DNA polymerization-independent functions of DNA polymerase epsilon in assembly and progression of the replisome in fission yeast.
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.
Graduate School of Science, Osaka University, Osaka, Japan.
Tetsuya
T
Handa
Mai
M
Kanke
Tatsuro S
TS
Takahashi
Takuro
T
Nakagawa
Hisao
H
Masukata
D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D004265:DNA Helicases / Q000378:metabolism; D004256:DNA Polymerase I / Q000378:metabolism; D004257:DNA Polymerase II / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism; D046912:Multiprotein Complexes / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D018741:Replication Origin; D012568:Schizosaccharomyces / Q000201:enzymology*; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion
C116395:CDC45 protein, S pombe; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D046912:Multiprotein Complexes; D009687:Nuclear Proteins; D021122:Protein Subunits; D029702:Schizosaccharomyces pombe Proteins; D004256:DNA Polymerase I; D004257:DNA Polymerase II; D004265:DNA Helicases