearranged DNA was double digested with XbaI and BsrBI resulting in a 4.36 kb fragment harboring most of met2, the integrated centromere (black circle), and the LEU2 marker (white rectangle). The absence of a bubble arc when probed for the 3′ end of MET2 and met2 (hashed rectangle) indicates that an origin is not present on either DNA fragment. The centromere in the rearranged construct was detected as a pause site (black arrowhead) visualized as a dot of relatively increased intensity on the descending Y-arc.