Schizosaccharomyces pombe Cells Lacking the Amino-Terminal Catalytic Domains of DNA Polymerase Epsilon Are Viable but Require the DNA Damage Checkpoint Control

Abstract

In Schizosaccharomyces pombe, the catalytic subunit of DNA polymerase epsilon (Pol ) is encoded by cdc20 ؉ and is essential for chromosomal DNA replication. Here we demonstrate that the N-terminal half of Pol that includes the highly conserved polymerase and exonuclease domains is dispensable for cell viability, similar to observations made with regard to Saccharomyces cerevisiae. However, unlike budding yeast, we find that fission yeast cells lacking the N terminus of Pol (cdc20 ⌬N-term ) are hypersensitive to DNA-damaging agents and have a cell cycle delay. Moreover, the viability of cdc20 ⌬N-term cells is dependent on expression of rad3 ؉ , hus1 ؉ , and chk1 ؉ , three genes essential for the DNA damage checkpoint control. These data suggest that in the absence of the N terminus of Pol , cells accumulate DNA damage that must be repaired prior to mitosis. Our observation that S phase occurs more slowly for cdc20⌬N-term cells suggests that DNA damage might result from defects in DNA synthesis. We hypothesize that the C-terminal half of Pol is required for assembly of the replicative complex at the onset of S phase. This unique and essential function of the C terminus is preserved in the absence of the N-terminal catalytic domains, suggesting that the C terminus can interact with and recruit other DNA polymerases to the site of initiation.

Schizosacchromyces pombeDpb2 Binds to Origin DNA Early in S Phase and Is Required for Chromosomal DNA Replication

“…Pol ⑀ has also been implicated in both DNA repair (Jessberger et al, 1993;Wang et al, 1993;Shivji et al, 1995;Holmes, 1999) and cell cycle checkpoint control in eukaryotic cells (Navas et al, 1995). Recently, we have shown that the DNA polymerase and exonuclease domains of Pol ⑀ are dispensable for cell viability in fission yeast (Feng and D'Urso, 2001). Similar observations have been made for the evolutionarily distant yeast, Saccharomyces cerevisiae (Kesti et al, 1999;Dua et al, 2000).…”

“…It should be noted that ChIP analysis is not sensitive enough to detect enrichment of Dpb2 or Mcm6 binding to origin DNA in exponentially growing populations where very few cells are in S phase. Therefore, considering that the amount of Dpb2 association to chromatin detected in cdc10-arrested cells is similar to the amount detected in either G2-arrested (cdc25) or exponentially growing 972 cells, we conclude that very little, if any, Dpb2 protein is bound to chromatin in the absence of Cdc10.In a previous study, we demonstrated that fission yeast cells are viable in the absence of the N-terminal, but not C-terminal half of the enzyme (Feng and D'Urso, 2001). This result was surprising because the N terminus of Pol ⑀ contains all the domains necessary for polymerase activity.…”

“…In a previous study, we demonstrated that fission yeast cells are viable in the absence of the N-terminal, but not C-terminal half of the enzyme (Feng and D'Urso, 2001). This result was surprising because the N terminus of Pol ⑀ contains all the domains necessary for polymerase activity.…”

“…Interestingly, the essential features of the Pol ⑀ catalytic subunit do not reside in the N-terminal half of the enzyme that contains the highly conserved DNA polymerase domains, but rather in the C-terminal half of the enzyme where there are no known catalytic functions (Fuss and Linn, 2002). The observation that mutants in Pol ⑀ arrest in late G1 or early S phase combined with the fact that the enzyme's polymerase activity is dispensable, led us to propose that Pol ⑀ might be important for assembly of the initiation complex (Feng and D'Urso, 2001).Consistent with this hypothesis, we demonstrate that both the C-terminal half of Pol ⑀, and its smaller putative subunit, Dpb2, bind preferentially to replication origins early in S phase. Furthermore, we demonstrate that like cdc20 mutants (Feng and D'Urso, 2001), a temperature-sensitive mutant of dpb2 arrests with a 1C DNA content after shift to the restrictive temperature, suggesting that Dpb2 is required for the onset of S phase.…”

“…The observation that mutants in Pol ⑀ arrest in late G1 or early S phase combined with the fact that the enzyme's polymerase activity is dispensable, led us to propose that Pol ⑀ might be important for assembly of the initiation complex (Feng and D'Urso, 2001).…”