Functional Centromeres Determine the Activation Time of Pericentric Origins of DNA Replication in Saccharomyces cerevisiae
(A) Cartoon depiction of chromosome XIV with a non-functional centromere (white circle) integrated at MET2. Chromosome XIV of WT cells was modified such that MET2 was disrupted with the same sequence used to disrupt MET2 in the rearranged strain (see Figure 1) except that the centromere was made inactive by mutating the essential CDEIII domain. This chromosome is maintained through its wild type centromere at the endogenous location (black circle). (B) Flow cytometry of cells with a non-functional centromere in the MET2 locus. Similar to the WT strain (see Figure 2B), cells from this cell line enter S-phase by 40 minutes and achieved 2C DNA content by 140 minutes. (C) Replication kinetic curves for met2::cen7 and ARS1410. As observed in the WT strain, the replication curves for met2::cen7 (green) and ARS1410 (blue) are positioned more closely to that of R11 (dotted line) than ARS306 (dashed line) (compare to Figure 2C). D) Replication indices for met2:cen7 (green diamond) and ARS1410 (blue diamond). RIs of ARS306 and R11 are indicated by black dashed and dotted arrows, respectively. met2:cen7 and ARS1410 had RIs of 0.81 and 0.74, respectively. (E) 2D gel analysis of ARS1410. Presence of a bubble arc (black arrow) for ARS1410 in the strain in which the non-functional centromere was integrated at MET2 (compare with Figure 1B) indicates that ARS1410 is a functional origin in this strain.