The DNA polymerase activity of Pol ε holoenzyme is required for rapid and efficient chromosomal DNA replication in Xenopus egg extracts

Koh Shikata,Taro Sasa-Masuda,Shou Waga,Akio Sugino,Yukiko Okuno

doi:10.1186/1471-2091-7-21

Abstract

BackgroundDNA polymerase ε (Pol ε) is involved in DNA replication, repair, and cell-cycle checkpoint control in eukaryotic cells. Although the roles of replicative Pol α and Pol δ in chromosomal DNA replication are relatively well understood and well documented, the precise role of Pol ε in chromosomal DNA replication is not well understood.ResultsThis study uses a Xenopus egg extract DNA replication system to further elucidate the replicative role(s) played by Pol ε. Previous studies show that the initiation timing and elongation of chromosomal DNA replication are markedly impaired in Pol ε-depleted Xenopus egg extracts, with reduced accumulation of replicative intermediates and products. This study shows that normal replication is restored by addition of Pol ε holoenzyme to Pol ε-depleted extracts, but not by addition of polymerase-deficient forms of Pol ε, including polymerase point or deletion mutants or incomplete enzyme complexes. Evidence is also provided that Pol ε holoenzyme interacts directly with GINS, Cdc45p and Cut5p, each of which plays an important role in initiation of chromosomal DNA replication in eukaryotic cells.ConclusionThese results indicate that the DNA polymerase activity of Pol ε holoenzyme plays an essential role in normal chromosomal DNA replication in Xenopus egg extracts. These are the first biochemical data to show the DNA polymerase activity of Pol ε holoenzyme is essential for chromosomal DNA replication in higher eukaryotes, unlike in yeasts.

Highlights

DNA polymerase ε (Pol ε) is involved in DNA replication, repair, and cell-cycle checkpoint control in eukaryotic cells
Isolation of cDNA encoding each subunit of Xenopus Pol ε holoenzyme A previous study reported cloning of a partial cDNA for the catalytic subunit (p260) of Xenopus Pol ε [24], and in this report, the corresponding full-length cDNA was cloned by 5' Rapid amplification of cDNA ends (5' Race)
Because the former enzymes are polymerase proficient, while p260∆Cat holoenzyme and p260 DN are polymerasedeficient, these preparations contained a small amount of DNA polymerase activity, and the last two subcomplexes do not contain the second essential subunit of Xenopus Pol ε (xPol ε) holoenzyme (Table 1), these results clearly demonstrate that the DNA polymerase activity of Pol ε holoenzyme is required for chromosomal DNA replication in Xenopus egg extracts

Summary

Introduction

DNA polymerase ε (Pol ε) is involved in DNA replication, repair, and cell-cycle checkpoint control in eukaryotic cells. Three structurally and functionally distinct DNA polymerases, known as DNA polymerases α, δ, and ε -ε, respectively), are required for chromosomal DNA replication in yeasts [1,2,3]. The complex structure of each Pol α, -δ, and -ε is well conserved from yeast to human [4],. DNA primase initiates DNA replication by synthesizing a short oligo-ribonucleotide primer which is immediately elongated by Pol α to form short RNA-DNA fragments on both leading and lagging strand of DNA. To carry out processive DNA synthesis in vitro, Pol δ requires PCNA and its loader, Replication Factor-C (RF-C) [5]. In cooperation with Fen1p (Rad27p), Dna2p and RPA, Pol δ plays a crucial role in processing RNA-linked Okazakifragments in budding yeast [5]

Methods

Results

Discussion

Conclusion