Ectopic induction of Clb2 in early G1 phase is sufficient to block prereplicative complex formation in Saccharomyces cerevisiae - PubMed (original) (raw)

Ectopic induction of Clb2 in early G1 phase is sufficient to block prereplicative complex formation in Saccharomyces cerevisiae

C S Detweiler et al. Proc Natl Acad Sci U S A. 1998.

Abstract

Eukaryotic cells ensure the stable propagation of their genome by coupling each round of DNA replication (S phase) to passage through mitosis (M phase). This control is exerted at the initiation of replication, which occurs at multiple origins throughout the genome. Once an origin has initiated, reinitiation is blocked until the completion of mitosis, ensuring that DNA is replicated at most once per cell cycle. Recent studies in several organisms have suggested a model in which S- and M-phase promoting cyclin-dependent kinases prevent reinitiation by blocking the repetition of an early step in the initiation reaction. In budding yeast, this regulation is thought to involve inhibition of prereplicative complex (pre-RC) formation at origins by S and M phase-promoting Clb kinases. To date, however, there has been no direct demonstration that these kinases can perform such an important function. In this report we provide such a confirmation by showing that ectopic induction in G1 phase of a mitotic Clb, Clb2, is sufficient to inhibit DNA replication and does so by preventing pre-RC formation. This inhibition requires that Clb2 be induced before Cdc6, an initiation protein required for pre-RC formation; once pre-RCs have formed, Clb2 can no longer inhibit initiation. These results support the notion that during the normal cell cycle reassembly of the pre-RC, and hence reinitiation at an origin, is directly inhibited by S and M phase-promoting cyclin-dependent kinases.

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Figures

Figure 1

Figure 1

Induction of Clb2db 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.

Figure 2

Figure 2

Induction of Clb2db in G1 before Cdc6 expression is sufficient to block pre-RC formation. Metp-CDC6 (yJL1169, lanes 1–4) and Metp-CDC6 Galp-clb2_db (yJL1167, lanes 5–7) cells were subjected to induction protocol 1 as described in Fig. 1_B. Samples for footprint analysis of the 2 micron replication origin were taken at the second alpha factor arrest before Clb2db induction (lanes 1, 2, 5, and 6) and after Cdc6 induction (lanes 3, 4, 7, and 8). Each sample was treated with two different concentrations of DNase I and loaded in adjacent lanes. The ORC-induced hypersensitive site characteristic of the the postreplicative footprint is indicated by the arrow.

Figure 3

Figure 3

Clb2db does not inhibit Cdc6 by preventing Cdc6 accumulation. _Metp-(HA)_6_-CDC6 GAL-clb2_db (yJL1172, lanes 2–10) and Metp-(HA)6_-CDC6 (yJL1173, lanes 11–19) cells were subjected to induction protocol I as depicted in Fig. 1_B but were not released from the arrest so that (HA)6-Cdc6 accumulation (arrowhead) could be monitored by Western blot analysis (lanes 2, 3, 7, and lanes 11, 12, 16). Thirty and sixty minutes after induction of the (HA)6-Cdc6, 2 mM methionine was added back to a portion of the cultures to repress _(HA)6_-CDC6 expression; proteins levels were monitored at 15 min intervals by immunoblotting (arrowhead, lanes 4–6, 8–10, 13–15, 17–19). Lane 1, CDC6 (YJL312) cells grown in SCR-Leu. Sec61 was immunoblotted as an internal loading control (arrow).

Figure 4

Figure 4

A model for Clb/Cdc28 kinase inhibition of rereplication. In G1 phase when Clb/Cdc28 is absent, Cdc6 and Mcm proteins join ORC to form the pre-RC at origins. Activation of Clb/Cdc28 kinase activity late in G1 triggers replication initiation and disassembly of the pre-RCs. The continual presence of Clb/Cdc28 during S, G2, and M phases also blocks reassembly of the pre-RC, preventing reinitiation until the Clbs are destroyed at the end of mitosis.

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