We describe a cell-free system from HeLa cells that initiates DNA repl
ication under cell cycle control. G1 but not G2 phase nuclei initiate
replication when coincubated with S phase nuclei in cytosolic extracts
from S phase but not from G1 or G2 phase HeLa cells. S phase nuclei o
r an S phase nuclear extract are required for the initiation of semico
nservative DNA replication in G1 nuclei but not for elongation in S ph
ase nuclei. S phase nuclear extract could be replaced by recombinant h
uman cyclins A and E complexed to Cdk2 but not by Cdk2 alone or by hum
an cyclin B1 complexed to CdcP. In S phase cytosol, cyclins A/Cdk2 and
E/Cdk2 triggered initiation synergistically.