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

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

“…Similar to all known initiation mutants reported, the plasmid loss of cdc14-1 cells could be suppressed by adding multiple copies of an ARS to the plasmid (Hogan & Koshland, 1992;Ma et al, 2010). Overexpression of the initiation protein Orc6p caused synthetic dosage lethality in cdc14-1 cells (Kroll et al, 1996), and synthetic lethality was observed when cdc14-1 was combined with cdc6-1, orc2-1 or orc5-1 (Loo et al, 1995;Kroll et al, 1996;Yuste-Rojas & Cross, 2000).…”

“…In an effort to identify novel factors that are involved in or regulate DNA replication initiation, our lab has carried out a yeast phenotypic screen with randomly mutagenized yeast cells and have obtained several new hypomorphic alleles of cdc14 mutants as well as mutants in other genes (Ma et al, 2010). These new cdc14 mutants, like the previous cdc14-1 (Hogan & Koshland, 1992) and all known replication-initiation mutants reported, have high rates of plasmid loss that can be suppressed by the presence of multiple copies of an ARS on the plasmid (Zou et al, 1997;Tye, 1999;Zhang et al, 2002;Ma et al, 2010). These results and the previous genetic data described above prompted us to further examine the role of Cdc14p in DNA replication licensing.…”

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 quantitative plasmid loss assay was performed as previously described (Ma et al, 2010;Zhang et al, 2002) with minor modifications. Cells containing p1ARS or p8ARSs were grown to early log phase (2610 6 cells/ml) in SCM -Trp -Leu medium at 25˚C, and plated on SCM -Trp or SCM -Trp -Leu for the initial time point.…”

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

“…In an effort to identify factors that are involved in or regulate DNA replication initiation, we have previously carried out a phenotypic screen with yeast cells that were randomly mutagenized (Cheng et al, 2010;Ma et al, 2010;Wang et al, 2010). From this screen, we isolated several new hypomorphic alleles of cdc14 mutants that, like cdc14-1 (Hogan and Koshland, 1992) and all known replication-initiation mutants previously tested (Loo et al, 1995;Zou et al, 1997;Tye, 1999;Zhang et al, 2002), have high rates of plasmid loss that can be rescued by adding multiple copies of an ARS to the plasmid (Ma et al, 2010).…”