DNA polymerization-independent functions of DNA polymerase epsilon in assembly and progression of the replisome in fission yeast

Mol Biol Cell. 2012 Aug;23(16):3240-53. doi: 10.1091/mbc.E12-05-0339. Epub 2012 Jun 20.

Abstract

DNA polymerase epsilon (Pol ε) synthesizes the leading strands, following the CMG (Cdc45, Mcm2-7, and GINS [Go-Ichi-Nii-San]) helicase that translocates on the leading-strand template at eukaryotic replication forks. Although Pol ε is essential for the viability of fission and budding yeasts, the N-terminal polymerase domain of the catalytic subunit, Cdc20/Pol2, is dispensable for viability, leaving the following question: what is the essential role(s) of Pol ε? In this study, we investigated the essential roles of Pol ε using a temperature-sensitive mutant and a recently developed protein-depletion (off-aid) system in fission yeast. In cdc20-ct1 cells carrying mutations in the C-terminal domain of Cdc20, the CMG components, RPA, Pol α, and Pol δ were loaded onto replication origins, but Cdc45 did not translocate from the origins, suggesting that Pol ε is required for CMG helicase progression. In contrast, depletion of Cdc20 abolished the loading of GINS and Cdc45 onto origins, indicating that Pol ε is essential for assembly of the CMG complex. These results demonstrate that Pol ε plays essential roles in both the assembly and progression of CMG helicase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cdc20 Proteins
  • Cell Cycle Proteins / chemistry
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism
  • Chromosomal Proteins, Non-Histone / metabolism
  • DNA Helicases / metabolism
  • DNA Polymerase I / metabolism
  • DNA Polymerase II / genetics
  • DNA Polymerase II / metabolism
  • DNA Polymerase II / physiology*
  • DNA Replication
  • DNA-Binding Proteins / metabolism
  • Multiprotein Complexes / metabolism
  • Nuclear Proteins / metabolism
  • Protein Binding
  • Protein Interaction Domains and Motifs
  • Protein Multimerization*
  • Protein Subunits / genetics
  • Protein Subunits / metabolism
  • Protein Subunits / physiology
  • Replication Origin
  • Schizosaccharomyces / enzymology*
  • Schizosaccharomyces pombe Proteins / chemistry
  • Schizosaccharomyces pombe Proteins / genetics
  • Schizosaccharomyces pombe Proteins / metabolism
  • Sequence Deletion

Substances

  • CDC45 protein, S pombe
  • Cdc20 Proteins
  • Cell Cycle Proteins
  • Chromosomal Proteins, Non-Histone
  • DNA-Binding Proteins
  • Multiprotein Complexes
  • Nuclear Proteins
  • Protein Subunits
  • Schizosaccharomyces pombe Proteins
  • DNA Polymerase I
  • DNA Polymerase II
  • DNA Helicases