Studies on Human DNA Polymerase ε and GINS Complex and Their Role in DNA Replication

In eukaryotic cells, DNA replication is carried out by the coordinated action of three DNA polymerases (Pols), Pol alpha, delta, and epsilon. In this report, we describe the reconstitution of the human four-subunit Pol epsilon and characterization of its catalytic properties in comparison with Pol alpha and Pol delta. Human Pol epsilon holoenzyme is a monomeric complex containing stoichiometric subunit levels of p261/Pol 2, p59, p17, and p12. We show that the Pol epsilon p261 N-terminal catalytic domain is solely responsible for its ability to catalyze DNA synthesis. Importantly, human Pol (hPol) epsilon was found more processive than hPol delta in supporting proliferating cell nuclear antigen-dependent elongation of DNA chains, which is in keeping with proposed roles for hPol epsilon and hPol delta in the replication of leading and lagging strands, respectively. Furthermore, GINS, a component of the replicative helicase complex that is composed of Sld5, Psf1, Psf2, and Psf3, was shown to interact weakly with all three replicative DNA Pols (alpha, delta, and epsilon) and to markedly stimulate the activities of Pol alpha and Pol epsilon. In vivo studies indicated that siRNA-targeted depletion of hPol delta and/or hPol epsilon reduced cell cycle progression and the rate of fork progression. Under the conditions used, we noted that depletion of Pol epsilon had a more pronounced inhibitory effect on cellular DNA replication than depletion of Pol delta. We suggest that reduction in the level of Pol delta may be less deleterious because of its collision-and-release role in lagging strand synthesis.