Ctf4/AND-1 is a highly conserved gene product required for both DNA replication 
and the establishment of sister chromatid cohesion. In this report, we examined 
the mechanism of action of human Ctf4 (hCtf4) in DNA replication both in vitro 
and in vivo. Our findings show that the purified hCtf4 exists as a dimer and 
that the hCtf4 SepB domain likely plays a primary role determining the dimeric 
structure. hCtf4 binds preferentially to DNA template-primer structures, 
interacts directly with the replicative DNA polymerases (alpha, delta, and 
epsilon), and markedly stimulates the polymerase activities of DNA polymerases 
alpha and epsilon in vitro. Depletion of hCtf4 in HeLa cells by small 
interfering RNA resulted in G(1)/S phase arrest. DNA fiber analysis revealed 
that cells depleted of hCtf4 exhibited a rate of DNA replication slower than 
cells treated with control small interfering RNA. These findings suggest that in 
human cells, hCtf4 plays an essential role in DNA replication and its ability to 
stimulate the replicative DNA polymerases may contribute to this effect.