Identification and cloning of two putative subunits of DNA polymerase epsilon 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.

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Faculty Opinions recommendation of Yeast DNA polymerase epsilon participates in leading-strand DNA replication.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1088313.541348 ◽

2007 ◽

Author(s):

John Rouse

Keyword(s):

Dna Replication ◽

Dna Polymerase ◽

Dna Polymerase Epsilon ◽

Leading Strand ◽

Polymerase Epsilon

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Effects of the anticancer drug cis-diamminedichloroplatinum(II) on the activities of calf thymus DNA polymerase .epsilon.

Biochemistry ◽

10.1021/bi00054a015 ◽

1993 ◽

Vol 32 (3) ◽

pp. 841-848 ◽

Cited By ~ 17

Author(s):

Lin Huang ◽

John J. Turchi ◽

Alan F. Wahl ◽

Robert A. Bambara

Keyword(s):

Dna Polymerase ◽

Anticancer Drug ◽

Calf Thymus Dna ◽

Dna Polymerase Epsilon ◽

Calf Thymus ◽

Polymerase Epsilon

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DNA Polymerase Epsilon Catalytic Subunit A

10.32388/nhd93x ◽

2020 ◽

Author(s):

Keyword(s):

Dna Polymerase ◽

Catalytic Subunit ◽

Subunit A ◽

Dna Polymerase Epsilon ◽

Polymerase Epsilon

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Analysis of APOBEC-induced mutations in yeast strains with low levels of replicative DNA polymerases

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1922472117 ◽

2020 ◽

Vol 117 (17) ◽

pp. 9440-9450 ◽

Cited By ~ 6

Author(s):

Yang Sui ◽

Lei Qi ◽

Ke Zhang ◽

Natalie Saini ◽

Leszek J. Klimczak ◽

...

Keyword(s):

Dna Polymerase ◽

Dna Polymerases ◽

Induced Mutations ◽

Single Stranded Dna ◽

Dna Polymerase Epsilon ◽

Dna Polymerase Alpha ◽

Yeast Strains ◽

Polymerase Epsilon ◽

Low Levels ◽

Lagging Strand

Yeast strains with low levels of the replicative DNA polymerases (alpha, delta, and epsilon) have high levels of chromosome deletions, duplications, and translocations. By examining the patterns of mutations induced in strains with low levels of DNA polymerase by the human protein APOBEC3B (a protein that deaminates cytosine in single-stranded DNA), we show dramatically elevated amounts of single-stranded DNA relative to a wild-type strain. During DNA replication, one strand (defined as the leading strand) is replicated processively by DNA polymerase epsilon and the other (the lagging strand) is replicated as short fragments initiated by DNA polymerase alpha and extended by DNA polymerase delta. In the low DNA polymerase alpha and delta strains, the APOBEC-induced mutations are concentrated on the lagging-strand template, whereas in the low DNA polymerase epsilon strain, mutations occur on the leading- and lagging-strand templates with similar frequencies. In addition, for most genes, the transcribed strand is mutagenized more frequently than the nontranscribed strand. Lastly, some of the APOBEC-induced clusters in strains with low levels of DNA polymerase alpha or delta are greater than 10 kb in length.

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