GINS Is a DNA Polymerase ϵ Accessory Factor during Chromosomal DNA Replication in Budding Yeast

Takashi Seki,Masaki Akita,Yoichiro Kamimura,Sachiko Muramatsu,Hiroyuki Araki,Akio Sugino

doi:10.1074/jbc.m603482200

Abstract

GINS is a protein complex found in eukaryotic cells that is composed of Sld5p, Psf1p, Psf2p, and Psf3p. GINS polypeptides are highly conserved in eukaryotes, and the GINS complex is required for chromosomal DNA replication in yeasts and Xenopus egg. This study reports purification and biochemical characterization of GINS from Saccharomyces cerevisiae. The results presented here demonstrate that GINS forms a 1:1 complex with DNA polymerase epsilon (Pol epsilon) holoenzyme and greatly stimulates its catalytic activity in vitro. In the presence of GINS, Pol epsilon is more processive and dissociates more readily from replicated DNA, while under identical conditions, proliferating cell nuclear antigen slightly stimulates Pol epsilon in vitro. These results strongly suggest that GINS is a Pol epsilon accessory protein during chromosomal DNA replication in budding yeast. Based on these results, we propose a model for molecular dynamics at eukaryotic chromosomal replication fork.

Highlights

Three structurally and functionally distinct DNA polymerases, known as DNA polymerases ␣, ␦, and ⑀, are required for chromosomal DNA replication in budding yeast [1, 2]
Dpb2p, Mcm10p, Cdc45p, and Mcm2p co-immunoprecipitated with 3ϫ FLAGtagged Psf2p (Fig. 1B). These results suggest that Pol ⑀, Cdc45p, Mcm2p, and Mcm10p form a stable complex with GINS in vivo during S phase
This study shows that purified GINS forms a 1:1 complex with Pol ⑀, but not other replicases, and that GINS stimulates Pol ⑀-catalyzed DNA synthesis in vitro

Summary

Introduction

Three structurally and functionally distinct DNA polymerases, known as DNA polymerases ␣, ␦, and ⑀, are required for chromosomal DNA replication in budding yeast [1, 2]. The results presented here demonstrate that GINS forms a 1:1 complex with DNA polymerase ⑀ (Pol ⑀) holoenzyme and greatly stimulates its catalytic activity in vitro.

Results

Conclusion