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How asymmetric DNA replication achieves symmetrical fidelity

Nature Structural & Molecular Biology volume 28, pages 1020–1028 (2021)Cite this article

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Abstract

Accurate DNA replication of an undamaged template depends on polymerase selectivity for matched nucleotides, exonucleolytic proofreading of mismatches, and removal of remaining mismatches via DNA mismatch repair (MMR). DNA polymerases (Pols) δ and ε have 3′–5′ exonucleases into which mismatches are partitioned for excision in cis (intrinsic proofreading). Here we provide strong evidence that Pol δ can extrinsically proofread mismatches made by itself and those made by Pol ε, independently of both Pol δ’s polymerization activity and MMR. Extrinsic proofreading across the genome is remarkably efficient. We report, with unprecedented accuracy, in vivo contributions of nucleotide selectivity, proofreading, and MMR to the fidelity of DNA replication in Saccharomyces cerevisiae. We show that extrinsic proofreading by Pol δ improves and balances the fidelity of the two DNA strands. Together, we depict a comprehensive picture of how nucleotide selectivity, proofreading, and MMR cooperate to achieve high and symmetrical fidelity on the two strands.

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Fig. 1: URA3 and CAN1 reporter gene mutation rates and hotspot mutation rates.
Fig. 2: Genome mutation rates from mutation accumulation experiments.
Fig. 3: Repair efficiency and specificity of Pol δ extrinsic PR.
Fig. 4: Pol δ extrinsically repairs its own errors.
Fig. 5: Determinants of DNA replication fidelity.

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Data availability

All whole genome sequencing data is available through Sequence Read Archive accession number PRJNA689775. Source data are provided with this paper.

Code availability

Muver suite is available via GitHub44 or upon request.

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Acknowledgements

We thank D. Gordenin and R. Schaaper for critical reading of and thoughtful comments on the manuscript. We thank P. Mieczkowski and others from the High Throughput Sequencing Facility of UNC Chapel Hill for performing Illumina sequencing. This study was supported by Project Z01 ES065070 to T.A.K from the Division of Intramural Research of the NIH, NIEHS.

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Authors and Affiliations

  1. Genome Integrity & Structural Biology Laboratory, NIH/NIEHS, DHHS, Research Triangle Park, Durham, NC, USA

    Zhi-Xiong Zhou, Scott A. Lujan, Jordan St. Charles, Joseph Dahl, Corinne E. Farrell, Jessica S. Williams & Thomas A. Kunkel

  2. Integrative Bioinformatics Support Group, NIH/NIEHS, DHHS, Research Triangle Park, Durham, NC, USA

    Adam B. Burkholder

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Contributions

Z.-X.Z. and T.A.K. conceived the project. Z.-X.Z. performed most of the experiments. Z.-X.Z. and S.A.L. analyzed the genome-wide mutation data. A.B.B. performed mutation calling using the muver pipeline. J.A.S. contributed to mutation accumulation experiments. J.D. purified the Pol ε holoenzyme. C.F. and J.S.W. contributed to Supplementary Table 3.

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Correspondence to Thomas A. Kunkel.

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Peer review information Nature Structural & Molecular Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available. Beth Moorefield was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Genetic interaction between pol2-M644G and pol3-exo-.

Reporter gene assays were performed similar to Fig. 1. n > =15 independent cultures were used for fluctuation analysis for each genotype.

Source data

Extended Data Fig. 2 pol3-x mutant does not support colony growth.

Tetrad dissection from a heterozygous pol3-x/POL3-WT diploid strain. Plate was incubated at °C for 5 days.

Supplementary information

Supplementary Information

Supplementary Note, Figures 1–7, Tables 1–9, and Supplementary Source Data.

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Supplementary Data

Source data for Supplementary Table 3 and Supplementary Fig. 6.

Source data

Source Data Fig. 1

Individual data points from fluctuation analysis.

Source Data Fig. 2

Substitution mutation rates for each genomic isolate.

Source Data Fig. 3

Rates of individual substitution type for each isolate.

Source Data Fig. 3

Uncropped image of PAGE gel.

Source Data Fig. 4

Substitution mutation rates for each genomic isolate; individual data points from fluctuation analysis.

Source Data Extended Data Fig. 1

Individual data points from fluctuation analysis.

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Zhou, ZX., Lujan, S.A., Burkholder, A.B. et al. How asymmetric DNA replication achieves symmetrical fidelity. Nat Struct Mol Biol 28, 1020–1028 (2021). https://doi.org/10.1038/s41594-021-00691-6

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