Journal: Biomolecules

Article Title: EGFP Reporters for Direct and Sensitive Detection of Mutagenic Bypass of DNA Lesions

doi: 10.3390/biom10060902

Figure Lengend Snippet: Transfection-based reporter assay for direct detection of transcriptional mutagenesis (TM) and mutagenic translesion DNA synthesis (TLS) templated by synthetic DNA lesions incorporated into vector DNA: ( a ) Scheme of the enhanced green fluorescent protein (EGFP) Q205* vector and of the derived constructs containing synthetic apurinic/apyrimidinic (AP) lesions (X) at the specified positions; ( b ) Detection of TM by flow cytometry of HeLa cells analyzed 24 h after transfection with constructs containing dA, THF and S-THF in double stranded DNA at the nucleotide 613 in TS. Scatter plots were gated by the DsRed fluorescence to generate the EGFP fluorescence distribution plots and calculate the median FL1-H. Grouped plots on the left show data of a representative experiment. Bar chart of the right shows quantification of the EGFP expression gain for n = 3 independent experiments (mean±SD); ( c ) Detection of mutagenic TLS. Cells transfected with constructs containing dT or THF at the nucleotide 613 in NTS opposite to a 18-nt gap were analyzed by flow cytometry 24 h post transfection. Mutant frequency was calculated as a ratio of EGFP-positive cells (in the upper right quadrant, UR) to total transfected cell count (UR+UL) for n = 3 independent experiments (mean ± SD).

Article Snippet: The resulting gap in the NTS was used to accommodate synthetic oligonucleotide 5′-TGAGCACC[THF]AGTCCGCCC-3′ containing the THF AP lesion (BioSpring GmbH) or the respective 5′-TGAGCACCTAGTCCGCCC-3′ unmodified oligonucleotide (Eurofins Genomics).

Techniques: Transfection, Reporter Assay, Mutagenesis, DNA Synthesis, Plasmid Preparation, Derivative Assay, Construct, Flow Cytometry, Fluorescence, Expressing, Cell Counting

Journal: Biomolecules

Article Title: EGFP Reporters for Direct and Sensitive Detection of Mutagenic Bypass of DNA Lesions

doi: 10.3390/biom10060902

Figure Lengend Snippet: Detection of TM by AP lesion in the EGFP A207P (c.619G>C) reporter: ( a ) Scheme of the EGFP A207P reporter indicating position of the lesion (X) at the nucleotide 619 of the TS and possible outcomes of the transcriptional bypass; ( b ) Verification of incorporation of the lesions into the vector DNA. Upper gel: proof of incorporation of synthetic DNA strands containing dG or the indicated AP lesions (THF, S-THF) into the TS. The vector containing the single-stranded 18-nt gap in the TS migrates as open circular form (oc). Efficient ligation of synthetic oligonucleotides into the gap is confirmed by reconstitution of the covalently closed circular form (cc). Lower gel: verification of incision of the THF lesion by human APE1 and inhibited incision of the S-THF; ( c ) TM visualized by fluorescence microscopy and quantitative flow cytometry as a reversal of the non-fluorescent EGFP phenotype. HeLa cells were transfected with the indicated EGFP constructs in the presence of DsRed-Monomer as a transfection marker.

Article Snippet: The resulting gap in the NTS was used to accommodate synthetic oligonucleotide 5′-TGAGCACC[THF]AGTCCGCCC-3′ containing the THF AP lesion (BioSpring GmbH) or the respective 5′-TGAGCACCTAGTCCGCCC-3′ unmodified oligonucleotide (Eurofins Genomics).

Techniques: Plasmid Preparation, Ligation, Fluorescence, Microscopy, Flow Cytometry, Transfection, Construct, Marker

Journal: Biomolecules

Article Title: EGFP Reporters for Direct and Sensitive Detection of Mutagenic Bypass of DNA Lesions

doi: 10.3390/biom10060902

Figure Lengend Snippet: Detection of mutagenic TLS over the synthetic AP lesion in the EGFP A207P (c.619G>C) reporter by reversion to the fluorescent phenotype: ( a ) verification of the presence of gaps in the TS opposite to the specified nucleotides in the TLS reporter constructs by inhibited ligation and its reconstitution with matching synthetic 18-mer oligonucleotides. THF lesion at the nucleotide 619 is marked with “X”; ( b ) Repair of the 18-nt gap by re-synthesis of the missing TS fragment in the construct encoding the functional EGFP (c.619G) and detection of mutagenic TLS on the EGFP A207P template containing no modification (c.619C) or AP lesion (c.619THF) at the nucleotide 619. HeLa cells were transfected with the indicated constructs together with the DsRed-Monomer expression vector as a transfection marker. The fractions of EGFP-positive cells (UR/(UR+UL)×100%) were used as an estimate of the repair and mutation rates, as in ( n = 2 independent experiments, mean ± range).

Article Snippet: The resulting gap in the NTS was used to accommodate synthetic oligonucleotide 5′-TGAGCACC[THF]AGTCCGCCC-3′ containing the THF AP lesion (BioSpring GmbH) or the respective 5′-TGAGCACCTAGTCCGCCC-3′ unmodified oligonucleotide (Eurofins Genomics).

Techniques: Construct, Ligation, Functional Assay, Modification, Transfection, Expressing, Plasmid Preparation, Marker, Mutagenesis