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DNA Polymerase Δ Proofreads Errors Made by DNA Polymerase E

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DNA Polymerase Δ Proofreads Errors Made by DNA Polymerase E DNA polymerase δ proofreads errors made by DNA polymerase e Chelsea R. Bulocka, Xuanxuan Xinga,1, and Polina V. Shcherbakovaa,2 aEppley Institute for Research in Cancer and Allied Diseases, Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE 68198 Edited by Sue Jinks-Robertson, Duke University School of Medicine, Durham, NC, and approved February 5, 2020 (received for review October 9, 2019) − During eukaryotic replication, DNA polymerases e (Pole) and δ analogous Pole-exo variant (2, 17–25). Furthermore, haploid (Polδ) synthesize the leading and lagging strands, respectively. In yeast deficient in Polδ proofreading do not survive when DNA a long-known contradiction to this model, defects in the fidelity of mismatch repair (MMR) is also inactivated with the death at- Pole have a much weaker impact on mutagenesis than analogous tributed to an excessive level of mutagenesis (26). In contrast, yeast Polδ defects. It has been previously proposed that Polδ contributes lacking both proofreading by Pole and MMR are viable, and while more to mutation avoidance because it proofreads mismatches the mutation rate in these strains is high, it does not reach the created by Pole in addition to its own errors. However, direct lethal threshold (19, 21, 22, 25, 27). Similarly, when identical ty- evidence for this model was missing. We show that, in yeast, rosine to alanine substitutions were made in the conserved region the mutation rate increases synergistically when a Pole nucleotide III of the polymerase domains (Polδ-Y708A and Pole-Y831A), the selectivity defect is combined with a Polδ proofreading defect, dem- Polδ variant produced a much stronger mutator effect than the onstrating extrinsic proofreading of Pole errors by Polδ. In contrast, analogous Pole variant (28). To explain the controversy between combining Polδ nucleotide selectivity and Pole proofreading defects the accepted fork model and the disparity of Polδ and Pole effects produces no synergy, indicating that Pole cannot correct errors on mutagenesis, a hypothesis has been entertained that Polδ made by Polδ. We further show that Polδ can remove errors made proofreads errors made by Pole in addition to its own errors, thus, by exonuclease-deficient Pole in vitro. These findings illustrate the contributing more significantly to mutation avoidance. This hy- complexity of the one-strand–one-polymerase model where synthe- pothesis, discussed in multiple publications (2, 29–31), stems from sis appears to be largely divided, but Polδ proofreading operates on the original observation by Morrison and Sugino that the combi- both strands. δ e nation of Pol and Pol proofreading defects results in a syner- GENETICS gistic increase in mutation rate (19). The synergy implies that the DNA replication | extrinsic proofreading | DNA polymerase δ | exonucleases of Pole and Polδ act on the same pool of replication DNA polymerase e errors and could potentially mean Pole correcting errors made by Polδ,Polδ correcting errors made by Pole, or both polymerases he most widely accepted model of eukaryotic DNA replica- proofreading for each other. In general, the possibility of extrinsic Ttion proposed in the 1990s suggests that Polα-primase syn- proofreading has been demonstrated in multiple in vivo and thesizes short RNA–DNA primers at the origins and at the in vitro studies. Initial experiments showed that errors made by beginning of the Okazaki fragments, Pole synthesizes the leading purified calf thymus Polα could be corrected by the e subunit of strand, and Polδ completes the lagging strand (1). During the Escherichia coli DNA polymerase III or by Polδ (32, 33). Several three decades that passed since the landmark publication by mammalian autonomous exonucleases have also been shown to Morrison et al., numerous reports have contributed evidence for e δ the participation of Pol and Pol in leading and lagging strand Significance replication, respectively. Genetic studies detected strand-specific increases in mutagenesis in yeast and human cells carrying inac- Polδ and Pole are the two major replicative polymerases in curate Pole or Polδ variants (2–6). More sensitive assays moni- e δ eukaryotes, but their precise roles at the replication fork re- toring ribonucleotide incorporation into DNA by Pol or Pol main a subject of debate. A bulk of data supports a model variants with relaxed sugar selectivity confirmed ribonucleotide ac- where Pole and Polδ synthesize leading and lagging DNA cumulation in the leading strand in Pole mutants and in the lagging δ δ e strands, respectively. However, this model has been difficult to strand in Pol mutants (7, 8). Pol but not Pol was shown to δ α reconcile with the fact that mutations in Pol have much proofread errors made by Pol (9) and participate in the maturation stronger consequences for genome stability than equivalent of Okazaki fragments on the lagging strand (10, 11). At the same mutations in Pole. We provide direct evidence for a long- time, Pole but not Polδ interacts with the Cdc45-MCM-GINS δ e δ entertained idea that Pol can proofread errors made by Pol helicase on the leading strand (12). While the roles of Pol in the in addition to its own errors, thus, making a more prominent synthesis of the leading strand near replication origins and termi- contribution to mutation avoidance. This paper provides an nation zones have recently been detected (13–16), these stretches of δ essential advance in the understanding of the mechanism of Pol synthesis appear to account for a relatively minor fraction of eukaryotic DNA replication. the leading strand (∼18%, ref. 16). Overall, a bulk of evidence e supports the originally proposed division of labor with Pol and Author contributions: P.V.S. designed research; C.R.B. and X.X. performed research; Polδ predominantly replicating opposite DNA strands. C.R.B., X.X., and P.V.S. analyzed data; and C.R.B., X.X., and P.V.S. wrote the paper. In contradiction to this model, Polδ fidelity defects have long The authors declare no competing interest. been known to have a greater impact on mutagenesis than This article is a PNAS Direct Submission. e e δ analogous Pol defects. Both Pol and Pol contribute to mu- This open access article is distributed under Creative Commons Attribution-NonCommercial- tation avoidance via their intrinsic nucleotide selectivity con- NoDerivatives License 4.0 (CC BY-NC-ND). ferred by the polymerase domain and the proofreading activity 1Present address: Comprehensive Cancer Center, Ohio State University College of Medi- located in a separate exonuclease domain. The exonuclease ac- cine, Columbus, OH 43210. tivity of both polymerases can be abolished by alanine substitutions 2To whom correspondence may be addressed. Email: [email protected]. at the conserved carboxylate residues in the ExoI motif FDIET/C This article contains supporting information online at https://www.pnas.org/lookup/suppl/ − (17, 18). The resulting mutator phenotype of the Polδ-exo variant doi:10.1073/pnas.1917624117/-/DCSupplemental. is an order of magnitude stronger than the phenotype of the www.pnas.org/cgi/doi/10.1073/pnas.1917624117 PNAS Latest Articles | 1of7 Downloaded by guest on October 1, 2021 increase the fidelity of Polα in vitro (34–36). Both E. coli and yeast deficient in MMR and harboring pol3-D520V are not viable eukaryotic replicative polymerases can excise nucleotides incor- (41); therefore, we assessed the epistatic relationship between pol3- porated by translesion synthesis polymerases at sites of DNA D520V and MMR deficiency in diploid strains, which can tolerate a damage (37, 38). With respect to the extrinsic proofreading ca- higher level of mutagenesis. We used the MSH6 deletion to in- pabilities of Polδ and Pole in vivo, several studies have been illu- activate MMR as the Msh6-dependent pathway is primarily re- minating. As already mentioned, Polδ but not Pole has been shown sponsible for the repair of single-base mismatches (42), which is the to proofread errors made by an error-prone Polα variant in yeast predominant type of replication errors generated by exonuclease- (9). Furthermore, Polδ exonuclease defects are almost completely deficient Polδ and Pole (43–45). Diploids homozygous for both pol3- recessive, indicating that wild-type Polδ can efficiently proofread D520V and msh6 mutations showed a strong synergistic increase in − errors created by Polδ-exo (18, 26, 31). On the other hand, the mutation rate as compared with the single pol3-D520V and msh6 − mutant allele encoding Pole-exo is semidominant, suggesting that mutants (SI Appendix,TableS2). A similar synergistic increase in wild-type Pole does not correct errors in trans (31, 39). Flood et al. mutagenesis in pol3-D520V/pol3-D520V msh6/msh6 diploids was (31) further investigated extrinsic proofreading by Polδ and Pole observed by Flood et al. for base substitutions at a single nucleotide using transformation of yeast cells with oligonucleotides that, when position in the TRP5 gene (31). We recapitulate and expand these annealed, create a 3′-terminal mismatch. These experiments earlierfindingsbyusingtheforward mutagenesis reporter CAN1 showed that Polδ but not Pole can proofread in trans and that the that can detect a variety of base substitutions and indels in many exonuclease of Polδ can act on oligonucleotides annealed to both DNAsequencecontextsaswellasthehis7-2 frameshift reporter that leading and lagging strands. However, it remained unknown is particularly sensitive to MMR defects. Together, these data whether the exonuclease of Polδ could proofread errors gener- demonstrate that pol3-D520V does not confer a MMR defect. Thus, ated by Pole during normal chromosomal replication. the synergy between pol2-4 and pol3-D520V indicates proofreading To answer this question, we used yeast strains harboring a of the same errors by Pole and Polδ. It also shows that the pol3- nucleotide selectivity defect in one polymerase, Polδ or Pole, and D520V allele provides an adequate model for the extrinsic proof- a proofreading defect in the other.
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