Catalytic and noncatalytic functions of DNA polymerase κ in translesion DNA synthesis

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Acknowledgement

We thank J. Gautier (Columbia University) for sharing Polκ-expressing constructs, D. Durocher (University of Toronto) for the RPE1-hTERT p53^−/− cells and staff of the CPR/ReNew Genomics Platform for support: H. Wollmann, M. Michaut and A. Kalvisa. We also thank members of the Duxin laboratory and J. Walter for feedback on the manuscript. The Novo Nordisk Foundation Center for Protein Research is supported financially by the Novo Nordisk Foundation (grant agreement NNF14CC0001). This project has received funding from the European Research Council under the European Union’s Horizon 2020 research and innovation program (grant agreement 715975) and from the Novo Nordisk Foundation (grant NNF22OC0074140). B.B. is supported by the European Molecular Biology Organization (grant agreement no. ALTF 1149-2020). Work in J.N.’s lab was supported by a Novo Nordisk Fonden distinguished investigator grant (NNF23OC0082227) and a grant from the Danish Cancer Society (R269-A15586-B71). T.C.R.M. was supported by a Novo Nordisk Fonden Hallas-Møller emerging investigator grant (NNF22OC0073571), the Danish National Research Foundation (DNRF115) and the Carlsberg Foundation (CF21-0571). J.P.D. is part of the European doctoral network (Replifate, grant agreement #101072903). We also extend our gratitude to J. Lukas for his exceptional leadership, which has elevated the Center for Protein Research to the forefront of biological research.

Contributions

These authors contributed equally: Sara M. Ambjørn, Alberto Carli, Ivo A. Hendriks.

S.S.B. performed all experiments unless stated otherwise. A.C. generated the AlphaFold models in Figs. 1c and 7e,f and Extended Data Figs. 1b,c and 7c under the supervision of T.M. S.M.A. and B.M. performed the CRISPR base-editor tiling screen (Fig. 1b and Extended Data Fig. 1a) under the supervision of J.N. E.P.T.H. provided technical advice on the base-editor screen. N.D. designed the CRISPR library and performed statistical analysis of the sequencing data. I.A.H. performed MS analysis on CHROMASS samples (Fig. 6 and Extended Data Fig. 6) under the supervision of M.L.N. L.S and B.B. generated the p3d-Phen-A minor groove DNA substrate under the supervision of S.S. and J.P.D. I.G. performed the experiment in Fig. 6d and preliminary experiments for this project. D.S. provided the AP-ICL-containing plasmid. S.A.G. generated the POLK-KO cells. S.M.A. performed the colony assay experiments (Fig. 7a–d). A.Z. assisted with recombinant Polκ protein purification. S.S.B. and J.P.D. designed and analyzed the experiments. S.S.B. and J.P.D. prepared the manuscript with feedback and input from all authors of the manuscript.

Data Availability

The MS proteomics data were deposited to the ProteomeXchange Consortium through the PRIDE⁹⁸ partner repository with the dataset identifier PXD044258. Source data are provided with this paper.

Supplemental Material

Supplementary Tables 1–3: Supplementary Table 1. POLK base-editor tiling screen results and analysis (Fig. 1 and Extended Data Fig. 1). Note that the library was generated for both ABE and CBE but only ABE was used. The significance and fold change were derived from limma (Methods). Supplementary Table 2. MS analysis of protein recruitment to UV-treated sperm chromatin in mock, Rev1-depleted or Polκ-depleted extracts (Fig. 6 and Extended Data Fig. 6). ΔMock, mock extracts incubated in undamaged chromatin; ΔMock + UV, mock extracts incubated with UV-treated chromatin; ΔRev1 + UV, Rev1-depleted extracts incubated with UV-treated chromatin; ΔPolκ + UV, Polκ-depleted extracts incubated with UV-treated chromatin. For quantification purposes, all LFQ-normalized protein intensity values were log₂-transformed and filtered for presence in eight of eight replicates in at least one experimental condition. Missing values were inputted below the global experimental detection limit at a downshift of 1.8 and a randomized width of 0.15 (in log₂ space). Statistical significance of differences was, in all cases, tested using two-tailed Student’s two-sample t-testing with a q value of <1%. Proteins not enriched over the no-DNA control in at least one CHROMASS condition (FDR < 1%, s₀ =&thinsp;1 and 2,500 rounds of randomization) were removed from the analysis, after which previously inputted values were reinputted on the basis of the new total matrix. Final biological differences were determined using two-tailed Student’s two-sample t-testing (FDR < 1%, s₀ =&thinsp;0.5 and 2,500 rounds of randomization) on the remaining CHROMASS-enriched proteins. Supplementary Table 3. PCR primers used during base-editor tiling screen (Fig. 1, Extended Data Fig. 1 and Methods).