Supporting Information

Supporting Information Nagel et al. 10.1073/pnas.1401182111 N × MFI SI Materials and Methods F = ; [S1] S Human Cells. A total of 48 human cell lines derived from 39 individuals were obtained from the Coriell Institute or the where N is the total number of live cells appearing in the pos- – American Type Culture Collection (Table 1). Epstein Barr itive region for that fluorophore, MFI is the mean fluorescence virus-transformed lymphoblastoid cell lines were maintained intensity of the N cells, and S is the total number of live cells. in log phase in Gibco RPMI 1640 supplemented with 15% The fluorescence signal from the reporter protein expressed (vol/vol) FBS and 2 mM L-glutamine, penicillin, and strepto- from an undamaged plasmid, included in all transfections to mycin. Primary skin fibroblasts and glioblastoma cell lines were control for transfection efficiency, was designated FE.Thenor- cultured in Gibco DMEM/F12 medium supplemented similarly malized fluorescence signal for a given reporter FO was calcu- but with 10% (vol/vol) FBS and nonessential amino acids for the lated using Eq. S2: glioblastoma cells. Fibroblasts and glioblastoma cells were subcultured by trypsinization. F FO = : [S2] FE Rodent Cells. Mouse embryonic fibroblasts (MEFs) were prepared − − from wild-type C57BL/6J and previously described Ogg1 / Normalized reporter expression from a damaged reporter plas- O mouse models (3). Primary MEFs were immortalized by viral mid, Fdam, and that from the same reporter plasmid in the O infection with a large T-antigen–expressing vector. Hamster cells absence of damage, Fun, were used to compute the percent re- V79 (WT) and xrs6 (Ku80 deficient) were a gift from Prof. Penny porter expression (%R.E.) using Eq. S3: Jeggo (University of Sussex, Brighton, UK). VC8 hamster cells FO (deficient for BRCA2) were a gift from Prof. Ryan Jensen (Yale % : : = dam × : [S3] R E O 100 University, New Haven, CT). Immortalized MEFs and all ham- Fun ster cells were maintained in Gibco DMEM medium supplemented with 10% (vol/vol) FBS, 2 mM L-glutamine, penicillin, Plasmids. The AmCyan, EGFP, mOrange, and mPlum and tagBFP and streptomycin and were subcultured by trypsinization. reporter genes were subcloned into the pmaxCloning Vector (Lonza) between the KpnI and SacI restriction sites in the × 6 Transfection. For transfection, 2 10 lymphoblastoid cells in multiple cloning site. The Kozak translation initiation consensus μ μ 100 L complete medium were combined with 9 g of reporter sequence and an additional NheI restriction site were introduced plasmid mixture using the mixtures described in Table 2. Cells at the 5′ end of each reporter, and a HindIII restriction site was were electroporated using a 96-well Bio-Rad MXcell gene added to the 3′ end. The pmaxCloning Vector places reporter μ pulser, with an exponential waveform at 260 V and 950 F. genes under the CMV intermediate-early promoter. Plasmids Following electroporation, a multichannel pipette was used to were amplified using Escherichia coli DH5α (Invitrogen) and μ add 100 L complete medium to each well in the electroporation purified using Qiagen endotoxin-free Maxi and Giga kits. Con- plate. After mixing, a multichannel pipette was used to transfer structs were confirmed by DNA sequencing and restriction en- μ 50 L of transfected cells from the 96-well electroporation plate zyme digestion. to the corresponding wells of a 96-well cell culture plate prefilled with 200 μL of complete medium. Thus, in total, cells were di- UV-Irradiated Substrates. Plasmids were irradiated in TE buffer (10 luted 10-fold following electroporation. Two additional replicate mM Tris·HCl, 1 mM EDTA, pH 7.0) at a DNA concentration of 96-well culture plates were prepared identically using the re- 50 ng/μL in a volume of 1.5 mL in 10-cm polystyrene Petri dishes maining cells in the electroporation plate. The transfected cells (without lids) with UV-C light generated by a Stratalinker 2000 then were incubated at 37 °C, 5% CO2, and one plate was re- box. Following treatment, reporter plasmids were combined in moved for analysis at each time point (18 and 40 h); the third the following ratio: 1 part tagBFP, 10 parts AmCyan, 1 part GFP, plate was reserved as a backup. At a minimum, each transfection 2 parts mOrange, and 4 parts mPlum; these proportions were was performed in duplicate, on three separate days. Fibroblasts used to compensate for weaker fluorescence intensities observed were trypsinized, resuspended in culture medium, and trans- for some of the reporters. The same plasmid mixture without UV fected using the same conditions. The transfected cells then were irradiation was prepared as described above, except without UV transferred to a unique well in a six-well culture plate in which treatment. Data obtained following transfections with the plas- each well was prefilled with 3 mL of culture medium. mid mixture containing irradiated plasmids have been labeled Electroporation yielded consistently high transfection effi- “damaged,” and those from untreated plasmids have been la- ciency, ranging between 10% and 50% across all cells studied, as beled “undamaged”; however, every transfection included an judged by flow cytometry (see below). Chromatin immunopre- undamaged reporter. The undamaged transfection reporter was cipitation of DNA isolated from transfected cells using anti- used to normalize transfection efficiency. Further details re- bodies to histone H3 or H4 confirmed the chromatinization of garding the UV-C dose delivered to each plasmid are available reporter plasmids (Fig. S1B). The same transfection procedures in Table 2. Plasmid mixtures were ethanol precipitated, washed were used for samples analyzed by sequencing. To increase the with 70% (vol/vol) ethanol, and redissolved in TE buffer at ∼1.5 yield of RNA, a larger quantity of cells was transfected; for each μg/μL; damaged and undamaged plasmid mixtures were adjusted condition (damaged or undamaged), four 100-μL transfections to the same final concentration, confirmed using a NanoDrop were combined after electroporation and diluted into a single spectrophotometer. 4-mL culture. Substrates Containing a G:G Mismatch. Substrates were prepared Calculation of Percent Fluorescent Reporter Expression. Fluores- using a method based on a previously published protocol (4). The cence signal (F) was computed using Eq. S1: pmax:mOrange plasmid was nicked with Nb.BstI (New England Nagel et al. www.pnas.org/cgi/content/short/1401182111 1of10 Biolabs) to generate a single-strand break in the transcribed using the following oligo with ssDNA prepared from the WT strand (Fig. S2B). The nicked strand then was digested with mOrange plasmid: 5′-GTAGGCCTTGGAGCCGTAGGTG- exonuclease III, and the remaining single-stranded circular DNA AACTGAGG-3′. (ssDNA) was purified by using a 1% agarose gel. Twenty mi- crograms of the ssDNA was combined with 40 μg of G299C Substrates Containing an A:C Mismatch. Substrates were prepared mutant pmax:mOrange plasmid linearized with NheI (New using a method similar that described above for preparation of the England Biolabs); the mixture was denatured by addition of 0.3 N O6-MeG– and 8-oxoG–containing plasmids (Fig. S3A). A non- sodium hydroxide, and then returned to neutral pH to facilitate fluorescent GFP variant (C289T) was identified. The protein annealing between WT ssDNA and the complementary strand of expressed from this construct lacks a conserved arginine required the linearized mutant sequence to yield a heteroduplex containing for chromophore maturation. Single-stranded DNA was pre- a G:G mismatch at position 299 of the mOrange gene. Sub- pared from this plasmid as described above, except the nicking sequently, reactions were cleaned up using a Qiagen PCR cleanup enzyme was Nt.BspQI, which nicks the nontranscribed strand. kit, and unwanted linear and single-stranded DNA side products Thus, after ExoIII digest, the remaining ssDNA comes from the were digested with Plasmid-Safe ATP-Dependent DNase (Epi- transcribed strand. The following 5′-phosphorylated oligonucle- centre). Nicked plasmid was purified by using a 1% agarose gel otide was annealed to the ssDNA: 5′-P-GGCTACGTCCAG- and ligated with 800 units T4 DNA ligase (New England Biolabs). GAGCGCACCATCTTCTTC-3′. Primer extension was carried Finally, a second gel purification was performed to isolate closed out as described above, except the extension temperature was circular products (Fig. S2C). Homoduplex DNA was prepared by lowered to 61 °C to increase yield. The resulting substrate is using the same procedure, except that linearized DNA was pre- a heteroduplex in which the transcribed strand has the mutant pared from the WT pmax:mOrange. sequence (A) and the nontranscribed strand has the WT sequence (C) at position 289. Mismatch repair activity restores the Substrates Containing a Site-Specific O6-Methylguanine. A non- WT sequence to the transcribed strand and leads to GFP ex- fluorescent variant of the pmax:mPlum reporter (T208C) was pression. A WT homoduplex was prepared identically using WT identified. This construct was modified further with a mutation plasmid DNA as the starting material, and the substrates were that does not change the encoded protein sequence (C207G) to validated using the MT1 and TK6 cell lines (Fig. S3B). generate a unique recognition site (GGGCCC) for the restriction enzyme PspOMI (New England Biolabs). Substrates were pre- Substrates Containing a Blunt-End Double-Strand Break. A unique pared based on a previously described method (4), with minor ScaI restriction site was inserted into the 5′ untranslated region of modifications (Fig. S2E). Single-stranded DNA was prepared as the pmax:BFP reporter plasmid, immediately 5′ of the reporter described above. Five picomoles of a phosphorylated O6-meth- gene (Fig. S3C). Plasmids were linearized with ScaI restriction ylguanine (O6-MeG)–containing oligonucleotide (5′-CACG- enzyme and purified by phenol/chloroform extraction and eth- TAGGCCTTGGXCCCGTACATGATCTG-3′, where X = O6- anol precipitation, and digest completeness was confirmed by gel MeG) was combined with 2.5 μg of single-stranded pmax: electrophoresis.

Load more