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Apn2 resolves blocked 3′ ends and suppresses Top1-induced mutagenesis at genomic rNMP sites

Nature Structural & Molecular Biology volume 26, pages 155–163 (2019)Cite this article

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Abstract

Ribonucleoside monophosphates (rNMPs) mis-incorporated during DNA replication are removed by RNase H2-dependent excision repair or by topoisomerase I (Top1)-catalyzed cleavage. The cleavage of rNMPs by Top1 produces 3′ ends harboring terminal adducts, such as 2′,3′-cyclic phosphate or Top1 cleavage complex (Top1cc), and leads to frequent mutagenesis and DNA damage checkpoint induction. We surveyed a range of candidate enzymes from Saccharomyces cerevisiae for potential roles in Top1-dependent genomic rNMP removal. Genetic and biochemical analyses reveal that Apn2 resolves phosphotyrosine–DNA conjugates, terminal 2′,3′-cyclic phosphates, and their hydrolyzed products. APN2 also suppresses 2-base pair (bp) slippage mutagenesis in RNH201-deficient cells. Our results define additional activities of Apn2 in resolving a wide range of 3′ end blocks and identify a role for Apn2 in maintaining genome integrity during rNMP repair.

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Fig. 1: Terminal phosphate adducts block Polδ activities.
Fig. 2: apn2 is synthetic lethal in pol2-M644G rnh201 cells.
Fig. 3: Apn2 clips dinucleotide at the 2′,3′-cyclic phosphate-terminated end.
Fig. 4: Apn2 exonuclease activity on a 2′,3′-cyclic phosphate-terminated end is stimulated by PCNA and permits Polδ-catalyzed primer extension.
Fig. 5: Top1cc removal by Apn2.
Fig. 6: exo1∆ suppresses the lethality of pol2-M644G rnh201Δ apn2Δ.

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The data that support the findings in this work are available from the corresponding authors upon request.

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Acknowledgements

We are grateful to P. Sung, N. Hollingsworth, N. Kim, H. Klein, T. Kunkel, and S. Jinks-Robertson for providing plasmids and yeast strains, H. Bedwell for technical support, S. Bell and B. Calvi for critical reading of the manuscript. This work was supported by William and Ella Owens Medical Research Foundation, Nathan Shock Center Pilot grant, and NIH research grant GM71011 (to S.E.L.), ThriveWell Foundation (to E.Y.S.), and GM124765 (to H.N.).

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  1. These authors contributed equally: Fuyang Li, Quan Wang.

Authors and Affiliations

  1. Department of Molecular Medicine, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA

    Fuyang Li, Ja-Hwan Seol & Sang Eun Lee

  2. Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN, USA

    Quan Wang, Xiaoyu Lu & Hengyao Niu

  3. Department of Radiation Oncology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA

    Jun Che, Eun Yong Shim & Sang Eun Lee

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Contributions

F.L., Q.W., E.Y.S., S.E.L., and H.N. designed the experiments. F.L., J.-H.S., and J.C. constructed the yeast strains and performed the genetic assays. Q.W. purified all the proteins and conducted the biochemical experiments. X.L. assisted with protein purifications. F.L., Q.W., J.-H.S., J.C., E.Y.S., S.E.L., and H.N. analyzed the data and wrote the paper.

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Correspondence to Sang Eun Lee or Hengyao Niu.

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Supplementary Figure 1 SDS-PAGE analysis of purified protein factors.

a. Purified Polδ complex (~ 0.3 µg of protein in total) was fractionated in 4–20% SDS-PAGE and stained by coomassie blue G-250. b. Purified RFC complex (~ 2 µg of protein in total) and PCNA (~ 0.5 µg) were fractionated in 4–20% SDS-PAGE and stained by coomassie blue G-250. c. Purified Top1 and top1-T722A (~ 0.5 µg each) were fractionated in 4–20% SDS-PAGE and stained by coomassie blue G-250. d. Purified Apn1, Apn2, Tdp1 and Tpp1 from E. coli (~ 1 µg each) were fractionated in 10% SDS-PAGE and stained by coomassie blue G-250. e. Purified Apn2 and apn2-E59A from yeast (~ 0.5 µg each) were fractionated in 4–20% SDS-PAGE and stained by coomassie blue G-250.

Supplementary Figure 2 Analyses of mutants of RAD1, TPP1, TDP1 and APN1 in synthetic lethality with pol2-M644G rnh201∆.

Tetrad analysis results of pol2-M644G rnh201Δ apn1Δ (a), pol2-M644G rnh201Δ rad1Δ (b), pol2-M644G rnh201Δ slx4Δ top1Δ (c), pol2-M644G rnh201Δ tdp1Δ (d), pol2-M644G rnh201Δ tpp1Δ (e), pol2-M644G rnh201Δ pol3-01 (f), pol2-M644G rnh201Δ rad1Δ tdp1Δ (g), pol2-M644G rnh201Δ mre11Δ top1Δ (h), heterozygote diploid cells. Spores of indicated genotypes are marked with solid and dotted circles, squares, triangles, or pentagons.

Supplementary Figure 3 Apn2 does not directly cleave the embedded ribonucleotide but process the terminal cyclic phosphate derived from ribonucleotide cleavage by Top1.

a. Alkaline gel analysis of genomic DNA isolated from yeast strains including wild type, rad1∆, apn2∆, apn2∆ rad1∆, rnh201∆ and rnh201∆ apn2∆ to examine the presence of genomic ribonucleotide. b. Cleavage at the embedded rNMPs by RNase H2 (20 nM), Apn2 (100 nM) and Top1 (100 nM), top1-T722A (100 nM). c. Cleavage at embedded rNMPs by Top1 (20 nM) and top1-T722A (20 nM). d. Cleavage at an abasic site by purified Apn1 (20 nM) or Apn2 (500 nM). e. Processing of 2’, 3’ cyclic phosphate-terminated nicks by Tpp1 (0.01–0.05 nM) and Apn1 (20–100 nM). f. Apn2 (20 and 100 nM) catalyzed processing of phosphate-terminated ribonucleotide end present at a nick, a 3’- end of single-stranded DNA or a recessed 3’- end.

Supplementary Figure 4 Influence of PCNA on DNA end processing by Apn2, Apn1 and Tpp1.

a. Digestion of a recessed 3’-OH end by Apn2 (20 nM) in the absence or presence of PCNA on duplex DNA with either both or single DNA end occluded. b. Digestion of 3’- end harboring a ribonucleotide or a monophosphate attached ribonucleotide by Apn2 (20 nM) in the absence or presence of PCNA. c. Polδ/PCNA-catalyzed primer extension from a 2’, 3’ cyclic phosphate-terminated end in the absence and presence of Apn1 (20 nM) and/or Exo1 (1 nM). d. Polδ/PCNA-catalyzed primer extension from a 2’, 3’ cyclic phosphate-terminated end in the absence and presence of Tpp1 (0.05 nM) and/or Exo1 (1 nM). e. Polδ/PCNA-catalyzed primer extension from a terminal ribonucleotide with a monophosphate attached in the absence and presence of Apn2 (20 nM) or apn2-E59A (20 nM). f. Polδ/PCNA-catalyzed primer extension from a 3’-terminal ribonucleotide in the absence and presence of Apn2 (20 nM) or apn2-E59A (20 nM).

Supplementary Figure 5 A multi-faceted role for Apn2 in the error-free repair of Top1-induced lesions at genomic rNMP sites.

Mis-incorporated ribonucleotides (rU) is cleaved by Top1, which generates 2’, 3’ cyclic phosphate (Δ) and 5’-OH ends. The 2’, 3’ cyclic phosphate termini can be removed by the second Top1 cleavage at 2 bp proximal to the initial Top1 cleavage site. Top1cc generated by the secondary Top1 cleavage may be processed by Tdp1/Tpp1, or Apn2 to promote error-free repair. Otherwise, the Top1-catalyzed ligation across 2 bp gap may cause 2 bp deletion. Alternatively, Apn2 and Srs2-Exo1 process either 2’, 3’ cyclic phosphate termini or 5’-OH ends to initiate error-free gap repair events. Apn2-PCNA also remove 3’ monophosphate ends. Apn2 is a versatile and multi-functional enzyme involved in multiple steps of rNMP repair.

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Supplementary Figures 1–5, Supplementary Tables 1–3 and Supplementary Dataset Uncropped gels

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Li, F., Wang, Q., Seol, JH. et al. Apn2 resolves blocked 3′ ends and suppresses Top1-induced mutagenesis at genomic rNMP sites. Nat Struct Mol Biol 26, 155–163 (2019). https://doi.org/10.1038/s41594-019-0186-1

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