Xenopus egg extracts and DNA replication reactions

Xenopus egg extracts were prepared as described previously81. All experiments involving animals were approved by the Danish Animal Experiments Inspectorate and conform to relevant regulatory standards and European guidelines.

For plasmid DNA replication, plasmids were licensed in high-speed supernatant (HSS) at a final concentration of 7.5 ng µl−1 for 30 min at room temperature (RT). Replication was initiated by adding two volumes of nucleoplasmic egg extract (NPE). Gap-filling reactions were performed in nonlicensing extracts (extracts that do not support MCM2–MCM7 licensing), where one volume of HSS was premixed with two volumes of NPE before the addition of plasmid DNA (final concentration of 10 ng µl−1). For replication in the presence of LacI, plasmid DNA (150 ng µl−1) was incubated with an equal volume of 12 µM LacI for 1 h before licensing57. The ubiquitin E1 inhibitor MLN-7243 (Active Biochem) was supplemented to NPE at a final concentration of 200 µM 10 min before initiating the reaction. To visualize DNA replication intermediates, replication reactions were supplemented with [α-32P]dATP (Perkin Elmer). For each time point, 1 µl of the reaction mixture was added to 5 µl of stop buffer (5% SDS, 80 mM Tris pH 8.0, 0.13% phosphoric acid and 10% Ficoll), followed by the addition of 1 µl of proteinase K (20 mg ml−1) (Roche). The samples were incubated at 37 °C for 1 h and subsequently separated using 0.9% native agarose gel electrophoresis; results were visualized using a phosphor imager. The radioactive signal was quantified using ImageJ (National Institutes of Health).

Preparation of DNA constructs

pDPC and pDPC2×Lead were prepared as previously described56. Additionally, pDPCPK and pDPCssDNA-PK were prepared as previously described15 as pMHPK and pMHssDNA-PK, respectively. Moreover, pDPCLead and pDPCLag were prepared as previously described57 as pDPC-LTop (Lead) and pDPC-LBot (Lag), respectively.

To generate a plasmid containing p3d-Phen-A, we first removed the LacO array from pJSL3 (ref. 82) using the complementary overhangs of the BsrG1 and BsiWI restriction sites. Subsequently, the two Nb.BsrdI nicking sites were removed using mutagenesis. An A located at position 1557 of the plasmid was mutated to a C to remove the first Nb.BsrDI site using the following primers: 5′-CCACGATGCCTGTAGCCATGGCAACAACGTTGC-3′ and 5′- GCAACGTTGTTGCCATGGCTACAGGCATCGTGG-3′. Secondly, a T located at position 1740 of the plasmid was mutated to a C to remove the second Nb.BsrDI site using the following primers: 5′-GGTCTCGCGGTATCATCGCAGCACTGGGGCCAG-3′ and 5′-CTGGCCCCAGTGCTGCGATGATACCGCGAGACC-3′. Afterward, we used the PciI and BsaX1 restriction sites to clone in the oligo 5′-CATGGCTCTTCNACCTCAACTACTTGACCCTCCTCATTCATTGCTTG-3′ to introduce Nt.BspQ1 and Nb.BsrD1 nicking sites. Finally, to generate p3d-Phen-A, the vector was nicked using Nt.BspQ1 and Nb.BsrD1 and ligated with an excess of the following oligo containing 3d-Phen-A at position 15: 5′-ACCTCAACTACTTGACCCTCCTCATT-3′ (ref. 31). pAP-ICL was generated as previously described60.

Antibodies and immunodepletions

Antibodies to Rev1 (Rev1-N and Rev1-C)22, Rfwd3 (ref. 15), Polη15 and HMCES59 were described previously. Antibodies to Polκ, Rev3 and FancA 2 were raised by New England Peptide by immunizing rabbits with Ac-CPASKKSKPNSSKNTIDRFFK-OH, Ac-CLADLSIPQLD-OH and Ac-CSFKAPDDYDDLFFEPVF-OH, respectively. The antibody to FancA 1 was a kind gift of A. Sobeck83.

To immunodeplete Rev1 from Xenopus egg extracts, an equal volume of Protein A Sepharose fast flow (PAS) (GE Health Care) beads was bound to anti-Rev-N or anti-Rev1-C antibodies overnight at 4 °C. The beads were then washed twice with 500 µl of PBS, once with ELB (10 mM HEPES pH 7.7, 50 mM KCl, 2.5 mM MgCl2 and 250 mM sucrose), twice with ELB supplemented with 0.5 M NaCl and twice with ELB. One volume of precleared HSS or NPE was then depleted by mixing with 0.2 volumes of antibody-bound beads and then incubated at RT for 15 min, before being isolated. For HSS, the depletion procedure was performed once with Rev1-N coupled beads and once with Rev1-C coupled beads. For NPE, the depletion procedure was performed twice with Rev1-N coupled beads and once with Rev1-C coupled beads. To immunodeplete Polκ, Polη or Rfwd3 from Xenopus egg extracts, one volume of PAS beads was bound to five volumes of affinity-purified antibody (1 mg ml−1). The beads were washed as described above and one volume of precleared HSS or NPE was then depleted by mixing with 0.2 volumes of antibody-bound beads for 15 min at RT. The depletion procedure was performed once for HSS and three times for NPE. For HMCES and Polκ combined depletion, one volume of beads was bound to eight volumes of each affinity-purified antibody (1 mg ml−1). The beads were washed and depletion was performed as described for Polκ immunodepletion.

Immunoprecipitations

For the FancA and Polκ immunoprecipitation experiments, 5 μl of PAS beads were incubated with 10 μg of the respective affinity-purified antibody for 1 h at RT. The Sepharose beads were subsequently washed twice with PBS and three times with IP buffer (10 mM HEPES pH 7.7, 50 mM KCl, 2.5 mM MgCl2 and 0.25% NP-40). Next, 5 μl of NPE was diluted with 20 μl of IP buffer and incubated with antibody-prebound beads for 1 h at RT. The beads were then washed three times with IP buffer and resuspended in 50 μl of 2× Laemmli sample buffer before analysis by western blotting.

Nascent leading-strand analysis

For nascent leading-strand analysis, 3–4 µl of replication reaction was added to ten volumes of transparent stop buffer (50 mM Tris-HCl pH 7.5, 0.5% SDS and 25 mM EDTA). The replication intermediates were purified as previously described84,85. The DNA was digested with the indicated restriction enzymes and subsequently supplemented with 0.5 volumes of denaturing PAGE gel loading buffer II (Life technologies). The digested DNA products were separated on a 6% polyacrylamide sequencing gel.

Protein expression and purification

Full-length Xenopus laevis Polκ with an N-terminal 6xHis-tag was amplified from pCMV-Sport.ccdb-Polκ36 and cloned into pET28b (Novagen) using primers A and B and restriction enzymes BamHI and XhoI. Xenopus Polκ C-ter with an N-terminal 6xHis-tag was cloned into pET28b using primers B and C and restriction enzymes BamHI and XhoI. Polκ amino acid substitutions were introduced by Quikchange mutagenesis and confirmed by Sanger sequencing.

Plasmids containing WT Polκ, mutant Polκ or Polκ C-ter were transformed into BL21 Escherichia coli competent cells. Cells were grown at 37 °C to an optical density of 0.6–0.8 in Luria–Bertani broth and were subsequently induced with 0.5 mM IPTG for 4 h. Bacteria were harvested by centrifugation and resuspended in 20 ml of lysis buffer (50 mM Tris pH 7.5, 300 mM NaCl, 2 mM MgCl2, 1 mM DTT and 1× Roche EDTA-free cOmplete protease inhibitor cocktail). Suspensions were sonicated and cleared by high-speed centrifugation at 15,000 r.p.m. in a F15-8x50cy rotor for 1 h at 4 °C. The soluble fraction was collected and incubated with 2 ml of Ni-NTA Superflow affinity resin (Qiagen), previously equilibrated with lysis buffer, for 2 h at 4 °C. The resin was then washed three times with 20 ml of wash buffer (50 mM Tris pH 7.5, 300 mM NaCl, 2 mM MgCl2, 1 mM DTT, 0,1% Triton-X and 10 mM imidazole). Then, 6xHis-tagged Polκ was eluted with elution buffer (50 mM Tris pH 7.5, 300 mM NaCl, 2 mM MgCl2, 1 mM DTT, 10% glycerol and 10 mM imidazole). Elution fractions containing the target proteins were pooled and dialyzed against dialysis buffer (50 mM Tris pH 7.5, 300 mM NaCl, 2 mM MgCl2, 1 mM DTT and 10% glycerol) at 4 °C overnight. After dialysis, protein fractions were concentrated to 100 μl using centrifugal filters with a molecular weight cutoff of 30,000 (Amicon) and subsequently aliquoted, flash-frozen in liquid nitrogen and stored at −80 °C.

Primer A: 5′-ATGCGGATCCAATGGATAACAAGCAAGAAGCAGAG-3′

Primer B: 5′-ATGCCTCGAGCTACTTGAAGAATCTGTCGATGGTG-3′

Primer C: 5′-ATGCGGATCCAAAACATCACCAGAAGAGCATTACTAG-3′

Plasmids for expressing X. laevis WT and CD Polκ in rabbit reticulocytes were kind gifts from J. Gautier36. Briefly, 2 μg of pCMV-Sport-Polκ was incubated with 100 µl of TnT Sp6 Quick master mix (Promega) supplemented with 4 μl of 1 mM methionine for 90 min at 30 °C. The reaction volume was subsequently adjusted to 400 μl with PBS and DNA was precipitated by the addition of 0.06% polymin-P and incubation for 30 min at 4 °C with rotation. The mixture was then centrifuged at 14,000g for 30 min and the proteins in the supernatant were precipitated with saturated ammonium sulfate to a final concentration of 55% for 30 min at 4 °C with rotation, followed by centrifugation at 16,000g for 30 min. The protein pellet was subsequently resuspended in 15 μl of ELB, dialyzed for 3 h at 4 °C in ELB. As a negative control, a reaction without DNA was performed. The Polκ protein preparations obtained using this method were used for gap-filling synthesis experiments (Extended Data Fig. 1d).

Plasmid pulldown

Plasmid pulldowns were performed as described previously22. Briefly, 6 μl of streptavidin-coupled magnetic beads (Dynabead M-280, Invitrogen) per pulldown reaction were equilibrated with wash buffer 1 (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA pH 8 and 0.02% Tween-20) and then incubated with 12 pmol of biotinylated LacI at RT for 40 min. The beads were washed four times with pulldown buffer 1 (10 mM HEPES pH 7.7, 50 mM KCl, 2.5 mM MgCl2, 250 mM sucrose, 0.25 mg ml−1 BSA and 0.02% Tween-20), resuspended in 40 μl and stored on ice until used. At the indicated time points, 10 μl of reaction was added to the beads and rotated for 30 min at 4 °C. The beads were subsequently washed twice in wash buffer 2 (10 mM HEPES pH 7.7, 50 mM KCl, 2.5 mM MgCl2, 0.25 mg ml−1 BSA and 0.03% Tween-20) and resuspended in 40 μl of 2× Laemmli sample buffer.

Chromatin spin down

Demembranated Xenopus sperm chromatin was prepared as described previously86 and stored at −80 °C at a concentration of 100,000 sperm chromatin per µl (320 ng µl−1). For analysis of UV-damaged chromatin, sperm chromatin was diluted to 25,000 sperm chromatin per µl in ELB, deposited on parafilm and irradiated with 2,000 J m−2 (for nonreplicating reactions) or 20 J m−2 (for replicating reactions) of UV-C. For nonreplicating reactions, HSS and NPE were premixed at a 1:2 ratio to block licensing. Subsequently, undamaged or UV-damaged sperm chromatin was added at a final concentration of 16 ng µl−1. For replicating reactions, sperm chromatin was licensed in one volume of HSS for 30 min followed by the addition of two volumes of NPE. At the indicated time points, 8 µl of replication reaction was stopped with 60 µl of ELB supplemented with 0.2% Triton-X. The mixture was carefully layered on top of a sucrose cushion (10 mM HEPES pH 7.7, 50 mM KCl, 2.5 mM MgCl2 and 500 mM sucrose) and spun for 1 min at 6,800g in a swing-bucket centrifuge at 4 °C. The chromatin pellet was carefully washed twice with 200 µl of ice-cold ELB and resuspended in 2× Laemmli buffer.

AlphaFold model generation

Molecular models were predicted using AlphaPulldown 0.30.0 (ref. 77), running AlphaFold 2.3.1 (ref. 78). AlphaPulldown parameters were as follows: cycles = 3, models = 5 and predictions per model = 1. Structure predictions were generated for X. laevis Q6DFE4 (POLK), P18248 (PCNA), Q6NRK6 (REV1), D0VEW8 (REV3), Q8QFR4 (REV7), O93610 (POLD2), Q76LD3 (POLD3) and P62972 (UBIQP), individually and as complexes of either full-length proteins or protein fragments.

Models were evaluated on their predicted local distance difference test78, interface predicted template modeling77,87, predicted template modeling78 and predicted aligned error88 scores. From each prediction, the best model as determined by AlphaPulldown was selected for inclusion in the final complex models.

Model building was performed using UCSF ChimeraX89,90. The catalytic complex was modeled on a scaffold of human Polκ holoenzyme with Ub-PCNA (Protein Data Bank (PDB) 7NV1 (ref. 55)).

The noncatalytic complex was modeled on a scaffold of the yeast Polζ (PDB 6V93 (ref. 69)). To establish the relative position of Polζ to PCNA, a structure of processive human Polδ holoenzyme was used (PDB 6TNY (ref. 91)). The monoubiquitinated PCNA and scaffold DNA attached to the polymerase complex was modeled on a structure of monoubiquitinated PCNA (PDB 3TBL (ref. 80)).

CHROMASS

CHROMASS experiments were performed as previously described62. Briefly, isolated sperm chromatin was either untreated or treated with 2,000 J m−2 of UV-C. Each reaction was performed in quadruplicate. The sperm chromatin was then incubated at a final concentration of 16 ng µl−1 in nonlicensing extracts that were mock-treated, Polκ-depleted or Rev1-depleted. Reactions were stopped after 45 min. Specifically, 10 µl of replication reaction was stopped with 60 µl of ELB supplemented with 0.2% Triton-X and chromatin spin down performed as described above. The chromatin pellet was then resuspended in 100 µl of denaturation buffer (9 M urea and 100 mM Tris-HCl pH 8) and transferred to a new low-binding tube. Cysteines were reduced (1 mM DTT for 15 min at RT) and alkylated (0.55 M chloroacetamide for 40 min at RT protected from light). Proteins were first digested with 0.5 µg of LysC (2.5 h at RT) and then with 0.5 µg of trypsin at 30 °C overnight. Peptides were acidified with 10% trifluoroacetic acid (pH < 4), followed by the addition of 400 mM NaCl, and purified by StageTip (C18 material). For this, StageTips were first activated in 100% methanol, then equilibrated in 80% acetonitrile in 0.1% formic acid and finally washed twice in 0.1% formic acid. Samples were loaded onto the equilibrated stage tips and washed twice with 50 µl of 0.1% formic acid. StageTip elution was performed with 80 μl of 25% acetonitrile in 0.1% formic acid; eluted samples were dried to completion in a SpeedVac at 60 °C, dissolved in 10 μL 0.1% formic acid and stored at −20 °C until MS analysis.

MS data acquisition

All MS samples were analyzed on an EASY-nLC 1200 system (Thermo) coupled to an Orbitrap Exploris 480 MS instrument (Thermo). Of the n = 4 biochemical replicates, 50% were analyzed per run (R1–R4). Afterward, an additional n = 4 technical replicates were performed by mixing 25%:25% of R1:R2 (R5), R2:R3 (R6), R3:R4 (R7) and R4:R1 (R8), totaling n = 8 technical replicates. Separation of peptides was performed using 20-cm columns (75-μm internal diameter) packed in house with ReproSil-Pur 120 C18-AQ 1.9-μm beads (Dr. Maisch). Elution of peptides from the column was achieved using a gradient ranging from buffer A (0.1% formic acid) to buffer B (80% acetonitrile in 0.1% formic acid), at a flow of 250 nl min−1. The gradient length was 80 min per sample, including ramp up and wash out, with an analytical gradient of 58 min ranging from 7% B to 34% B. Analytical columns were heated to 40 °C using a column oven and ionization was achieved using a NanoSpray Flex NG ion source. Spray voltage was set to 2 kV, ion transfer tube temperature was set to 275 °C and RF funnel level was set to 40%. The full scan range was set to 300–1,300 m/z, MS1 resolution was set to 120,000, MS1 automated gain control (AGC) target was set to ‘200’ (2,000,000 charges) and MS1 maximum injection time was set to ‘auto’. Precursors with charges 2–6 were selected for fragmentation using an isolation width of 1.3 m/z and fragmented using higher-energy collision disassociation with a normalized collision energy of 25. Precursors were excluded from resequencing by setting a dynamic exclusion of 80 s. The MS2 AGC target was set to ‘200’ (200,000 charges), intensity threshold was set to 360,000 charges per second, MS2 maximum injection time was set to ‘auto’, MS2 resolution was set to 30,000 and number of dependent scans was set to 13.

MS data analysis

All MS RAW data were analyzed using the freely available MaxQuant software (version 1.5.3.30)92 in a single computational run. Default MaxQuant settings were used, with exceptions specified below. For the generation of theoretical spectral libraries, the X. laevis FASTA database was downloaded from UniProt on October, 3 2022. In silico digestion of proteins to generate theoretical peptides was performed with trypsin, allowing up to three missed cleavages. The minimum peptide length was set to six and maximum peptide mass was set to 6,000 Da. Allowed variable modifications were oxidation of methionine (default), protein N-terminal acetylation (default), deamidation of asparagine and glutamine, peptide N-terminal glutamine to pyroglutamate conversion, dioxidation of tryptophan and replacement of three protons by iron (cation Fe(III)) on aspartate and glutamate. These variable modifications were determined by an initial analysis of the RAW data using pFind version 3.1.6 in ‘open search’ mode89 to unbiasedly determine any known modifications (from the Unimod database) affecting >0.5% of peptide–spectrum matches (PSMs) across all samples. The maximum number of variable modifications per peptide was set to three. Label-free quantification (LFQ) using MaxLFQ was enabled93 with ‘fast LFQ’ disabled. Matching between runs was enabled, with an alignment window of 20 min and a match time window of 1 min. A stringent MaxQuant 1% false discovery rate (FDR) control was applied at the PSM, protein and site-decoy levels (default).

MS data annotation and quantification

The X. laevis FASTA databases downloaded from UniProt lacked comprehensive gene name annotations. Missing or uninformative gene names were, when possible, semiautomatically curated, as described previously15. Quantification of the MaxQuant output files (‘proteinGroups.txt’) and all statistical handling were performed using Perseus software (version 1.5.5.3)94. In total, n = 8 technical replicates (derived from n = 4 biochemical replicates) were analyzed. 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). The statistical significance of differences was in all cases tested using two-tailed Student’s two-sample t-testing, with permutation-based FDR control applied to ensure a corrected P value (that is, q value) of <1%. Proteins not enriched over the no-DNA control in at least one CHROMASS condition (FDR < 1%, s0 = 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 = 0.5 and 2,500 rounds of randomization) on the remaining CHROMASS-enriched proteins.

Cell culture

Cells were cultured in high-glucose DMEM with glutaMAX Supplement and pyruvate (Gibco) supplemented with 10% FBS (Gibco) and 100 U per ml of penicillin–streptomycin (Gibco) at 37 °C with 5% CO2.

Generation of U2OS Flp-In T-REx POLK-KO cells

U2OS Flp-In T-REx cells were a kind gift from H. Piwnica-Worms. Four different gRNAs targeting different regions of POLK (5′-TAGGTTCAACACACCTGACG-3′, 5′-ATACATATAGATACCTCGTC-3′, 5′-ATACCGAGCTGTGAGTAAAG-3′ and 5′-AGGACAGGAAACACCAACAA-3′) were cloned into pSpCas9(BB)-2A-Puro (PX459) V2.0 (Addgene, 62988). sgRNA-containing plasmids were transfected into U2OS Flp-In T-REx cells using Dharmacon 1 (Horizon Discovert T-2005-01) transfection reagent according to the manufacturer’s protocol. After 24 h of incubation, transfected cells were selected with 1 µM puromycin for 48 h and plated sparsely to isolate single colonies. Single colonies were screened by qPCR for a lack of POLK mRNA using a primer pair (forward, 5′-TTGGGTCTAGGTTCAACACACC-3′; reverse, 5′-GCAAGCTCACTGCAAAGTTCT-3′). To perform the qPCR, total RNA was extracted using Qiagen RNeasy Mini (Qiagen 74104) according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized from total RNA using the iScript cDNA synthesis kit (BioRad, 1708890) according to the manufacturer’s instructions. qPCR was performed in 96-well plates using the mentioned primers and Brilliant III ultrafast SYBR green qPCR master mix (Agilent, 600882) in a Stratagene Mx3005P machine using standard thermocycling conditions.

Cloning of pcDNA5/FRT/TO/Venus–POLK constructs

Human WT and CD POLK (harboring D198A and E199A substitutions) cDNA sequences were a kind gift from O. Scharer. BamHI and NotI restriction sites were added using PCR (forward primer, 5′-ATGCGGATCCATG GATAGCACAAAGGAGAAGTGTGAC-3′; reverse primer, 5′-TATAGCGGCCGCTTACTTAAAAAATATATCAAGGGTATGTTTGGG-3′) and cloned into pcDNA5/FRT/TO-Venus. Constructs were sequence-verified.

Generation of POLK-KO cells stably expressing Venus–Polκ

To generate stable cell lines in the Flp-In system, U2OS Flp-In T-REx POLK-KO cells were cotransfected with the Flp recombinase-encoding plasmid pOG44 (Invitrogen) and a pcDNA5/FRT/TO plasmid encoding Venus–WT Polκ or Venus–CD Polκ at a 10:1 ratio using the jetOPTIMUS transfection reagent (Polyplus). Then, 48 h after transfection, cells were selected in medium supplemented with 5 μg ml−1 blasticidin S HCl and 200 μg ml−1 hygromycin B (Gibco) for 2–3 weeks. Expression from the Tet-ON inducible promoter in U2OS Flp-In T-REx cells was induced with 20 ng ml−1 doxycycline.

Colony formation assays

The cells were trypsinized, resuspended in medium and counted. A total of 200 cells were seeded per well in six-well plates with three wells per condition. Venus–Polκ-expressing Flp-In T-REx cells were induced with 20 ng ml−1 doxycycline. Then, 24 h after seeding, cells were treated with the indicated compound (31.25 pg ml−1 illudin S or 1 μM cisplatin) or left untreated. After seven additional days of growth, formed colonies were fixed and stained in a methyl violet solution (0.5% methyl violet and 25% methanol) and the number of colonies was quantified on a GelCount (Oxford Optronix). The survival after treatment with a given compound was calculated as the average number of colonies after treatment divided by the average number of colonies in the untreated condition multiplied by 100%. The experiments were performed three times independently and analyzed in PRISM (GraphPad). One-way analyses of variance (ANOVAs) with Tukey’s multiple-comparisons tests were performed to test for statistical significance.

Western blot analysis of cell lysates

Cells were harvested by trypsinization, lyzed on ice in radioimmunoprecipitation assay buffer (10 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS) supplemented with 1 mM DTT and cOmplete protease inhibitor cocktail (Roche) and sonicated with Bioruptor Plus (Diagenode). The lysate was cleared by centrifugation at 20,000g at 4 °C for 30 min and a BCA assay (Pierce) was used to measure protein concentrations. Samples were analyzed by SDS–PAGE and western blotting using anti-Polκ (Bethyl laboratories, A301-975A) and anti-tubulin (Abcam, ab6160) antibodies.

Base-editor tiling screen and analysis

An sgRNA library targeting the coding sequence of Polκ was designed. The gRNA oligonucleotide pool was synthesized by GenScript as an 83-nt oligonucleotide sequence, following a previously published design48. The oligonucleotide sequence consisted of primer sites for amplification with overhang sequences with Esp3I recognition sites and the gRNA: 5′-[forward primer (20 nt)]CGTCTCACACCG[sgRNA (20 nt)]GTTTCGAGACG[reverse primer (20 nt)]. The gRNA oligonucleotide pool was amplified using NEBNext Ultra II Q5 master mix (New England BioLabs) and the primers (forward, 5′-GTGTAACCCGTAGGGCACCT-3′; reverse, 5′-GTCGAGAGCAGTCCTTCGAC-3′). Amplicons were cloned into the Abe8e-Cas9-SpG lentiviral vector pRDA_479 (ref. 48) using Golden Gate cloning with Esp3I and T7 ligase. pRDA_479 was a gift from J. Doench and D. Root (Addgene, plasmid 179099). The ligated plasmid library was purified by PCR (NucleoSpin gel and PCR Clean‑Up, Macherey-Nagel) and isopropanol precipitation and electroporated into Endura electrocompetent cells (Lucigen), which were grown at 30 °C for 16 h on agar with 100 μg ml−1 carbenicillin. Plasmid DNA was prepared from the colonies on the plates (NucleoBond Xtra Maxi, Macherey-Nagel). To confirm library representation, the gRNA inserts were amplified from the plasmid library using NEBNext Ultra II Q5 master mix and the primers D506_F and D702_R_PAGE_BE (Supplementary Table 3). Gel-purified amplicons were sequenced on a NextSeq2000 (Illumina). The Polκ tiling library was part of a larger adenosine base editing (ABE) and cytosine base editing (CBE) tiling library, for which only Polκ with ABE is analyzed here. Note that some CBE guides also score, most likely because of low-frequency editing beyond the optimal 4–8-nt editing window. A lentiviral library was produced by cotransfection of HEK293T/17 cells (American Type Culture Collection, CRL-11268) with the sgRNA plasmid library and lentiviral packaging plasmids pMD2.G and psPAX2 using Lipofectamine 3000 (Invitrogen) in Opti-MEM medium (Gibco). pMD2.G (Addgene, plasmid 12259) and psPAX2 (Addgene, plasmid 12260) were gifts from D. Trono. Then, 6 h after transfection, the medium was exchanged for DMEM GlutaMax supplemented with 10% FBS, 100 U per ml of penicillin–streptomycin and 1% BSA. Next, 48 h after transfection, the lentiviral supernatant was collected and filtered through a 0.45-μm syringe filter before storing at −80 °C. RPE1-hTERT p53−/− cells (a kind gift from D. Durocher) were cultured in DMEM GlutaMax supplemented with 10% FBS and 100 U per ml of penicillin–streptomycin and passaged every 3 days. The screen was performed as a duplicate (two separate transductions) at a coverage of >500-fold sgRNA representation, which was maintained throughout the screen. Cells were transduced with the lentiviral library at a low multiplicity of infection (0.3–0.4) and transductions were caried out by treating cells with 8 μg ml−1 polybrene and lentiviral supernatant for 24 h. Transduced cells were selected by treatment with 20 μg ml−1 puromycin for 24 h followed by trypsinization and reseeding in the same plates with 20 μg ml−1 puromycin for another 24 h. After selection, cells were passaged for 6 days before splitting into untreated or illudin-S-treated fractions, where they were passaged for an additional 12 days in medium with or without a low dose of illudin S (1.4 ng ml−1). The dose of illudin S corresponds to a 20% reduction in cell numbers (LD20) compared to the untreated condition in uninfected cells, which was determined in a titration experiment. Genomic DNA was extracted from cell pellets harvested after selection, which we consider the start of the screen (t0) and at the final time point (t18). The genomic DNA region containing the integrated sgRNA was amplified by PCR using NEBNext Ultra II Q5 master mix with the LCV2_forward and LCV2_reverse primers (Supplementary Table 3). A second PCR reaction introduced i5 and i7 multiplexing barcodes (Supplementary Table 3) and gel-purified PCR products were sequenced on Illumina NextSeq2000. Sequencing data of t0, untreated t18 and illudin-S-treated t18 samples were converted to gRNA sequencing counts by MAGeCK95 and mapping was performed to gRNAs tiling POLK and control gRNAs (essential splice sites, nontargeting and intergenic)48. Low-abundance gRNAs were removed (counts < 30) and raw sequencing counts were normalized per condition replicate to log2 transcripts per million (log2TPM). The log2TPM values were compared using limma96 for three sample pairs (t0 versus untreated t18, t0 versus illudin-S-treated t18 and untreated t18 versus illudin-S-treated t18) and the fold change, P value and q value were collected for each gRNA. gRNA editing outcomes were predicted on the basis of the editing of all adenines within the editing window of positions 4–8 (ref. 97).

Reproducibility

A minimum of two independent experiments were conducted for each experimental result shown in this manuscript.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.