Abstract
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.
Highlights
Which interact to form an active DNA helicase complex at origins that support duplex unwinding as well as the recruitment of DNA polymerases (Pol) and auxiliary factors required for DNA synthesis
We conclude that the SepB domain contributes importantly to the dimeric structure of human Ctf4 (hCtf4)
The size of DNA chains produced was increased significantly. These findings suggest that hCtf4, which binds to both primer-template ends and Pol ␣, may facilitate and/or stabilize the interaction of the polymerase with primer ends, resulting in enhanced processivity
Summary
Which interact to form an active DNA helicase complex at origins that support duplex unwinding as well as the recruitment of DNA polymerases (Pol) and auxiliary factors required for DNA synthesis. We describe the isolation of human Ctf and characterization of a number of its biochemical properties We show that it exists as a homodimer and interacts with primedtemplate junction DNA as well as with all three replicative polymerases, albeit with different affinities. This association, was not detected when cells progressed through mitosis but re-occurred when cells exited anaphase and entered telophase These findings indicate that hCtf plays an important and direct role in replication at the fork, most likely through its interactions with key replication proteins and replicative DNA polymerases
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