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Evidence for Interplay Among Yeast Replicative DNA Polymerases Alpha, Delta and Epsilon From Studies of Exonuclease and Polymerase Active Site Mutations

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Evidence for Interplay Among Yeast Replicative DNA Polymerases Alpha, Delta and Epsilon From Studies of Exonuclease and Polymerase Active Site Mutations

Youri I Pavlov et al. BMC Biol.

Erratum in

  • BMC Biol. 2007;5:27

Abstract

Background: DNA polymerase epsilon (Pol epsilon) is essential for S-phase replication, DNA damage repair and checkpoint control in yeast. A pol2-Y831A mutation leading to a tyrosine to alanine change in the Pol epsilon active site does not cause growth defects and confers a mutator phenotype that is normally subtle but strong in a mismatch repair-deficient strain. Here we investigate the mechanism responsible for the mutator effect.

Results: Purified four-subunit Y831A Pol epsilon turns over more deoxynucleoside triphosphates to deoxynucleoside monophosphates than does wild-type Pol epsilon, suggesting altered coordination between the polymerase and exonuclease active sites. The pol2-Y831A mutation suppresses the mutator effect of the pol2-4 mutation in the exonuclease active site that abolishes proofreading by Pol epsilon, as measured in haploid strain with the pol2-Y831A,4 double mutation. Analysis of mutation rates in diploid strains reveals that the pol2-Y831A allele is recessive to pol2-4. In addition, the mutation rates of strains with the pol2-4 mutation in combination with active site mutator mutations in Pol delta and Pol alpha suggest that Pol epsilon may proofread certain errors made by Pol alpha and Pol delta during replication in vivo.

Conclusions: Our data suggest that Y831A replacement in Pol epsilon reduces replication fidelity and its participation in chromosomal replication, but without eliminating an additional function that is essential for viability. This suggests that other polymerases can substitute for certain functions of polymerase epsilon.

Figures

Figure 1
Figure 1
Purified four-subunit Y831A Pol ε. SDS-PAGE analysis of peak fraction from final SMART MonoS column (see Methods). 1 μl of collected fraction 15 was mixed with Invitrogen loading buffer and run on denaturing 4–12% NUPAGE Bis-Tris polyacrylamide gel for 50 minutes at 200 V in MOPS buffer. The gel was stained with SimpleStain colloidal blue stain as recommended by the vendor (Invitrogen). Lane 1, Pol ε Y831A; lane 2, the Benchmark His-tagged protein standards (Invitrogen).
Figure 2
Figure 2
Elevated nucleotide turnover by Y831A Pol ε. DNA synthesis and dTTP turnover by Pol ε was measured on poly(dA)/oligo(dT) substrate. Reactions were performed as described in Methods, using 0.13 U of each enzyme for 20 μl reactions. (A) Analysis of polymerase reaction by TLC in 1 M LiCl running buffer. Lanes 1, 2, 3, 4: wild-type Pol ε at 0, 3, 7 and 15 minutes of reaction, respectively. Lanes 5, 6, 7: reactions with Y831A Pol ε at 3, 7 and 15 minutes, respectively. Positions of unincorporated label, label in DNA and dTMP are shown by arrows. (B) Analysis of polymerase reaction by TLC in 0.4 M LiCl running buffer. Lane assignment is the same as in (A). (C) Plot of time-course of DNA synthesis and dTMP turnover by wild-type Pol ε. Open circles connected by solid line represent dTMP retained into DNA; open rectangles connected by a dashed line represent excised dTMP. (D) Plot of time-course of DNA synthesis and dTMP turnover by Y831A Pol ε. Symbols are the same as in (C).
Figure 3
Figure 3
Models explaining results on the genetic interaction of mutants with defects in the active site and proofreading of DNA polymerase ε. Thickness of arrows indicates the relative probability of a depicted pathway. See text for explanations. (A) Dissociation-proofreading. The newly synthesized DNA is in red. 'M' stands for incorrectly inserted nucleotide. DNA polymerase is drawn as octagonal oval; an exonuclease is drawn as rectangle. (B) Three distinct roles of Pol ε in replication.

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