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.