ATP-dependent Pre-replicative Complex Assembly Is Facilitated by Adk1p in Budding Yeast

Abstract

Pre-replicative complex (pre-RC) assembly is a critical part of the mechanism that controls the initiation of DNA replication, and ATP binding and hydrolysis by multiple pre-RC proteins are essential for pre-RC assembly and activation. Here, we demonstrate that Adk1p (adenylate kinase 1 protein) plays an important role in pre-RC assembly in Saccharomyces cerevisiae. Isolated from a genetic screen, adk1 G20S cells with a mutation within the nucleotide-binding site were defective in replication initiation. adk1⌬ cells were viable at 25°C but not at 37°C. Flow cytometry indicated that both the adk1-td (temperature-inducible degron) and adk1 G20S mutants were defective in S phase entry. Furthermore, Adk1p bound to chromatin throughout the cell cycle and physically interacted with Orc3p, whereas the Adk1 G20S protein had a reduced ability to bind chromatin and Orc3p without affecting the cellular ATP level. In addition, Adk1p associated with replication origins by ChIP assay. Finally, Adk1-td protein depletion prevented pre-RC assembly during the M-to-G 1 transition. We suggest that Adk1p regulates ATP metabolism on pre-RC proteins to promote pre-RC assembly and activation.The initiation of DNA replication governs the genome duplication in eukaryotes, which occurs once and only once per cell cycle. In Saccharomyces cerevisiae, replication initiation is critically controlled by pre-replicative complex (pre-RC) 3 assembly, which includes sequential loading of origin recognition complex (ORC) (1-3), Noc3p (4), Cdc6p (5-8), Cdt1p (9), and minichromosome maintenance (MCM) proteins (Mcm2-7p) (10, 11) onto autonomously replicating sequence (ARS) elements. In addition, ATP binding to and hydrolysis by Cdc6p and several subunits of ORC are essential for pre-RC assembly (12-15). At least 10 components (Orc1p, Orc4p, Orc5p, Cdc6p, and Mcm2-7p) of pre-RCs are ATPases (16) containing the Walker A and B motifs required for ATP binding and hydrolysis.Mutation analysis showed that all conserved ATP-binding motifs of these proteins are essential for cell viability (12,17,18).There have been numerous reports on the roles of binding and hydrolysis of ATP in pre-RC assembly. In S. cerevisiae, ATP binding of Orc1p activates ORC assembly and ORC binding to replication origins (1). Mutation within the Orc1p ATP-binding site prevents ORC binding to DNA in vitro and is lethal in vivo (19). Other studies indicated that mutations in the Walker A motif eliminate the ATP-binding and hydrolysis activities of Orc1p (20,21). When ATP binds to Orc1p, an initial round of chromatin loading of MCM proteins is permitted, whereas ATP hydrolysis is required for other rounds of MCM loading in vitro (13,22). Human ORC assembly in vitro is dependent on ATP binding and impaired by mutations in Orc4p or Orc5p ATP-binding sites (23,24). In S. cerevisiae, Cdc6p binding to ORC, which is dependent on the Cdc6p ATPase activity, changes the ORC structure and contributes to pre-RC assembly (25). Cdc6p mutated in the Walker B motif cannot interact with Orc1p in vitro ...

Mitotic entry in the presence of DNA damage is a widespread property of aneuploidy in yeast

“…Replication-initiation defects can be detected by comparing the frequency of loss of a plasmid carrying one origin of replication (ARS) with that of a plasmid carrying eight ARS sequences ( Hogan and Koshland, 1992 ; Cheng et al ., 2010 ). Indeed, cells harboring a temperature-sensitive allele in the gene encoding the replication initiation factor Cdc6 lose plasmids with a single ARS at a much higher frequency than plasmids with eight ARSs ( Figure 3A ; Hogan and Koshland, 1992 ).…”

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.…”

The Rix1 (Ipi1p-2p-3p) complex is a critical determinant of DNA replication licensing independent of their roles in ribosome biogenesis

“…4,[24][25][26][27][28][29] However, the genetic background (ade2 ADE3) of the td strains is not suitable for the (ade2 ade3)-based colony color assay that we used to screen for p1ARS plasmid loss mutants in another strain background. [26][27][28][29] Therefore, we developed a colony size assay to identify candidates with high rates of plasmid loss. Since the td strains are leucine auxotrophic due to the leu2 mutation and hence cannot grow on SCM-Leu (synthetic complete medium lacking leucine) plates without the LEU2-containing p1ARS or p8ARSs plasmid, replication initiation mutants should form small colonies on SCM-Leu plates when they carry p1ARS but bigger colonies with p8ARSs.…”