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.
Ma
Lijuan
L
Sun Yat-sen University, Guangzhou, China.
Zhai
Yuanliang
Y
Feng
Daorong
D
Chan
Tsz-choi
TC
Lu
Yongjun
Y
Fu
Xinrong
X
Wang
Jiafeng
J
Chen
Yanhong
Y
Li
Jianna
J
Xu
Ke
K
Liang
Chun
C
D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D016681:Genome, Fungal; D018386:Kinetochores / Q000378:metabolism; D026341:Minichromosome Maintenance 1 Protein; D009154:Mutation; D051738:Origin Recognition Complex / Q000378:metabolism; D016196:S Phase; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism
C092693:CTF18 protein, S cerevisiae; C078338:CTF4 protein, S cerevisiae; D018797:Cell Cycle Proteins; C444703:Ctf3 protein, S cerevisiae; D004268:DNA-Binding Proteins; C498030:MCM1 protein, S cerevisiae; D026341:Minichromosome Maintenance 1 Protein; D051738:Origin Recognition Complex; D029701:Saccharomyces cerevisiae Proteins; C499592:TAH11 protein, S cerevisiae; D014157:Transcription Factors