Fig 2: Model of Hrq1/RECQL4 function during replication-associated intrastrand crosslink repair.When the replication fork stalls, PCNA (yellow trimer) is monoubiquitylated (Ub, purple circle) by Rad6-Rad18 on lysine 164 (K164). Monoubiquitylation of PCNA recruits the error-prone translesion synthesis polymerases, i.e. Rev1 to bypass the lesion. Alternatively, PCNA is further poly-ubiquitylated by Rad5-Ubc13-Mms2. Polyubiquitylation of PCNA mediates error-free repair through template switching, which is a homology directed process. Hrq1/RECQL4 (blue oval) facilitates a template switch to bypass the lesion and then is subsequently ubiquitylated by Rad16 and degraded. Degradation of Hrq1 by the proteasome prevents aberrant recombination and subsequent mutation. Once the lesion is bypass, the NER machinery facilitates repair. Created using biorender.com.

Fig 3: RECQL4 functions in a separate pathway from REV1 and is degraded following DNA crosslinks.(A) RECQL4 levels decrease following cisplatin treatment. U2OS cells were incubated with 50 μg/ml cycloheximide in the presence or absence of 50 μg/ml cisplatin. Protein extracts were analyzed by western blot for endogenous RECQL4 protein levels (α-RECQL4) or a loading control, Tubulin (γ-Tubulin), at the indicated time points. Quantification of the proportion of RECQL4 remaining relative to time 0 (before CHX addition) and the loading control, Tubulin, are plotted on the graph in log scale. Each experiment was performed in triplicate with standard error plotted (Raw densitometry data in Sheets A-C in S6 Data). (B) RECQL4 protein levels decrease following acetaldehyde treatment. U2OS cells were incubated with 50 μg/ml cycloheximide in the presence or absence of 50 μg/ml acetaldehyde. Protein extracts were analyzed by western blot for endogenous RECQL4 protein levels (α-RECQL4) or a loading control, GAPDH (α-GAPDH), at the indicated time points. Quantification of the proportion of RECQL4 remaining relative to time 0 (before CHX addition) and the loading control, GAPDH, are plotted on the graph in log scale. Each experiment was performed in triplicate with standard error plotted (Raw densitometry data in Sheets D-F in S6 Data). (C) Cisplatin exposed U2OS cells exhibit decreased viability when both RECQL4 and REV1 are knocked down by siRNA. MTS assay was performed on after 72 hr exposure to the indicated dose of cisplatin. Western is shown to confirmed knockdown of proteins, note since RECQL4 and REV1 are of similar size, the same samples were loaded twice to detect RECQL4 and REV1. Each experiment was performed three times with mean and standard errors plotted (Unprocessed data in S7 Data). (D) Decreased cell viability following cisplatin exposure when both RECQL4 and REV1 are knocked down. Clonogenic survival assay with siControl (scrambled siCon), siRECQL4, siREV1, and siRECQL4 siREV1 treated cells with or without cisplatin. Each condition was plated in triplicate for each experiment and the experiment was performed three times with mean and standard errors plotted (Colony quantification in S8 Data).

Fig 4: Generation of REV1 and UNG knockout cell line clones.a) Schematic of UNG locus. Position of exon 1, targeting sgRNAs (sgUNG #1, sgUNG #2 and sgUNG#3) and primers for PCR screening (JM1093 and JM1094) are indicated. b) PCR amplicons generated using primers JM1093 and JM1094 and genomic DNA templates prepared from the indicated cell lines. n = 2 experiments. c) Plots depict a percentage of sequenced amplicons generated as in b that contain deletions (purple). d) Schematic of REV1 locus. Position of exon 4, targeting sgRNAs (sgREV1 #1, sgREV1 #2 and sgREV1 #3), and primers for PCR screening (KC35 and KC36) are indicated. e) PCR amplicons generated using primers KC35 and KC36 and genomic DNA templates prepared from the indicated cell lines. n = 2 experiments. f) Plots depict a percentage of sequenced amplicons generated as in e that contain deletions (purple). g) Schematic of SMUG1 locus. Position of exon 2, targeting sgRNAs (sgSMUG1 #1, sgSMUG #2 and sgSMUG #3), and primers for PCR screening (KC23 and KC21) are indicated. h) PCR amplicons generated using primers KC23 and KC21 and genomic DNA templates prepared from the indicated cell lines. n = 2 experiments. i) Plots depict a percentage of sequenced amplicons generated as in h that contain deletions (purple). j) Immunoblotting with anti-APOBEC3A (01D05), anti-APOBEC3 (04A04), anti-UNG, anti-REV1, anti-SMUG1, and anti-actin antibodies in the indicated cell lines. n = 3 experiments.

Fig 5: The presence of functional Rev1 determines the types of mutations near a quadruplex sequence. (A) A schematic representation of the DNA sequence surrounding the supF gene in the pSP189-G4 plasmid is shown. The region analyzed for mutations was divided into four ‘zones’: Zone I (dotted line) includes 26 bp to the 5′-side of the G4 sequence, Zone II (green) includes the G4 sequence derived from the c-Myc promoter, Zone III (yellow) includes the 85 bp supF tRNA coding sequence, and Zone IV (dotted line) 30 bp sequence on the 3′-side of the supF gene. The sequences of the mutated plasmids harvested from white colonies are reported in supplementary information (Supplementary Figures S6 and S7). The distribution of mutations (base substitutions and deletions) on the G4 plasmid obtained for (B) HAP-1 cells and (C) REV1KO cells (±0.5 μM PDS) is reported for each zone. Ten plasmids extracted from white colonies from three replicates from each experimental condition were sequenced. A multiple sequence alignment of all the DNA sequences, including the original G4-pSP189 plasmid was performed using T-Coffee (21). At each position, the type of mutation - substitutions (blue) or deletions (red) were marked and summed over all 10 sequences per condition. Non-mutated positions (grey) were also counted similarly. Each pie-chart represents the percentage distribution of substitutions, deletions or non-mutated bases for each of the four zones