Daorong Feng

Cdc48p is required for the cell cycle commitment point at Start via degradation of the G1-CDK inhibitor Far1p

The budding yeast Cdc48p and its mammalian homologue p97 are involved in many important cellular activities. Because previous cdc48 mutants have exclusive G2/M arrest, Cdc48p was thought to play an essential role only during mitosis. We found that Cdc48p is required for the execution of Start (a yeast cell cycle commitment point equivalent to the restriction point in mammalian cells) in both a normal mitotic cell cycle and cell cycle reentry after mating pheromone withdrawal through degradation of the G1–cyclin-dependent kinase inhibitor Far1p. Our work is the first to uncover novel roles of Cdc48p as a critical cell cycle regulator in G1, and to shed new light on cell cycle regulation of Far1p, which is the first cyclin-dependent kinase inhibitor shown to be a substrate of an essential proteolysis event mediated by Cdc48p.

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

DNA replication in eukaryotic cells is tightly regulated to ensure faithful inheritance of the genetic material. While the replicators, replication origins and many replication-initiation proteins in Saccharomyces cerevisiae have been identified and extensively studied, the detailed mechanism that controls the initiation of DNA replication is still not well understood. It is likely that some factors involved in or regulating the initiation of DNA replication have not been discovered. To identify novel DNA replication-initiation proteins and their regulators, we developed a sensitive and comprehensive phenotypic screen by combining several established genetic strategies including plasmid loss assays with plasmids containing a single versus multiple replication origins and colony color sectoring assays. We isolated dozen of mutants in previously known initiation proteins and identified several novel factors, including Ctf1p Ctf3p, Ctf4p, Ctf18p, Adk1p and Cdc60p, whose mutants lose plasmid containing a single replication origin at high rates but lose plasmid carrying multiple replication origins at lower rates. We also show that overexpression of replication initiation proteins causes synthetic dosage lethality or growth defects in ctf1 and ctf18 mutants and that Ctf1p and Ctf18p physically interact with ORC, Cdt1p and MCM proteins. Furthermore, depletion of both Ctf1p and Ctf18p prevents S phase entry, retards S phase progression, and reduces pre-RC formation during the M-to-G1 transition. These data suggest that Ctf1p and Ctf18p together play important roles in regulating the initiation of DNA replication.