Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Pei Zhou ( peizhou@biochem.duke.edu ).

Six- to eight-week-old, female NCRNU-F nude mice (immunodeficient; nomenclature: CrTac:NCr-Foxn1 nu ; genotype: homozygous sp/sp) were purchased from Taconic Biosciences for experimentation. All mice were drug and test naive and were not involved in any previous procedure. All mice were housed in micro-isolator cages in the animal research facility of the MIT Division of Comparative Medicine (DCM), which is fully accredited by the AAALAC (Animal Welfare Assurance number A-3125) and meets NIH standards as set forth in the “Guide for Care and Use of Laboratory Animals” (DHHS). The MIT animal facility is maintained under specific pathogen free (SPF) conditions. The animal protocol was reviewed and approved by the MIT DCM Committee on Animal Care.

HT1080 cells (male, fibrosarcoma epithelial cells purchased from ATCC) were grown at 37°C with 5% COin RPMI 1640 (GIBCO), 10% (v/v) FBS (HyClone), and 1% Penicillin-Streptomycin antibiotic (Corning). A375 cells (female, malignant melanoma; kindly gifted by Oliver Jonas, Koch Institute, MIT), KP cells (female, mouse Kras;p53lung adenocarcinoma; kindly gifted by Tyler Jacks Lab, Koch Institute, MIT), and MEF (Mouse Embryonic Fibroblasts, sex unspecified in the original publication) wild-type (Rev1) and Rev1 knockout (Rev1) cells () were grown at 37°C with 5% COin DMEM (GIBCO), 10% (v/v) FBS (HyClone), and 1% Penicillin-Streptomycin antibiotic (Corning). LNCap cells (male, human prostate adenocarcinoma; kindly gifted by Michael Yaffe Lab, Koch Institute, MIT) were also grown at 37°C with 5% COin RPMI 1640 (-phenol) (GIBCO), 10% (v/v) FBS (HyClone), and 1% Penicillin-Streptomycin antibiotic (Corning). AG01522 cells (male, human primary cells purchased from Coriell Institute) were grown at 37°C with 5% COin MEM (-Glutamine; +Earle’s Salts; +Non-Essential Amino Acids) (GIBCO) and 20% (v/v) FBS (HyClone). All cells were trypsinized using 0.25% Trypsin-EDTA (ThermoFisher) for passaging.

BL21 Star (DE3) E. coli (ThermoFisher Scientific) and K12 JM109 E. coli (New England Biolabs) cells were used for protein expression and the gapped plasmid TLS assay, respectively. Selection and growth of E. coli was performed in the Lysogeny Broth (LB) medium supplemented with the appropriate antibiotics [100 μg/mL Ampicillin for the pET15b (Novagen/Sigma-Aldrich) or pUC19 (NEB) vectors or 50 μg/mL Streptomycin for the pCDFDuet-1 (Novagen/Sigma-Aldrich) vector] at 37°C.

Method Details

Molecular cloning and protein purification Wojtaszek et al., 2014 Wojtaszek J.L.

Wang S.

Kim H.

Wu Q.

D’Andrea A.D.

Zhou P. Ubiquitin recognition by FAAP20 expands the complex interface beyond the canonical UBZ domain. 10 -GB1 ( Zhou et al., 2001 Zhou P.

Lugovskoy A.A.

Wagner G. A solubility-enhancement tag (SET) for NMR studies of poorly behaving proteins. 600 of 0.5 with 0.1 mM isopropyl 1-thio-β-D-galactopyranoside (IPTG) at 18°C overnight. Harvested cells were lysed in a buffer containing 50 mM sodium phosphate (pH 8.0), 300 mM sodium chloride, and 0.1% β-mercaptoethanol using a French Pressure cell at 1250 psi. His 10 -GB1-tagged REV1 proteins were purified using Ni-NTA affinity chromatography (HisPur Ni-NTA, Pierce Biotechnology) and eluted with the lysis buffer containing 300 mM imidazole. Elution fractions were combined and exchanged into the FPLC buffer containing 25 mM HEPES (pH 7.5), 100 mM KCl, and 2 mM tris(2-carboxyethyl)phosphine (TCEP). Following TEV protease cleavage (1:20 molar ratio, 4 hours at room temperature) and a second Ni-NTA column to remove the His 10 -GB1 tag, POL κ RIR-REV1 CTD and its FLAG-tagged counterpart were further purified to homogeneity by size-exclusion chromatography (Superdex 200, GE Healthcare Life Sciences) in the FPLC buffer. The gene encoding the mouse POL κ RIR (K564-N577), a di-glycine linker, and the mouse REV1 CTD (F1150-T1249) was synthesized and cloned into a modified pET15b vector () as the C-terminal fusion protein to the solubility tag His-GB1 () separated by a TEV protease site. The FLAG-tagged chimeric POL κ RIR-REV1 CTD was generated by inserting the FLAG tag immediately after the TEV protease site. Both expression constructs were verified by DNA sequencing. The chimeric POL κ RIR-REV1 CTD and FLAG-tagged POL κ RIR-REV1 CTD were expressed in BL21 Star (DE3) E. coli cells (ThermoFisher Scientific). Cells were induced at O.D.of 0.5 with 0.1 mM isopropyl 1-thio-β-D-galactopyranoside (IPTG) at 18°C overnight. Harvested cells were lysed in a buffer containing 50 mM sodium phosphate (pH 8.0), 300 mM sodium chloride, and 0.1% β-mercaptoethanol using a French Pressure cell at 1250 psi. His-GB1-tagged REV1 proteins were purified using Ni-NTA affinity chromatography (HisPur Ni-NTA, Pierce Biotechnology) and eluted with the lysis buffer containing 300 mM imidazole. Elution fractions were combined and exchanged into the FPLC buffer containing 25 mM HEPES (pH 7.5), 100 mM KCl, and 2 mM tris(2-carboxyethyl)phosphine (TCEP). Following TEV protease cleavage (1:20 molar ratio, 4 hours at room temperature) and a second Ni-NTA column to remove the His-GB1 tag, POL κ RIR-REV1 CTD and its FLAG-tagged counterpart were further purified to homogeneity by size-exclusion chromatography (Superdex 200, GE Healthcare Life Sciences) in the FPLC buffer. 8 -REV7 containing a stabilizing R124A mutation and mouse REV3L (L1845-D1895) were synthesized, cloned into the pCDFDuet-1 vector, and verified by DNA sequencing ( Wojtaszek et al., 2012a Wojtaszek J.

Lee C.J.

D’Souza S.

Minesinger B.

Kim H.

D’Andrea A.D.

Walker G.C.

Zhou P. Structural basis of Rev1-mediated assembly of a quaternary vertebrate translesion polymerase complex consisting of Rev1, heterodimeric polymerase (Pol) ζ, and Pol κ. 8 -tagged REV7/3 was expressed in BL21 Star (DE3) cells (ThermoFisher Scientific). Cells were induced at O.D. 600 of 0.5 with 1 mM IPTG at 37°C for 6 hours. After lysing cells in a buffer containing 50 mM sodium phosphate (pH 8.0), 300 mM sodium chloride, and 0.1% β-mercaptoethanol using a French Pressure cell at 1250 psi, the His 8 -REV7/3 complex was purified by Ni-NTA chromatography (HisPur Ni-NTA, Pierce Biotechnology) and eluted with the lysis buffer containing 300 mM imidazole. The eluted His 8 -REV7/3 complex was further purified to homogeneity by size-exclusion chromatography (Superdex 200; GE Healthcare Life Sciences) in a buffer containing 25 mM HEPES (pH 7.5), 100 mM KCl and 2 mM TCEP. Codon-optimized genes encoding mouse His-REV7 containing a stabilizing R124A mutation and mouse REV3L (L1845-D1895) were synthesized, cloned into the pCDFDuet-1 vector, and verified by DNA sequencing (). His-tagged REV7/3 was expressed in BL21 Star (DE3) cells (ThermoFisher Scientific). Cells were induced at O.D.of 0.5 with 1 mM IPTG at 37°C for 6 hours. After lysing cells in a buffer containing 50 mM sodium phosphate (pH 8.0), 300 mM sodium chloride, and 0.1% β-mercaptoethanol using a French Pressure cell at 1250 psi, the His-REV7/3 complex was purified by Ni-NTA chromatography (HisPur Ni-NTA, Pierce Biotechnology) and eluted with the lysis buffer containing 300 mM imidazole. The eluted His-REV7/3 complex was further purified to homogeneity by size-exclusion chromatography (Superdex 200; GE Healthcare Life Sciences) in a buffer containing 25 mM HEPES (pH 7.5), 100 mM KCl and 2 mM TCEP.

Compound screening using the ELISA assay The ELISA assay for probing the REV1 CTD-REV7 interaction was carried out by immobilizing 50 nM His 8 -tagged REV7/3 in 200 μL phosphate-buffered saline (PBS, GIBCO) containing 0.2% BSA in a Ni-NTA coated 96-well plate (HisSorb, QIAGEN) for 30 minutes. Unbound His 8 -tagged REV7/3 was removed by washing the wells four times with PBS containing 0.05% Tween-20. In parallel, 80 nM FLAG-tagged POL κ RIR-REV1 CTD was pre-incubated with 10 μM small molecules in 200 μL PBS containing 2% DMSO for 30 minutes before transferring to the His 8 -REV7/3 coated wells. After incubation for 30 minutes, the wells were washed four times with PBS containing 0.05% Tween-20 to remove the unbound FLAG-tagged POL κ RIR-REV1 CTD. A solution of the anti-FLAG horseradish peroxidase (HRP)-conjugated antibody (Sigma-Aldrich) in PBS containing 0.2% BSA was then added to the wells. After incubating for 1 hour, the antibody was washed off four times with PBS containing 0.05% Tween-20. The 3,3′,5,5′-tetramethylbenzedine (TMB) substrate (SureBlue TMB, Seracare) was added to the wells. After incubation of 20-30 minutes, the reaction was quenched with 1 M HCl. A SpectraMax plate reader (Molecular Devices) was used to measure absorbance at 450 nm. The ELISA assay was used to screen several compound libraries, including the LOPAC library (Sigma-Aldrich) of 1,280 pharmacologically active compounds, the PRESTWICK library (Prestwick Chemical) of 1,200 FDA-approved drugs, and ∼8,000 compounds from the Korea Chemical Bank that are structural representatives of ∼430,000 diverse compounds. Among several hit compounds, JH-RE-06 was selected for further characterization due to its potency.

Chemical synthesis of JH-RE-06 (8-chloro-2-((2,4-dichlorophenyl)amino)-3-(3-methyl-butanoyl)-5-nitroquinolin-4(1H)-one) and JH-RE-25 (8-chloro-3-(3-methylbutanoyl)-2-morpholino-5-nitroquinolin-4(1H)-one) Choi et al., 2003 Choi E.B.

Yon G.H.

Lee H.K.

Yang H.C.

Yoo C.Y.

Pak C.S. Synthesis of β-lactam from acyl(arylcarbamoyl)-S,S-bis(alkylketene) dithioacetal: revised structure of the product from thermal cyclization of acyl(arylcarbamoyl)-S,S-bis(alkylketene) dithioacetal. Pak et al., 1992 Pak C.S.

Yang H.C.

Choi E.B. Aminolysis of 5-acyl-2,2-dimethyl-1,3-dioxane-4,6-diones (acyl meldrum’s acids) as a versatile method for the synthesis of β-oxo carboxamides. 9) was efficiently prepared from the commercially available Meldrum’s acid (1). The synthesis started with the preparation of the acyl Meldrum’s acid (3), which was reacted with 2-chloro-5-nitroaniline (4) under reflux. The reaction took place with the evolution of CO 2 to provide the β-oxo amide (5). Subsequently, the β-oxo amide (5) was transformed into the acyl(arylcarbamoyl)-ketene dithioacetal (6) by using CS 2 and Me 2 SO 4 in the presence of K 2 CO 3 . Thermal cyclization in 1,2-dichlorobenzene followed by oxidation using H 2 O 2 afforded the sulfoxide intermediate (7). Coupling of the sulfoxide intermediate (7) with 2,4-dichloroaniline (8) completed the synthesis of JH-RE-06 (9). In a similar manner, the sulfoxide intermediate (7) was coupled to morpholine to afford JH-RE-25 (10). The chemical identity and purity of the prepared compounds were verified by LC/MS and NMR. Upon identification of JH-RE-06 as a hit compound in the REV1 CTD-REV7/3 ELISA assay, we developed a modular synthetic route of the molecule ( Figure S2 A). Following literature reports on the synthesis of the 1,4-dihydroquinolinone scaffold (), JH-RE-06 () was efficiently prepared from the commercially available Meldrum’s acid (). The synthesis started with the preparation of the acyl Meldrum’s acid (), which was reacted with 2-chloro-5-nitroaniline () under reflux. The reaction took place with the evolution of COto provide the β-oxo amide (). Subsequently, the β-oxo amide () was transformed into the acyl(arylcarbamoyl)-ketene dithioacetal () by using CSand MeSOin the presence of KCO. Thermal cyclization in 1,2-dichlorobenzene followed by oxidation using Hafforded the sulfoxide intermediate (). Coupling of the sulfoxide intermediate () with 2,4-dichloroaniline () completed the synthesis of JH-RE-06 (). In a similar manner, the sulfoxide intermediate () was coupled to morpholine to afford JH-RE-25 (). The chemical identity and purity of the prepared compounds were verified by LC/MS and NMR.

Dose-dependent inhibition of the REV1 CTD-REV7/3 interaction by the AlphaScreen assay The FLAG-tagged mouse POL κ-REV1 CTD was diluted in PBS containing 1 mM Tris(2-carboxyethyl)phosphine (TCEP) and 0.005% Tween-20 at a final protein concentration of 1 nM and transferred to individual wells of a 96-well, half-area, white opaque plate (PerkinElmer). Serially diluted JH-RE-06 stock solutions in 50% DMSO were added to the wells to yield final inhibitor concentrations of 0-25 μM in 2% DMSO. After 30 min incubation, anti-FLAG Donor Beads (PerkinElmer) were added to a final concentration of 20 ng/μL to individual wells and incubated for an hour. His 8 -tagged mouse REV7/3 was subsequently added to the reaction mixture to a final concentration of 10 nM and incubated for 30 min. Anti-His Acceptor Beads (PerkinElmer) were added to a final concentration of 20 ng/μL and incubated for an hour. The chemiluminescent signals were observed with a PerkinElmer Enspire Reader at the excitation wavelength of 680 nm and detection wavelength of 615 nm.

Isothermal titration calorimetry Isothermal titration calorimetry measurements were carried out using a MicroCal VP-ITC instrument at 25°C, with the chimeric REV1 CTD protein (300 μM) in the syringe and compound (15 μM) in the cell. Compounds were initially dissolved in 50% MPD. Protein and compound samples were diluted in a buffer containing 50 mM HEPES pH 7.5, 50 mM KCl, 2 mM TCEP, 2% DMSO and 0.1% MPD. Microcal Origin 7 software was used to analyze the data.

X-ray crystallography Apo POL κ RIR-REV1 CTD A sample of 0.6 mM chimeric mouse POL κ-REV1 CTD in 25 mM HEPES pH 7.2, 100 mM KCl, 30 mM CHAPS, and 2 mM TCEP was mixed with the mother liquor containing 0.1 M sodium acetate, 25% w/v PEG 4000, 8% w/v isopropanol at a 1:1 drop ratio and crystallized upon incubation at 20°C. Crystals were flash frozen in liquid nitrogen without additional cryoprotectants. The POL κ RIR-REV1 CTD/JH-RE-06 complex A random micro-seed matrix screen was performed using a sample solution containing 0.6 mM of the chimeric POL κ RIR-REV1 CTD and 4 mM JH-RE-06 NaOH salt in a crystallization buffer of 25 mM HEPES pH 7.0, 100 mM KCl, 16.7% MPD and 0.1% β-mercaptoethanol and crystal seeds derived from apo protein crystals, yielding diffracting crystals in a mother liquor containing 20% PEG 3350, and 0.2 M magnesium formate. High-quality crystals were obtained through repeated seeding, and the final crystallization conditions contain 12.5 mM HEPES (pH 7.5), 50 mM KCl, 8.35% MPD, 0.05% β-mercaptoethanol, 10% PEG 3350 and 0.1 M magnesium formate. The crystals were harvested and cryo-protected with the mother liquor containing 15% MPD and 1.88 mM JH-RE-06 NaOH salt. Kabsch, 2010 Kabsch W. XDS. Emsley and Cowtan, 2004 Emsley P.

Cowtan K. Coot: model-building tools for molecular graphics. Adams et al., 2002 Adams P.D.

Grosse-Kunstleve R.W.

Hung L.W.

Ioerger T.R.

McCoy A.J.

Moriarty N.W.

Read R.J.

Sacchettini J.C.

Sauter N.K.

Terwilliger T.C. PHENIX: building new software for automated crystallographic structure determination. X-ray diffraction datasets were collected on the SER-CAT 22-ID beamline at Argonne National Laboratory and processed with XDS (). The structures of the apo POL κ RIR-REV1 CTD and the POL κ RIR-REV1 CTD/JH-RE-06 complex were determined by molecular replacement using the coordinate of the mouse REV1 CTD and POL κ RIR components of our previously determined quaternary complex crystal structure (PDB: 4FJO ) as the search model. The final coordinates were constructed by iterative cycles of model building with COOT ()) and refinement with PHENIX () and were deposited to the Protein Data Bank with accession numbers of 6C59 and 6C8C for the apo protein and the inhibitor-bound complex, respectively.

In vitro DSS-crosslinking experiment Chimeric FLAG-tagged POL κ RIR-REV1 CTD in a buffer containing 25 mM HEPES (pH 7.0), 100 mM KCl, and 4 mM TCEP was mixed with either MPD (control) or JH-RE-06 NaOH salt in MPD to yield a reaction solution containing 1 μM protein, 5% MPD, and either 0 or 100 μM compound. Appropriate dilutions of DSS in DMSO were added to the reaction mixture to yield DSS-to-protein molar ratios of 0:1, 0.5:1, 1:1, 5:1, 10:1, and 50:1 and a final DMSO concentration of 5% (v/v). The reaction mixture was incubated for 30 min at room temperature and then quenched by addition of 1 M Tris (pH 8.5). The SDS-loading dye containing 4 mM TCEP and 10.8 mM iodoacetamide (to block free cysteines) was added to each reaction mixture, and the samples were loaded onto a Pre-cast 4%–20% gradient SDS-PAGE gel (Bio-Rad). The gel samples were transferred to a 0.45 μm nitrocellulose membrane (Bio-Rad) for western blotting with the anti-FLAG primary antibody M2 (Sigma-Aldrich) and the HRP-conjugated secondary antibody (LI-COR) and imaging with the LI-COR Odyssey system.

Clonogenic survival assay Mochizuki and Furukawa, 1987 Mochizuki Y.

Furukawa K. Application of coomassie brilliant blue staining to cultured hepatocytes. 300 cells were plated in triplicate in 6-well plates for 24 hours. Cisplatin (cis-diammineplatinum(II) dichloride, Sigma-Aldrich) was added to relevant wells for 24 hours. All plates were incubated at 37°C for 24 hours. Media were changed the next day and in fresh media JH-RE-06 (at 1.5 μM concentration) was added to untreated or cisplatin-treated cells for another 24 hours. Media were changed at the end of these combination treatments, and cells were allowed to recover for 7 days. To stain the resulting colonies, media were aspirated and the fixative (50% methanol and 10% glacial acetic acid) was added for 10 minutes, followed by the addition of 0.02% Coomassie brilliant blue R-250 stain (ThermoFisher) in methanol: acetic acid: water in a ratio of 46.5:7:46.5 (v/v/v) (). Colonies that stained blue and contained at least 40 cells were counted. Relative cell survival or colony formation was calculated by dividing colony counts from treated samples by the DMSO or untreated controls.

Viability assay Relative viability of cells in response to JH-RE-06 and DNA-damaging agents was assessed by the CellTiter-Glo Luminescence cell viability assay (Promega) that determines the number of viable cells based on the relative amount of ATP in the culture, which is directly proportional to the number of metabolically active cells. Briefly, 10,000 cells were plated in each well of a 96-well, white, clear flat bottom plate (Corning). Increasing doses of drugs in various combinations—JH-RE-06 alone or in combination with DNA-damaging agents—were added into the plates after 24 hours. The JH-RE-06 compound was dissolved in 0.1% DMSO and other drugs were dissolved in solvents ascribed by the manufacturer. In all cases, DMSO controls were run in parallel to the drug treatments. The relative viability of cells was monitored after 24 hours of drug treatment by adding CellTiter-Glo Luminescence stain to an equilibrated plate per the manufacturer’s instructions. Luminescence was measured on the plate reader (Tecan Spark 10M). Relative luminescence, which is indicative of relative survival of metabolically active cells, was calculated by dividing the luminescence of treated samples with DMSO controls.

HPRT mutagenesis assay - cells. Colonies were fixed (50% methanol and 10% glacial acetic acid), stained (0.02% Coomassie brilliant blue R-250 stain in methanol: acetic acid: water in a ratio of 46.5:7:46.5 (v/v/v)), and counted after 14-20 days. The HPRT mutation frequency was calculated as the ratio of the number of HPRT- colonies in 6-TG media to the number of surviving colonies plated in complete media to determine clonal efficiency ( Silva et al., 2005 Silva M.J.

Costa P.

Dias A.

Valente M.

Louro H.

Boavida M.G. Comparative analysis of the mutagenic activity of oxaliplatin and cisplatin in the Hprt gene of CHO cells. For the hypoxanthine-guanine phosphoribosyl transferase (HPRT) mutagenesis assay, cells were first grown in HAT (complete media with 100 μM Hypoxanthine, 0.4 μM Aminopterin and 16 μM Thymidine) media (ThermoFisher) for 14 days to weed out any spontaneous HPRT mutants. After HAT selection, cells were exposed to cisplatin at the 0.5 μM concentration for 24 hours. Then, in fresh media, JH-RE-06 at a concentration of 1.5 μM was added to cells. After 24 hours of drug treatment, cells were trypsinized and washed with PBS. While 200-600 cells were plated in complete media in triplicates in 6-well plates to determine clonal efficiency, the rest of the cells were plated in complete media to allow the expression of the phenotype for 8 days. Then, 500,000 cells per treatment were plated in sextuplicate in 10 cm dishes in 6-TG media to allow the proliferation of mutated HPRTcells. Colonies were fixed (50% methanol and 10% glacial acetic acid), stained (0.02% Coomassie brilliant blue R-250 stain in methanol: acetic acid: water in a ratio of 46.5:7:46.5 (v/v/v)), and counted after 14-20 days. The HPRT mutation frequency was calculated as the ratio of the number of HPRTcolonies in 6-TG media to the number of surviving colonies plated in complete media to determine clonal efficiency ().

Nucleofection REV1 was knocked down by transiently transfecting SMARTpool: ON-TARGETplus REV1 siRNA by nucleofection. The siRNA was mixed with the nucleofection buffer Mouse/Rat Hepatocyte Nucleofector Kit (Lonza) and electroporated using the Nucleofector™ 2b device. Full-length mouse Rev1 on the pC3 plasmid (Clontech) was nucleofected using the same buffers and device into Rev1−/− cells to complement the REV1 function.

Synthesis of the 16-mer oligonucleotide containing a cisplatin 1,2-GG lesion and construction of the gapped plasmid The 16-mer oligonucleotide containing a cisplatin 1,2-GG lesion was synthesized as described below. The platination reaction was carried out with aquated derivatives of the platinum complexes to facilitate their reaction with a 16-mer oligonucleotide containing a 1,2-GG sequence (5′-CTCTCTCGGCCTTCTA-3′). The aquated complexes were obtained by overnight stirring in the dark at room temperature of a solution containing cisplatin and 1.98 equivalent of silver nitrate. The precipitated silver chloride was removed by a 0.2 μm syringe filter. DNA was mixed with aquated platinum complex in a 1:2 ratio at 37°C for 2 hours. The target 16-mer oligonucleotide containing the cisplatin 1,2-GG lesion was purified by reverse-phase HPLC with a C18 column (5um, 100Å, 150X4.6 mm, Phenomenex). The molecular weight and lesion location were characterized by LC-MS (AB Sciex). The double stranded plasmid pUC19 with ampicillin resistance was modified to include the oligonucleotide containing cisplatin 1,2-GG lesion on one strand and a gapped region across it. Briefly, the 16-mer cisplatin-containing oligonucleotide was flanked by two 21-mer regular DNA strands (5′-GCCCGTCGTAGCGCGCATGCA-3′ on the 5′ end and 5′-TCTCGAGTGTTCCGTCAGCAC-3′ on the 3′ end) and elongated to a 58-mer lesion-containing single strand DNA. After linearizing the plasmid by restriction endonucleases BstAPI and BspQI (New England Biolabs), the 58-mer oligonucleotide was mixed with two scaffolds (5′-TGCATGCGCGCTACGACG-3′ and 5′-AGCGTGCTGACGGAACACTCGAGA-3′) and ligated with the linear pUC19 plasmid to build up a circular plasmid containing a site-specific cisplatin 1,2-GG lesion and a 16-nucleotide gap on the opposite strand.

The quantitative assay of the gapped plasmid containing a cisplatin 1,2-GG lesion A competitor gapped plasmid that was three bases longer (started from a 19-mer oligonucleotide 5′- CTCTCTAGGCTCACTTCTA −3′) than the lesion-containing plasmid was used as the internal control. The cells were pre-treated with either DMSO or JH-RE-06 (1.5, 3.0 and 15.0 μM) for 24 hours. Gapped-lesion plasmid (200 ng) and competitor plasmid (50 ng) were transfected in a 4:1 ratio into 300,000 HT1080 cells using Lipofectamine 3000 (ThermoFisher). Transfected cells were incubated at 37°C for 4 hours. Next, the cells were trypsinized (0.25% trypsin-EDTA), and DNA was extracted using the QIAGEN DNA isolation kit. The isolated DNA was then transformed into the recA- E. coli strain, JM109, to propagate fully closed plasmids obtained from the mammalian cells. After 16 hours, total plasmid DNA was isolated from the E. coli cells and the region encompassing the cisplatin lesion from both the cisplatin and competitor plasmids was amplified by PCR (forward primer: 5′-TTGTACTGAGAGTGCACCATGCCCGT-3′, reverse primer: 5′-GAGTCAGTGAGCGAGGAAGCGTGCTG-3′). Two restriction endonucleases XhoI and SphI (New England Biolabs, Ipswich, MA) were used to digest the PCR products into short DNA pieces, 20-mer for the cisplatin plasmid and 23-mer for the competitor plasmid. The digestion products were chromatographed on a PolarAdvantage C18 column (250 X 2.1 mm, 3μm, 120Å, ThermoFisher) eluted at 0.1 mL/min with a methanol gradient (15% - 50%, 400 mM hexafluoro-2-propanol), followed by the ESI triple quadrupole time-of-flight mass spectrometry (AB Sciex 4600) to detect the final nucleotide signal in the negative ion mode. The input ratio is the initial 4:1 ratio of the cisplatin 1,2-GG lesion and the competitor plasmid that were used to transfect mammalian cells. The gap-filling efficiency by TLS was calculated by dividing the output ratio obtained from HPLC-MS with the input ratio (4:1) and the results were normalized to 100%.