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  • Supporting Information - PNAS...2009/10/12  · Supporting Information Yoon et al. 10.1073/pnas.0910121106 SI Text Construction of Plasmid Vectors Containing a cis-synTT Dimer. The
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Supporting Information Yoon et al. 10.1073/pnas.0910121106 SI Text Construction of Plasmid Vectors Containing a cis-syn TT Dimer. The pBluescript (/) vector (Stratagene) was the starting material for construction of the pSB and pBS vectors used in this study. The 16-mer oligonucleotides, containing a cis-syn TT dimer indicated in red in Fig. S1B, were synthesized and purified by the Synthetic Organic Chemistry Core Laboratory at the University of Texas Medical Branch in Galveston, TX. All primers used for PCR amplification were purchased from Integrated DNA Tech- nologies. PCR reactions were performed with pfu turbo DNA polymerase (Stratagene). The multiple cloning site within the lacZ gene of pBlusescript (/) vector was replaced with the specific target sequence by PCR amplification. The target se- quences of the resulting vectors contain BamHI, SbfI and SpeI sites (Fig. S1 A Right). A PCR fragment harboring the kanamy- cin-resistance gene containing the addition of one base at nucleotide position 544 was inserted into the vector. A DNA fragment of 700 bp containing the SV40 replication origin sequence was amplified from pSP189 (1) and inserted into the pSB and pBS vectors (Fig. S1 A). Final constructs were con- firmed by restriction enzyme digestion and sequence analysis, followed by transformation into XL1-Blue MRF bacterial strain (Stratagene). Both sense and antisense single-stranded DNAs were purified as described in ref. 2. Briefly, a single colony of XL1 blue MRF carrying the vector was grown in 200 mL Luria Bertani (LB) media containing ampicillin (100 g/mL) followed by the addition of M13KO7 helper phage (New England Bio- Labs). After 3h incubation, kanamycin (50 g/mL) was added, and cells were incubated overnight at 37 °C. Cells were pelleted by centrifugation and the supernatant was mixed with 6 g of NaCl and 10 g of polyethylene glycol (PEG)-8000. Precipitated phage particles were centrifuged and suspended in 10 mL TE buffer (pH 8.0) and 100 L 10% sodium dodecyl sulfate (SDS). After phenol/chloroform extractions, DNA was ethanol precipitated and suspended in 550 mM NaCl and 50 mM MOPS (pH 7.0) solution. Single-stranded DNA was purified with Qiagen maxi prep kit according to the manufacturer’s protocols except for the final elution step, which was carried out with 15 ml of elution buffer containing 1M NaCl, 50 mM MOPS (pH 7.0), 4 M urea, and 30% ethanol. To synthesize the heteroduplex target vector (Fig. S1B), 20 g of purified sense or antisense ssDNA was annealed in 1X NEBuffer2 to 100 pmol of 5-end phosphorylated primers, one of which carried either no damage at the TT site or a cis-syn TT dimer, and the other of which carried the top strand of the wild type kanamycin gene (top strand: 5-GACGGC- GAGGATCTCGTCGT-3) for construction of the lagging strand (pSB) vector, or bottom strand of the kanamycin gene (complementary strand of top strand) for construction of the leading strand (pBS) vector. The extension and ligation were carried out with T 4 DNA polymerase (New England BioLabs) and T 4 DNA ligase (New England BioLabs) in the presence of 1 mM ATP, 50 g/mL BSA, and 600 M dNTPs (Fig. S1B). The resulting circular duplex DNA was treated with SpeI to remove any DNA generated from synthesis using only the kan primer. Closed circular heteroduplex DNA was purified by cesium chloride density gradient method, and after methylation by dam methylase (New England BioLabs), purified vector was verified by digestion with SpeI and DpnI(Fig. S1B). In Vivo Translesion Synthesis Assays in Human Cells. siRNAs used for knockdowns of human TLS Pols are shown in Table S4, and the efficiency of their knockdown was verified by RT PCR (Fig. S2), using the primers shown in Table S5. For siRNA treatment, cells were plated in 6-well plates at 70% confluence (3 10 5 cells per well) and transfected with 100 pmol of siRNAs. For the simultaneous siRNA knockdown of two genes, 100 pmol of siRNAs for each gene were mixed and transfected. After 48-h incubation, the heteroduplex target vector DNA (2 g) and 50 pmol of siRNA (second transfection) were cotransfected with Lipofectamine 2000 (Invitrogen) (Fig. S1B). After 30-h incuba- tion, plasmid DNA was rescued from cells by the alkaline lysis method (3) and digested with DpnI to remove unreplicated plasmid DNA (see Fig. S1B). The plasmid DNA was then transformed into E. coli XL1-Blue super competent cells (Strat- agene). Transformed bacterial cells were diluted in 1 mL SOC media and plated on both LB/amp (50 g/mL ampicillin; Sigma) and LB/kan (25 g/mL kanamycin; Sigma) plates containing 1 M isopropyl-1-thio--D-galactopyranoside (IPTG) (Roche) and 100 g/mL X-Gal (Roche) (Fig. S1B). After 16-h incubation at 37 °C, blue and white colonies were counted from both ampicillin and kanamycin plates. The actual TLS frequency was determined from the number of blue colonies out of total colonies growing on LB/kan plates. Mutational Analyses of TLS Products from Human Cells. Plasmid DNA obtained from the blue colonies was analyzed to determine the mutation frequency and mutational changes incorporated during TLS. A well isolated single blue colony was picked and mixed with 50 L PCR 1 NEB reaction buffer (New England BioLabs) containing two primers (for pBS vector: forward, 5-CGCCCAATACGCAAACC-3; reverse, 5-AACGTG- GACTCCAACGTC-3; for pSB vector: forward, 5-CGC- CCAATACGCAAACC-3; reverse, 5-TAGGGTGATGGT- TCACGTAGTGG-3); and 5 units of Taq polymerase (New England BioLabs) were added to the mixtures. The PCR was carried out with initial heating for 5.5 min at 95 °C, followed by 30 cycles of amplification at 95 °C for 30 s, at 45 °C for 40 s, and at 72 °C for 1 min. PCR products (5 L) were digested with MfeI (New England BioLabs) and analyzed on a 1.5% agarose gel (Fig. S1C). Uncut samples were purified with Qiagen PCR purification kit and their DNA sequence determined by auto- mated DNA sequencing. Cell Culture and siRNA Knockdown of Human Pols. Two synthetic and HPLC purified duplex siRNAs for human Pol (Table S4) (3) were purchased from Ambion. Predesigned two to four sets of HPLC purified duplex siRNAs for human Pol, Pol, Rev3, Rev7, and nonspecific negative control siRNAs (NC siRNA no. 1) were purchased from Ambion. Cell lines used were SV40- transformed normal MRC5 (GM637) cells (4), XPA deficient and XP12BE (GM4427) cells (5) provided by Dr. Gerd Pfeifer (City of Hope, CA), and XPV deficient XP30RO (GM3617) cells (4, 6) obtained from Dr. James Cleaver (University of California, San Francisco). Fibroblast cells were grown in DMEM (Invitro- gen) containing 10% FBS (Invitrogen) and penicillin/ streptomycin (Invitrogen). Cells were plated in 6-well plates at 70% confluence (3 10 5 cells per well) and transfected with 100 pmoles synthetic duplex siRNAs using Lipofectamine 2000 reagent (Invitrogen) following the manufacturer’s instructions. Cells were incubated for 24 h for RT-PCR analysis (Fig. S2) after which cellular extracts and total RNA were prepared. RT-PCR for Checking the Efficiency of siRNA Knockdown of TLS Pols in Human Cells. One hundred nanograms of total RNA extracted using the RNasy Extraction Kit (Qiagen) were used for RT-PCR Yoon et al. www.pnas.org/cgi/content/short/0910121106 1 of 14

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  • Supporting InformationYoon et al. 10.1073/pnas.0910121106SI TextConstruction of Plasmid Vectors Containing a cis-syn TT Dimer. ThepBluescript (�/�) vector (Stratagene) was the starting materialfor construction of the pSB and pBS vectors used in this study.The 16-mer oligonucleotides, containing a cis-syn TT dimerindicated in red in Fig. S1B, were synthesized and purified by theSynthetic Organic Chemistry Core Laboratory at the Universityof Texas Medical Branch in Galveston, TX. All primers used forPCR amplification were purchased from Integrated DNA Tech-nologies. PCR reactions were performed with pfu turbo DNApolymerase (Stratagene). The multiple cloning site within thelacZ� gene of pBlusescript (�/�) vector was replaced with thespecific target sequence by PCR amplification. The target se-quences of the resulting vectors contain BamHI, SbfI and SpeIsites (Fig. S1 A Right). A PCR fragment harboring the kanamy-cin-resistance gene containing the addition of one base atnucleotide position 544 was inserted into the vector. A DNAfragment of �700 bp containing the SV40 replication originsequence was amplified from pSP189 (1) and inserted into thepSB and pBS vectors (Fig. S1 A). Final constructs were con-firmed by restriction enzyme digestion and sequence analysis,followed by transformation into XL1-Blue MRF� bacterial strain(Stratagene). Both sense and antisense single-stranded DNAswere purified as described in ref. 2. Briefly, a single colony ofXL1 blue MRF� carrying the vector was grown in 200 mL LuriaBertani (LB) media containing ampicillin (100 �g/mL) followedby the addition of M13KO7 helper phage (New England Bio-Labs). After 3h incubation, kanamycin (50 �g/mL) was added,and cells were incubated overnight at 37 °C. Cells were pelletedby centrifugation and the supernatant was mixed with 6 g of NaCland 10 g of polyethylene glycol (PEG)-8000. Precipitated phageparticles were centrifuged and suspended in 10 mL TE buffer(pH 8.0) and 100 �L 10% sodium dodecyl sulfate (SDS). Afterphenol/chloroform extractions, DNA was ethanol precipitatedand suspended in 550 mM NaCl and 50 mM MOPS (pH 7.0)solution. Single-stranded DNA was purified with Qiagen maxiprep kit according to the manufacturer’s protocols except for thefinal elution step, which was carried out with 15 ml of elutionbuffer containing 1M NaCl, 50 mM MOPS (pH 7.0), 4 M urea,and 30% ethanol. To synthesize the heteroduplex target vector(Fig. S1B), 20 �g of purified sense or antisense ssDNA wasannealed in 1X NEBuffer2 to 100 pmol of 5�-end phosphorylatedprimers, one of which carried either no damage at the TT site ora cis-syn TT dimer, and the other of which carried the top strandof the wild type kanamycin gene (top strand: 5�-GACGGC-GAGGATCTCGTCGT-3�) for construction of the laggingstrand (pSB) vector, or bottom strand of the kanamycin gene(complementary strand of top strand) for construction of theleading strand (pBS) vector. The extension and ligation werecarried out with T4 DNA polymerase (New England BioLabs)and T4 DNA ligase (New England BioLabs) in the presence of1 mM ATP, 50 �g/mL BSA, and 600 �M dNTPs (Fig. S1B). Theresulting circular duplex DNA was treated with SpeI to removeany DNA generated from synthesis using only the kan primer.Closed circular heteroduplex DNA was purified by cesiumchloride density gradient method, and after methylation by dammethylase (New England BioLabs), purified vector was verifiedby digestion with SpeI and DpnI (Fig. S1B).

    In Vivo Translesion Synthesis Assays in Human Cells. siRNAs used forknockdowns of human TLS Pols are shown in Table S4, and theefficiency of their knockdown was verified by RT PCR (Fig. S2),

    using the primers shown in Table S5. For siRNA treatment, cellswere plated in 6-well plates at 70% confluence (�3 � 105 cellsper well) and transfected with 100 pmol of siRNAs. For thesimultaneous siRNA knockdown of two genes, 100 pmol ofsiRNAs for each gene were mixed and transfected. After 48-hincubation, the heteroduplex target vector DNA (2 �g) and 50pmol of siRNA (second transfection) were cotransfected withLipofectamine 2000 (Invitrogen) (Fig. S1B). After 30-h incuba-tion, plasmid DNA was rescued from cells by the alkaline lysismethod (3) and digested with DpnI to remove unreplicatedplasmid DNA (see Fig. S1B). The plasmid DNA was thentransformed into E. coli XL1-Blue super competent cells (Strat-agene). Transformed bacterial cells were diluted in 1 mL SOCmedia and plated on both LB/amp (50 �g/mL ampicillin; Sigma)and LB/kan (25 �g/mL kanamycin; Sigma) plates containing 1�M isopropyl-1-thio-�-D-galactopyranoside (IPTG) (Roche)and 100 �g/mL X-Gal (Roche) (Fig. S1B). After 16-h incubationat 37 °C, blue and white colonies were counted from bothampicillin and kanamycin plates. The actual TLS frequency wasdetermined from the number of blue colonies out of totalcolonies growing on LB/kan plates.

    Mutational Analyses of TLS Products from Human Cells. PlasmidDNA obtained from the blue colonies was analyzed to determinethe mutation frequency and mutational changes incorporatedduring TLS. A well isolated single blue colony was picked andmixed with 50 �L PCR 1� NEB reaction buffer (New EnglandBioLabs) containing two primers (for pBS vector: forward,5�-CGCCCAATACGCAAACC-3�; reverse, 5�-AACGTG-GACTCCAACGTC-3�; for pSB vector: forward, 5�-CGC-CCAATACGCAAACC-3�; reverse, 5�-TAGGGTGATGGT-TCACGTAGTGG-3�); and 5 units of Taq polymerase (NewEngland BioLabs) were added to the mixtures. The PCR wascarried out with initial heating for 5.5 min at 95 °C, followed by30 cycles of amplification at 95 °C for 30 s, at 45 °C for 40 s, andat 72 °C for 1 min. PCR products (5 �L) were digested with MfeI(New England BioLabs) and analyzed on a 1.5% agarose gel(Fig. S1C). Uncut samples were purified with Qiagen PCRpurification kit and their DNA sequence determined by auto-mated DNA sequencing.

    Cell Culture and siRNA Knockdown of Human Pols. Two synthetic andHPLC purified duplex siRNAs for human Pol� (Table S4) (3)were purchased from Ambion. Predesigned two to four sets ofHPLC purified duplex siRNAs for human Pol�, Pol�, Rev3,Rev7, and nonspecific negative control siRNAs (NC siRNA no.1) were purchased from Ambion. Cell lines used were SV40-transformed normal MRC5 (GM637) cells (4), XPA deficientand XP12BE (GM4427) cells (5) provided by Dr. Gerd Pfeifer(City of Hope, CA), and XPV deficient XP30RO (GM3617) cells(4, 6) obtained from Dr. James Cleaver (University of California,San Francisco). Fibroblast cells were grown in DMEM (Invitro-gen) containing 10% FBS (Invitrogen) and penicillin/streptomycin (Invitrogen). Cells were plated in 6-well plates at70% confluence (�3 � 105 cells per well) and transfected with100 pmoles synthetic duplex siRNAs using Lipofectamine 2000reagent (Invitrogen) following the manufacturer’s instructions.Cells were incubated for 24 h for RT-PCR analysis (Fig. S2) afterwhich cellular extracts and total RNA were prepared.

    RT-PCR for Checking the Efficiency of siRNA Knockdown of TLS Pols inHuman Cells. One hundred nanograms of total RNA extractedusing the RNasy Extraction Kit (Qiagen) were used for RT-PCR

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    http://www.pnas.org/cgi/data/0910121106/DCSupplemental/Supplemental_PDF#nameddest=SF1http://www.pnas.org/cgi/data/0910121106/DCSupplemental/Supplemental_PDF#nameddest=SF1http://www.pnas.org/cgi/data/0910121106/DCSupplemental/Supplemental_PDF#nameddest=SF1http://www.pnas.org/cgi/data/0910121106/DCSupplemental/Supplemental_PDF#nameddest=SF1http://www.pnas.org/cgi/data/0910121106/DCSupplemental/Supplemental_PDF#nameddest=SF1http://www.pnas.org/cgi/data/0910121106/DCSupplemental/Supplemental_PDF#nameddest=SF1http://www.pnas.org/cgi/data/0910121106/DCSupplemental/Supplemental_PDF#nameddest=ST4http://www.pnas.org/cgi/data/0910121106/DCSupplemental/Supplemental_PDF#nameddest=SF2http://www.pnas.org/cgi/data/0910121106/DCSupplemental/Supplemental_PDF#nameddest=ST5http://www.pnas.org/cgi/data/0910121106/DCSupplemental/Supplemental_PDF#nameddest=SF1http://www.pnas.org/cgi/data/0910121106/DCSupplemental/Supplemental_PDF#nameddest=SF1http://www.pnas.org/cgi/data/0910121106/DCSupplemental/Supplemental_PDF#nameddest=SF1http://www.pnas.org/cgi/data/0910121106/DCSupplemental/Supplemental_PDF#nameddest=SF1http://www.pnas.org/cgi/data/0910121106/DCSupplemental/Supplemental_PDF#nameddest=ST4http://www.pnas.org/cgi/data/0910121106/DCSupplemental/Supplemental_PDF#nameddest=SF2http://www.pnas.org/cgi/content/short/0910121106

  • analysis, performed with the Qiagen One Step RT-PCR Kitaccording to the manufacturer’s instructions. Primer sequencesused for RT-PCR are shown in Table S5. For GAPDH, ampli-fication was carried out at 95 °C for 30 s, 55 °C for 45 s, and 72 °Cfor 50 s for 24 cycles. Twenty-six cycles for human Pol�, Pol�,Pol�, and Rev7, and 32 cycles for human Rev3 were applied foramplification at 95 °C for 30 s, 55 °C for 45 s, and 72 °C for 1 min.RT PCR products were analyzed on 1.5% agarose gels (Fig. S2).

    Big Blue Transgenic Mouse Cell Line and siRNA Knockdown. The bigblue transgenic mouse embryonic fibroblast cells (BBMEF)expressing either the (6-4) PP photolyase or the neo-vectorcontrol (7) were grown in DMEM containing 10% FBS andantibiotics. HPLC purified duplex siRNAs for mouse poly-merases �, �, �, and Rev3 were purchased from Ambion. ThesiRNAs used for knockdowns of mouse TLS Pols are shown inTable S6, and the efficiency of their knockdown was verified byRT-PCR (Fig. S4) using the primers shown in Table S7. For thecII mutation assay, cells were plated on 100-mm plates at 50%confluence (�5 � 106 cells) and 500 pmoles synthetic duplexsiRNAs (Table S6) were transfected using 50 �L Lipofectamine2000 reagent (Invitrogen) following the manufacturer’s instruc-tions.

    UV Irradiation, Photoreactivation, and cII Mutational Assays in MouseCells. After 48 h of siRNA knockdown, cells were washed withHBSS buffer (Invitrogen) and irradiated at 5 J/m2 with UVC

    light, followed by photoreactivation for 3 h at room temperatureas previously described (7). Fresh growth medium was thenadded and cells were incubated for 24 h. After the 24 hincubation period, the second siRNA transfection was carriedout to maintain the siRNA knockdown of the target gene(s).Cells were incubated for an additional 4 days to allow formutation fixation. The mouse genomic DNA was isolated usingthe genomic DNA isolation kit (Qiagen). The LIZ shuttle vectorwas rescued from the genomic DNA by mixing DNA aliquots andtranspack packaging extract (Stratagene), and the cII assay wascarried out as described in ref. 7. The mutation frequency wascalculated by dividing the number of mutant plaques by thenumber of total plaques. For mutation analysis, the sequences ofPCR products of the cII gene from the mutant plaques wereanalyzed as described in ref. 7

    RT-PCR for Checking the Efficiency of siRNA Knockdown of TLS Pols inMouse Cells. Forty-eight hours after siRNA transfection, totalRNA was extracted using Qiagen RNasy extraction kit (Qiagen)and 100 ng of total RNA were used for RT-PCR analysis.RT-PCR was performed with Qiagen one-step RT-PCR kitfollowing the manufacturer’s protocols. Primer sequences forRT-PCR are given in Table S7. For GAPDH, amplification wascarried out at 95 °C for 30 s, 55 °C for 45 s, and 72 °C for 50 s for24 cycles. Twenty-six cycles for Pol�, Pol�, and Pol�, and 30cycles for Rev3 were applied for amplification at 95 °C for 30 s,55 °C for 45 s, and 72 °C for 1 min. RT-PCR products wereanalyzed on 1.5% agarose gel (Fig. S4).

    1. Canella KA, Seidman MM (2000) Mutation spectra in supF: Approaches to elucidatingsequence context effects. Mutat Res 450:61–73.

    2. Bregeon D, Doetsch PW (2004) Reliable method for generating double-stranded DNAvectors containing site-specific base modifications. BioTechniques 37:760–762.

    3. Choi JH, Pfeifer GP (2005) The role of DNA polymerase eta in UV mutational spectra.DNA Repair 4:211–220.

    4. Cleaver JE, et al. (1999) Increased ultraviolet sensitivity and chromosomal instabilityrelated to P53 function in the xeroderma pigmentosum variant. Cancer Res 59:1102–1108.

    5. Seetharam S, Kraemer KH, Waters HL, Seidman MM (1990) Mutational hotspot variabilityin an unltraviolet-treated shuttle vector plasmid propagated in xeroderma pigmentosumand normal human lymphoblasts and fibroblasts. J Mol Biol 212:433–436.

    6. Laposa RR, Feeney L, Cleaver JE (2003) Recapitulation of the cellular xerodermapigmentosum-variant phenotypes using short interfering RNA for DNA polymerase H.Cancer Res 63:3909–3912.

    7. You Y-H, et al. (2001) Cyclobutane pyrimidine dimers are responsible for the vastmajority of mutations induced by UVB irradiation in mammalian cells. J Biol Chem276:44688–44694.

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  • Fig. S1. In vivo TLS assays in human cells. (A) The shuttle vector used for the construction of the heteroduplex vector containing a site-specific cis-syn TT dimer.For the SV40 replication system, the SV40 replication origin was subcloned into the vector. The multiple cloning site in the lacZ� gene was replaced with a specifictarget sequence (Right). This target sequence contains an SpeI site that puts the lacZ� sequence out of frame. (B) Construction of heteroduplex target vectorscontaining a site-specific cis-syn TT dimer. (i) The single-stranded phagemid is annealed with two primers, a 16-mer oligonucleotide containing a cis-syn TT dimer(T∧T) and the wild type kanamycin 20-mer oligonucleotide (Kan�). (ii) The closed circular duplex DNA (ccDNA) is synthesized by T4 DNA polymerase/ligase. (iii)The cc plasmid is treated with SpeI to digest any plasmid lacking the TT dimer, purified by CsCl density gradient centrifugation, and then treated with dammethylase to methylate the GATC sequences. (iv) The purified cc DNA along with siRNA specific for a particular TLS Pol are cotransfected into human fibroblasts(HF) that had already been treated with the siRNA for 48 h. (v) After �30-h incubation, the rescued plasmid DNA is treated with DpnI to remove any unreplicatedplasmid and (vi) transformed into XL-1 blue E. coli. (vii) The TLS frequency is determined by phenotypic selection of transformed bacterial cells grown onLB/kan/X-gal plates. The heteroduplex target sequence within the modified lacZ� gene is shown (Left); in it, the lesion-containing strand is indicated in red andthe opposite strand has an SpeI site. T∧T indicates a cis-syn TT dimer site. Because the strand containing the TT dimer has an in-frame lacZ� sequence and the otherstrand containing the SpeI site has an out-of-frame lacZ� sequence, replication of the TT dimer-containing strand by TLS would result in blue colonies, whereasreplication of the TT dimer-containing strand by template switching would generate white colonies. (C) Sequence analysis of TLS products opposite a cis-syn TTdimer. Colony PCR is carried out with the blue colonies and the amplified PCR products of target sequence are digested by MfeI, which recognizes and cuts atthe AATT sequence. The uncut PCR products, which represent mutants, are sequenced to analyze the mutations generated during TLS.

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  • Fig. S2. Efficient siRNA knockdown of TLS Pols in XPA human fibroblasts; RT-PCR analysis of the efficiency of inhibition of TLS Pols by siRNAs. M, DNA sizemarker. Cells treated with siRNA: NC, negative control; E, Pol�; I, Pol�; K, Pol�; R3, Rev3; R7, Rev7. For each analysis, the effects of that siRNA were examined onGAPDH mRNA expression. In addition, for each analysis, we verified the specificity of the siRNA effect by showing the absence of any effect on the expressionof any other TLS Pol being studied.

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  • Fig. S3. Western blot analysis of siRNA knockdown of TLS Pols in XPA human fibroblasts. Forty-eight hours after siRNA transfection, cellular extracts wereisolated and the target protein expression was analyzed by Western blot. NC, negative control siRNA; E, human Pol eta siRNAs; I, human Pol iota siRNA; K, humanPol kappa siRNA. �-tubulin was used as the loading control (Lower).

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  • Fig. S4. RT-PCR analysis of the efficiency of siRNA inhibition of TLS Pols in (BBMEF) mouse cells. Total RNA was isolated 48 h after transfection with siRNA. RT-PCRwas carried out to analyze the efficiency of siRNA inhibition on target gene mRNA expression. The GAPDH gene was used as a negative control for RT-PCR. NC,negative control siRNA; E, mouse Pol� siRNA; I, mouse Pol� siRNA; K, mouse Pol� siRNA; R3, mouse Rev3 siRNA.

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  • Fig. S5. Roles of human and mouse TLS Pols in promoting replication through CPDs formed at TT, TC, and CC dipyrimidine sites. Whereas Pol� carries outpredominantly error-free synthesis, Pols � and � provide additional alternate pathways, wherein they promote error-prone TLS by extending from the nucleotideinserted opposite the 3�T or 3�C of the CPD by another Pol, whose identity remains to be determined.

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  • Table S1. Effects of siRNA knockdown of Pols �, �, �, and � on mutation frequencies and nucleotides inserted opposite a cis-syn TTdimer carried on the leading strand DNA template in NER defective XPA human cells

    Nucleotide inserted

    siRNANo. of Kan� blue

    colonies sequenced A G C TMutation

    frequency, %

    NC siRNA 238 (5)* 233 3 (3� T)† — 2 (5� T) 2.1Pol� siRNA 352 (15) 337 2 (5� T)

    3 (3� T)4 (3� T) 2 (5� T)

    4 (3� T)4.3

    Pol� siRNA 192 (4) 188 2 (3� T) — 2 (5� T) 2.1Pol� siRNA 320 (2) 318 1 (5� T) — 1 (3� T) 0.6Rev3 siRNA 277 (3) 274 3 (3� T) — — 1.1Rev7 siRNA 240 (2) 238 2 (3� T) — — 0.8Pol� � Pol� siRNA 288 (13) 275 1 (5� T)

    2 (3� T)6 (3� T) 1 (5� T)

    3 (3� T)4.5

    Pol� � Rev3 siRNA 300 (6) 294 4 (5� T)2 (3� T)

    — — 2.0

    Pol� � Rev3 siRNA 520 (0) 520 — — — 0.0Polk � Rev7 siRNA 490 (0) 490 — — — 0.0

    *Number of colonies where TLS occurred by insertion of a nucleotide other than an A opposite the TT dimer are shown in parentheses.†The site where mutation occurred, 3� T or the 5� T of the TT dimer, is indicated in parentheses.

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  • Table S2. Effects of siRNA knockdown of Pols �, �, �, and � on mutation frequencies and nucleotides inserted opposite a cis-syn TTdimer carried on the lagging strand DNA template in NER defective XPA human cells

    Nucleotide inserted

    siRNA No. of Kan� blue colonies sequenced A G C T Mutation frequency, %

    NC siRNA 285 (8)* 276 3 (5� T)†

    2 (3� T)— 3 (3� T) 2.8

    Pol� siRNA 264 (16) 248 3 (5� T)6 (3� T)

    1 (3� T) 2 (5� T)4 (3� T)

    6.1

    Pol� siRNA 144 (3) 141 1 (5� T)1 (3� T)

    — 1 (3� T) 2.1

    Pol� siRNA 258 (2) 256 1 (5� T)1 (3� T)

    — — 0.8

    Rev3 siRNA 242 (2) 240 1 (5� T)1 (3’ T)

    — — 0.8

    Rev7 siRNA 296 (3) 293 1 (5� T)2 (3’ T)

    — — 1.0

    Pol� � Rev3 siRNA 220 (6) 214 2 (5� T)2 (3� T)

    2 (3� T) — 2.7

    Pol� � Rev3 siRNA 482 (0) 482 — — — 0.0Pol� � Rev7 siRNA 454 (0) 454 — — — 0.0

    *Number of colonies where TLS occurred by insertion of a nucleotide other than an A opposite the TT dimer are shown in parentheses.†The site where mutation occurred, 3� T or the 5� T of the TT dimer, is indicated in parentheses.

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  • Table S3. Types of mutations induced by UV light in the cII gene in (BBMEF) mouse cells expressing (6-4) PP photolyase and treatedwith siRNAs for different TLS Pols

    siRNA � 5 J/m2 UVC � PR

    Mutational changeNo UV*

    � PR NC Pol� Pol� Rev3 Pol� � Rev3

    C to T (G to A) 27 (36.0)† 98 (64.9) 81 (69.2) 59 (62.8) 59 (65.5) 37 (43.5)C to A (G to T) 7 (9.3) 6 (4.0) 10 (8.5) 1 (1.1) 7 (7.7) 9 (10.6)C to G (G to C) 4 (5.3) 9 (6.0) 7 (6.0) 4 (4.3) 4 (4.4) 5 (5.9)A to T (T to A) 3 (4.0) 6 (4.0) 4 (3.4) 3 (3.2) 1 (1.1) 6 (7.1)A to C (T to G) 18 (24.0) 14 (9.3) 3 (2.6) 19 (20.2) 13 (14.4) 14 (16.5)A to G (T to C) 10 (13.3) 9 (6.0) 6 (5.1) 5 (5.3) 3 (3.3) 10 (11.8)Tandem mutation 0 (0) 7 (4.6) 4 (3.4) 0 (0) 1 (1.1) 0 (0)Insertion 3 (4.0) 2 (1.3) 0 (0) 1 (1.1) 2 (2.2) 2 (2.4)Deletion 2 (2.7) 0 (0) 2 (1.7) 2 (2.1) 0 (0) 2 (2.4)Total 75 (100) 151 (100) 117 (100) 94 (100) 90 (100) 85 (100)

    *Unirradiated cells were photoreactivated and treated with control (NC) siRNA.†Numbers in parentheses indicate %.

    Yoon et al. www.pnas.org/cgi/content/short/0910121106 10 of 14

    http://www.pnas.org/cgi/content/short/0910121106

  • Table S4. siRNA sequences used for knock down of human TLS Pols

    Gene siRNA sequence (sense)*

    Pol�-1† 5�-GUGGAGCAGCGGCAAAAUCTT-3�Pol�-2† 5�-UAAACCUUGUGCAGUUGUATT-3�Pol� 5�-GGAAAUUAUGAUGUGAUGATT-3�Pol� 5�-GCUCAAAUCACCAGCCAACTT-3�Rev3 5�-GCAAUUUUGAACCUUAUGGTT-3�Rev7 5�-CCCGGAGCUGAAUCAGUAUTT-3�

    *Only the sequence of one strand is shown for the duplex siRNAs used.†For Pol� knockdown, the combination of these two duplex siRNAs was used.

    Yoon et al. www.pnas.org/cgi/content/short/0910121106 11 of 14

    http://www.pnas.org/cgi/content/short/0910121106

  • Table S5. Primer sequences used for RT-PCR to verify the efficiency of siRNA knock down of different human TLSpolymerases

    Gene Primer sequence

    Pol� Forward; 5�-ACCCAGGCAACTACCCAAAAC-3�Reverse; 5�-GGGCTCAGTTCCTGTACTTTG-3�

    Pol� Forward; 5�-ATGATCAAGTGTTGCCCACAC-3�Reverse; 5�-GTGAAAAGGTTTGGAGATCAC-3�

    Pol� Forward; 5�-ACCCAGGCAACTACCCAAAAC-3�Reverse; 5�-GGGCTCAGTTCCTGTACTTTG-3�

    Rev3 Forward; 5�-GCCTCATGAAGCGCATATTCC-3�Reverse; 5�-TTGCTGTCCCATCTCTGGGAC-3�

    Rev7 Forward; 5�-TAGCGGGAAGGATGACCAC-3�Reverse; 5�-AGGCATCCTCCAAGCAGAC-3�

    Human GAPDH Forward; 5�-ACCACAGTCCATGCCATCAC-3�Reverse; 5�-TCCACCACCCTGTTGCTGTA-3�

    Yoon et al. www.pnas.org/cgi/content/short/0910121106 12 of 14

    http://www.pnas.org/cgi/content/short/0910121106

  • Table S6. siRNA sequences used for knockdown of mouse TLS Pols

    Gene siRNA sequence (sense)

    Pol� 5�-GCCCGAGCAUUUGGUGUCATT-3�Pol� 5�-CGUAGAUCUGGAUUGCUUUTG-3�Pol� 5�-GCAAGUCAAUCAACGGAUUTT-3�Rev3 5�-GGAAAGGUAAUGCAUCACATT-3�

    Yoon et al. www.pnas.org/cgi/content/short/0910121106 13 of 14

    http://www.pnas.org/cgi/content/short/0910121106

  • Table S7. Primer sequences used for RT-PCR to verify the efficiency of siRNA knockdown of mouse TLS Pols

    Gene Primer sequences

    Pol� Forward; 5�-GAAGCCCGAGCATTTGGTG-3�Reverse; 5�-GCCTCTCCTCAAGTTCCAG-3�

    Pol� Forward; 5�-GGGCAGTTTGCAGTCAAGG-3�Reverse; 5�-TGAGCTGCTGGATACGCTG-3�

    Pol� Forward; 5�-CAAAGCAGGCATGGAAGGG-3�Reverse; 5�-TCCTGCTGGGAAGGATCTG-]3�

    Rev3 Forward; 5�-GTGGTACGAGTCTTCGG-3�Reverse; 5�-TCTTGTGACTCGGGCTG-3�

    GAPDH Forward; 5�-ATTGTGCACATCCAGGCGG-3�Reverse; 5�-CCTCCTTCATGGACATGGG-3�

    Yoon et al. www.pnas.org/cgi/content/short/0910121106 14 of 14

    http://www.pnas.org/cgi/content/short/0910121106

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