Cdc14p resets the competency of replication licensing by dephosphorylating multiple initiation proteins during mitotic exit in budding yeast

Abstract

SummaryIn eukaryotes, replication licensing is achieved through sequential loading of several replication-initiation proteins onto replication origins to form pre-replicative complexes (pre-RCs), and unscheduled replication licensing is prevented by cyclin-dependent kinases (CDKs) through inhibitory phosphorylations of multiple initiation proteins. It is known that CDK inactivation during mitotic exit promotes pre-RC formation for the next cell cycle. However, whether the removal of the inhibitory phosphorylations on the initiation proteins is essential and the identity of the acting phosphatase(s) remain unknown. Here, we show that cell division cycle protein 14 (Cdc14p) dephosphorylates replication-initiation proteins Orc2p, Orc6p, Cdc6p and Mcm3p to restore their competence for pre-RC assembly in the budding yeast Saccharomyces cerevisiae. Cells without functional Cdc14p fail to dephosphorylate initiation proteins and to form pre-RCs -even when CDK activities are suppressed -and cannot replicate DNA in mitotic rereplication systems, whereas pulsed ectopic expression of Cdc14p in mitotic cells results in efficient pre-RC assembly and DNA rereplication. Furthermore, Cdc14p becomes dispensable for DNA rereplication in mitotic cells with combined non-phosphorylatable and/or phosphorylation-insensitive alleles of the initiation proteins. These data unravel the essential role of Cdc14p in replication licensing, beyond its established functions in mitotic exit, providing new insight into the intricate regulation of DNA replication through the interplay of CDKs and the Cdc14p phosphatase.

Cdt1p, through its interaction with Mcm6p, is required for the formation, nuclear accumulation and chromatin loading of the MCM complex

“…p1ARS contains one replication origin ARS1, and p8ARSs carries seven additional copies of H4-ARS inserted into p1ARS. It has been well documented that all known replication initiation mutants have a higher rate of p1ARS loss compared with p8ARSs (Hogan and Koshland, 1992;Loo et al, 1995;Zhang et al, 2002;Cheng et al, 2010;Ma et al, 2010;Wang et al, 2010;Zhai et al, 2010). In control cells containing the empty BD vector, the loss rates of p1ARS and p8ARSs were 1.2% and 0.5% per generation, respectively (Fig.…”

Cell Cycle Control of DNA Replication by Phosphorylation and Dephosphorylation of Replication-Initiation Proteins in Budding Yeast

“…Altogether, these events promote pre-RC assembly in a step-wise manner onto replication origins for replication initiation. Phosphorylated, inactive Cdc14p substrates are shaded gray, while their dephosphorylated, active forms are highlighted in red (substrates identified by us in Zhai et al, 2010) or orange (previously identified substrates). Modified from Zhai et al, 2010.…”

Identification of novel factors involved in or regulating initiation of DNA replication by a genome-wide phenotypic screen inSaccharomyces cerevisiae

“…27 In fact, we identified several additional idr mutants in CDC14 from the tertiary screen and further found that the phosphatase Cdc14p indeed plays an essential role in replication initiation by directly dephosphorylating several replication initiation proteins. 31 The idr mutants that could not be rescued by any of the known IDR genes were then transformed with a yeast genomic library 32 in order to clone the candidate IDR genes by complementation of the p1ARS plasmid loss phenotypes. Two methods were used to identify colonies containing the complementing library plasmids.…”

“…To further examine the genetic interactions of CTF1 and CTF18 with replication related genes, we utilized a genetic assay, synthetic dosage lethality, 31,40,41 to test cell growth defects in ctf1 and ctf18 mutants upon overexpression of individual known replication related genes, ORC1-6, MCM2-7, CDT1, CDC7, DBF4, CDC45, SLD2, SLD3, DPB11 and POL12. The results (see summary on Table 2 and data examples in Fig.…”