Figure 1.

Induction of Clb2^db in G₁ before Cdc6 expression is sufficient to block DNA replication. (A) Comparison of events in normal cell cycle (Upper) and experimentally manipulated cell cycle (Lower). In the normal cell cycle, disappearance of Clb kinase activity and induction of Cdc6 after mitosis leads to assembly of the pre-RC before START. Clb kinase activity is induced after START, contributing to the triggering of initiation, the entry into S phase, and the apparent disassembly of the pre-RC. To examine what happens if Clb kinase activity appears before pre-RC formation, Clb kinase was prematurely induced before Cdc6 induction in cells arrested before START. (B) Experimental protocol (see Materials and Methods for details). Met3p-CDC6 Gal1p-clb2db and Met3p-CDC6 cells were released from alpha factor arrest into medium containing methionine. When most of the cells were in G₂, alpha factor was added back to rearrest the cells in the next cell cycle. At this second arrest either galactose was added before transferring the cells to methionine-free medium (protocol 1) or cells were transferred to methionine-free medium before galactose was added (protocol 2). Following these inductions, cells released from the arrest by addition of pronase (Om). (C) Flow cytometry of cells sampled at times indicated by min (m) in B. Column a: YJL1169 (Metp-CDC6) induction protocol 1. Column b: YJL1167 (Metp-CDC6, Galp-clb2^db), induction protocol 1. Column c: YJL1167 (Metp-CDC6, Galp-clb2^db), induction protocol 2.