Whole genome CRISPR screen identifies N6-methyltransferases in repair of floxuridine-induced DNA lesions.

(A) Schematic of whole genome CRISPR screen in HT-29 cells reported in B. (B) Volcano plot displaying MAGeCK gene level log2(fold change) for each gene in treated and untreated arms versus -log10(p-value). Cut-off displays genes with log2(fold change) >|0.5| and -log10(p-value) >2. Genes whose loss sensitizes cells to floxuridine skew to the left. Non-targeting guides are shown in light gray and fall below cut-off values. Essential genes also performed as expected, dropping out at later time points (data not shown). (C) KEGG pathway analysis for genes that sensitize cells to floxuridine with log2(fold change) >|0.5| and -log10(p-value) >2. Flox, floxuridine; FDR, false discovery rate

Discovery-based proteomics identifies N6-methyltransferases at sites of UNG2-seeded condensates.

(A) Representative images from DLD-1 UNG KO cells expressing indicated mCherry-tagged cDNAs upon treatment with increasing concentrations of floxuridine at 64 hours post treatment.

(B) Quantification of experiment represented in A for percentage of cells with >5 mCherry foci.

Error bars, mean ± SEM; Ordinary one-way ANOVA with Dunnett’s multiple comparisons test with a single pooled variance, ∗∗∗p ≤ 0.001, n = 3 biological replicates. Statistical tests performed within individual groups, EV or UNG2, respectively. (C) PONDR VSL2 plot of disorder for UNG2.

(D) Representative schematic of mutant UNG2 cDNA constructs expressed in UNG KO DLD-1 cells in E. IDR only cDNA lacks amino acids 93, ΔIDR cDNA lacks amino acids 1-92, and IDR-C cDNA moved amino acids 1-92 to the C-terminus. (E) Representative images from DLD-1 UNG KO cells expressing indicated mCherry-Cry2-tagged cDNAs without or with stimulation of blue light for 60 seconds. While ΔIDR and EV images display cytoplasmic foci, these lack the distinct nuclear foci patterning observed for UNG2, IDR, and IDR-C constructs. (F) Schematic of proximity biotinylation of IDR interaction partners in cells. (G). Venn diagrams of factors identified by stable isotope labeling of amino acids in cell culture (SILAC)-based mass spectrometry (MS) with 1>log2(L/H), reflecting a 4-fold enrichment in UNG-IDR/Control, from two biological replicates. n.s., non-statistically significant; Flox, floxuridine; EV, empty vector; IDR, intrinsically disordered region

METTL3 deposits N6-methyladenonsine in DNA in response to agents that increase genomic uracil.

(A) Representative immunoblot images from DLD-1 cells nucleofected with ribonucleoproteins containing Cas9 and indicated guide RNAs (gRNA) as performed for B. α-tubulin represents loading control. (B) MTS cell viability assay in the presence of floxuridine. Error bars, mean ± SEM, n=2 biological replicates. (C) Growth curves in DLD-1 cells upon treatment with 15 μM METTL3 inhibitor and indicated drug concentration post treatment. Error bars, mean ± SEM, n=3 biological replicates. (D) Representative images from DLD-1 UNG KO upon treatment with floxuridine for 66 hours. Prior to staining with N6-methyladenosine antibody, indicated samples were treated with RNase A or DNase. (E) Quantification of experiment represented in D for percentage of cells with >5 N6-methyladenonsine foci. Error bars, mean ± SEM; Repeated measures one-way ANOVA with Dunnet’s multiple comparisons test with a single pooled variance, ∗p ≤ 0.05, n = 5 biological replicates. (F) Ratio of 6mA analyte to dA analyte as detected in DNA of DLD-1 cells upon treatment with 500 nM floxuridine or 500 nM raltitrexed using UPLC-MS/MS. ∗∗∗∗p ≤ 0.0001, n=3 biological replicates. (G) Representative images of 6mA staining in DLD-1 UNG KO cells upon treatment with 500 nM of floxuridine and 30 μM METTL3 inhibitor at 64 hours. (H) Quantification of experiment represented in G for percentage of cells with >10 6mA foci. Error bars, mean ± SEM; Ordinary one-way ANOVA with Dunnett’s multiple comparisons test with a single pooled variance, ∗p ≤ 0.05, n = 3 biological replicates. HPRT, cutting control targeting intronic region of HPRT gene; Flox, floxuridine; METTL3i, METTL3 inhibitor

6mA promotes uracil repair upstream of UNG2 in uracil base excision repair.

(A) Representative images of mCherry staining in DLD-1 UNG KO cells expressing UNG2-mCherry cDNAs upon treatment with 500 nM floxuridine and 30 μM METTL3 inhibitor at 64 hours.

(B) Quantification of experiment represented in A for percentage of cells with >5 mCherry foci.

Error bars, mean ± SEM; Ordinary one-way ANOVA with Tukey’s multiple comparisons test with a single pooled variance, ∗p ≤ 0.05, n = 3 biological replicates. (C) Real-time quantitative PCR from A, B for UNG2 transcript levels normalized to tubulin controls. Error bars, mean ± SEM; Mann-Whitney t-test for the following pairs: DMSO versus METTL3 inhibitor and floxuridine versus floxuridine + METTL3 inhibitor. ∗p ≤ 0.05, n = 3 biological replicates except for the floxuridine only condition which includes n=2 biological replicates. (D) Representative images of 6mA staining in DLD-1 UNG KO cells upon treatment with 500 nM floxuridine at 64 hours. HPRT indicates wild type cells. These cells were targeted with a cutting control targeting the intronic region of HPRT gene. (E) Quantification of experiment represented in D for percentage of cells with >5 6mA foci. Error bars, mean ± SEM; RM one-way ANOVA with Dunnet’s multiple comparisons test with a single pooled variance, ∗p ≤ 0.05, n = 5 biological replicates.

Flox, floxuridine; METTL3i, METTL3 inhibitor

6mA promotes genome repair of base damage beyond uracil incorporation.

(A, B, C) MTS cell viability in SW620 cells upon treatment with METTL3 inhibitor and indicated concentrations of drugs. Error bars, mean ± SEM, n=3, technical replicates, representative of 3 biological replicates. (D) Representative images of 6mA staining in DLD-1 UNG KO cells upon treatment with indicated DNA damaging agents at 64 hours. (E) Quantification of experiment represented in D for percentage of cells with >5 6mA foci. Error bars, mean ± SEM; Ordinary one-way ANOVA with Dunnet’s multiple comparisons test with a single pooled variance, ∗∗p ≤ 0.01, n = 3 biological replicates for all except for the HU condition which includes n=2 biological replicates. (F) Schematic of colony formation assay. SW620 cells, maintained in HAT media, were treated with 30 μM of METTL3 inhibitor for 7 days. 0.5×104 METTL3 inhibitor-treated cells seeded in the presence of 5 μM 6-Thioguanine (TG) and colony formation assay was assessed after 14 days. 0.5×102 METTL3 inhibitor-treated cells were seeded for untreated controls. (G) Representative dishes after 14 days of growth in 5 μM TG as described in F. (H) Quantitation of mutation frequency from G. Mutation frequency was calculated by normalizing to the untreated controls. Error bars, mean ± SEM; Paired t-test, ∗∗p ≤ 0.01, n = 3 biological replicates. HU, hydroxyurea; Gem, gemcitabine; MMC, mitomycin C; TG, 6-thioguanine; METTL3i, METTL3 inhibitor