Journal: Nucleic Acids Research

Article Title: Precision genome editing and in-cell measurements of oxidative DNA damage repair enable functional and mechanistic characterization of cancer-associated MUTYH variants

doi: 10.1093/nar/gkaf037

Figure Lengend Snippet: Overview of MUTYH structure and function. ( A ) ROS readily oxidize guanine (G) bases in DNA into 8-oxoG. In mammals, the glycosylases OGG1 and MUTYH initiate the BER process of recognizing, excising, and replacing 8-oxoG lesions. If 8-oxoG is still base-paired with cytosine (8-oxoG•C), OGG1 will recognize the lesion and initiate BER to convert it back to a canonical G•C base-pair. However, if the 8-oxoG•C is not repaired before DNA replication, 8-oxoG will preferentially base-pair with adenine (A) through Hoogsteen interactions to form an 8-oxoG•A mismatch. MUTYH recognizes 8-oxoG•A lesions and excises A to create an apurinic/apyrimidinic site (abasic site, AP site; [O]) across from 8-oxoG (8-oxoG•[O]), which is then further processed by downstream BER proteins back into 8-oxoG•C. If, however, MUTYH does not recognize or cannot repair 8-oxoG•A prior to an additional round of DNA replication, thymine (T) will be incorporated opposite the A, resulting in a permanent G•C to T•A mutation. ( B ) Structural model of MUTYH (AlphaFold model AF-Q9UIF7-F1), colored by domain, and with amino acid residues mutated in this study labeled. Amino acids are colored according to ClinVar classification (L111P and W131* are pathogenic or likely pathogenic, D271G is a variant of uncertain significance, and L296L is benign or likely benign). There are a Zn center and FeS cluster missing from the AlphaFold model, and the first 86 and last 51 amino acids are omitted for clarity, as the model confidence of these regions is low. ( C ) Domain map of isoform 5 of MUTYH is shown according to (B) and with mutations studied in this work indicated. Inset is showing a zoom-in of the first 100 amino acids and the optional regions due to alternative splicing; CTD, C-terminal domain; MTS, mitochondrial targeting signal; NTD, N-terminal domain.

Article Snippet: The membrane was then blocked with 5 ml of 5% nonfat dried milk in Tris-buffered saline with 0.1% Tween 20 (TBST; 20 mM Tris, 150 mM NaCl, and 0.1% Tween 20 from Thermo Fisher Scientific, #85114) for 1 h, and then incubated overnight at 4°C with the following primary antibodies diluted in 10 ml of TBST: MUTYH (C-6) (Santa Cruz Biotechnology #sc-374571, diluted 1:2500), APE1 (Thermo Fisher Scientific #PA5-29157, diluted 1:2500), and Normal Mouse IgG 3 (Cell Signaling #75952, diluted 1:3000).

Techniques: Mutagenesis, Labeling, Variant Assay, Alternative Splicing

Journal: Nucleic Acids Research

Article Title: Precision genome editing and in-cell measurements of oxidative DNA damage repair enable functional and mechanistic characterization of cancer-associated MUTYH variants

doi: 10.1093/nar/gkaf037

Figure Lengend Snippet: Generation of isogenic cell lines harboring clinically relevant MUTYH SNVs. ( A– C ) HEK293T cells were subjected to the work flow shown in . Shown are Sanger sequencing traces of the SNV loci after bulk editing (top left) and after isogenic cell line generation, with one representative isogenic cell line per genotype (null/unedited, heterozygous, and homozygous). See for the L111P mutation. For sequencing data of all 36 lines (three per genotype, for each of the four SNVs), see – . Note for the W131* variant, the intended target nucleotide is indicated by shading. ( D ) Quantification of western blot data (from – ) of isogenic cell line lysate for MUTYH. Bars represent the grand average of n = 2 technical replicates of n = 3 biological replicates (each data point represents the average of two technical replicates for each clone, with circles showing clone 1, triangles showing clone 2, and squares showing clone 3). Error bars represent the standard deviation of the grand averages of the three biological replicates repeated in duplicate experiments. Data were analyzed with unpaired, one-tailed, parametric t -tests (in which the heterozygous or homozygous lines were compared to their wild-type counterparts), and P -values are marked as follows: ns = P ≥ .05, not significant; * P ≤ .05, and ** P ≤ .01 are significant. ( E ) Mutants of interest in this study and their location within the N-terminal domain. The structures were generated by superimposing the crystallized N-terminal domain of MUTYH (PDB: 3N5N) and the MutY bacterial homolog structure (PDB: 1RRQ). The DNA harboring an 8-oxoG•A substrate is also shown. SNVs leading to amino acid changes and the premature stop codon for W131* are indicated. For W131*, amino acids after the STOP codon are transparent.

Article Snippet: The membrane was then blocked with 5 ml of 5% nonfat dried milk in Tris-buffered saline with 0.1% Tween 20 (TBST; 20 mM Tris, 150 mM NaCl, and 0.1% Tween 20 from Thermo Fisher Scientific, #85114) for 1 h, and then incubated overnight at 4°C with the following primary antibodies diluted in 10 ml of TBST: MUTYH (C-6) (Santa Cruz Biotechnology #sc-374571, diluted 1:2500), APE1 (Thermo Fisher Scientific #PA5-29157, diluted 1:2500), and Normal Mouse IgG 3 (Cell Signaling #75952, diluted 1:3000).

Techniques: Sequencing, Mutagenesis, Variant Assay, Western Blot, Standard Deviation, One-tailed Test, Generated

Journal: Nucleic Acids Research

Article Title: Precision genome editing and in-cell measurements of oxidative DNA damage repair enable functional and mechanistic characterization of cancer-associated MUTYH variants

doi: 10.1093/nar/gkaf037

Figure Lengend Snippet: Validation of 8-oxoG•A and 8-oxoG•[O] lesion-specific plasmid reporters. ( A ) Schematic diagram of generation and use of lesion-containing plasmids. The plasmid-based fluorescent reporters contain both a red fluorescent protein, mCherry, and an inactive EGFP. EGFP is inactive due to a sequence of DNA that frameshifts the EGFP and harbors an incorporated dual Type IIS restriction enzyme (BsaI) recognition site (referred to as a Golden Gate site or GG site), which allows restriction digestion to produce custom sticky ends . This construct enables various inserts with compatible sticky ends to be ligated into the digested plasmid backbone and produce an intact mCherry-P2A-EGFP plasmid with custom base-pairs at codon 34 of EGFP. To evaluate MUTYH DNA repair activity, an 8-oxoG•A or 8-oxoG•[O] lesion is incorporated into codon 34. Control plasmids in which G•C, T•A, or 8-oxo-G•C are incorporated into codon 34 were generated as well. Once transfected into live cells harboring either WT or mutated MUTYH , both genes are transcribed in a single mRNA transcript, but are translated into unique and separate fluorescent proteins via the self-cleaving P2A linker. mCherry fluorescence acts as a transfection marker, and EGFP fluorescence occurs only there is repair of 8-oxoG•A or 8-oxoG•[O] to 8-oxoG•C or G•C by MUTYH and downstream BER proteins. ( B ) Percentage of repair in living cells harboring wild-type MUTYH for the five different base pairs indicated in (A) are shown. Repair is quantified by calculating the percent of EGFP+ cells divided by the transfected, or mCherry+, cells. Bars represent the average of n = 3–10 biological replicates. Error bars represent the standard deviation of the biological replicates. Data were analyzed with unpaired, one-tailed, parametric t -tests (compared to the G•C control sample), and P -values are marked as follows: ns = P ≥ .05, not significant; ** P ≤ .01, and **** P ≤ .0001 are significant.

Article Snippet: The membrane was then blocked with 5 ml of 5% nonfat dried milk in Tris-buffered saline with 0.1% Tween 20 (TBST; 20 mM Tris, 150 mM NaCl, and 0.1% Tween 20 from Thermo Fisher Scientific, #85114) for 1 h, and then incubated overnight at 4°C with the following primary antibodies diluted in 10 ml of TBST: MUTYH (C-6) (Santa Cruz Biotechnology #sc-374571, diluted 1:2500), APE1 (Thermo Fisher Scientific #PA5-29157, diluted 1:2500), and Normal Mouse IgG 3 (Cell Signaling #75952, diluted 1:3000).

Techniques: Biomarker Discovery, Plasmid Preparation, Sequencing, Construct, Activity Assay, Control, Generated, Transfection, Fluorescence, Marker, Standard Deviation, One-tailed Test

Journal: Nucleic Acids Research

Article Title: Precision genome editing and in-cell measurements of oxidative DNA damage repair enable functional and mechanistic characterization of cancer-associated MUTYH variants

doi: 10.1093/nar/gkaf037

Figure Lengend Snippet: Repair of 8-oxoG•A using a MUTYH lesion-specific plasmid reporter. ( A ) Schematic diagram of the fluorescent reporter for 8-oxoG•A repair. ( B ) Representative flow cytometry plots and gating schemes to quantify 8-oxoG•A repair in live cells using matching L111P MUTYH mutant cell lines as an example. The plots show compensated red fluorescence intensity ( y -axis) versus compensated EGFP fluorescence intensity ( x -axis) for four representative samples, left to right: unedited HEK293T cells, HEK293T cells that were transfected with the L111P gRNA, but produced no editing at the target site (null clone), heterozygous L111P MUTYH clone 1, and homozygous L111P MUTYH clone 1. 8-oxoG•A repair is quantified by calculating the percent of EGFP+ cells divided by the transfected, or mCherry+, cells. Scatter gates were applied to remove nonviable cells and doublets as shown in . Quadrant boundaries for analysis were set by using unedited HEK293T cells that were transfected with mCherry only or EGFP only plasmids. The numbers in each quadrant represent the percentage of cells within that population. “+’’s in the quadrants indicate the median EGFP fluorescence intensity of EGFP-positive cells. ( C ) Percentage of 8-oxoG•A repair in living cells harboring various MUTYH mutants. Values calculated as described in (B). Bars represent the average of n = 3 biological replicates (circles show clone 1, triangles show clone 2, and squares show clone 3). Error bars represent the standard deviation of the three biological replicates. Data were analyzed with unpaired, one-tailed, parametric t -tests (in which the heterozygous or homozygous lines were compared to their wild-type counterparts), and P- values are marked as follows: ns = P ≥ 0.05, not significant: * P ≤ .05, ** P ≤ .01, and **** P ≤ .0001 are significant.

Article Snippet: The membrane was then blocked with 5 ml of 5% nonfat dried milk in Tris-buffered saline with 0.1% Tween 20 (TBST; 20 mM Tris, 150 mM NaCl, and 0.1% Tween 20 from Thermo Fisher Scientific, #85114) for 1 h, and then incubated overnight at 4°C with the following primary antibodies diluted in 10 ml of TBST: MUTYH (C-6) (Santa Cruz Biotechnology #sc-374571, diluted 1:2500), APE1 (Thermo Fisher Scientific #PA5-29157, diluted 1:2500), and Normal Mouse IgG 3 (Cell Signaling #75952, diluted 1:3000).

Techniques: Plasmid Preparation, Flow Cytometry, Mutagenesis, Fluorescence, Transfection, Produced, Standard Deviation, One-tailed Test

Journal: Nucleic Acids Research

Article Title: Precision genome editing and in-cell measurements of oxidative DNA damage repair enable functional and mechanistic characterization of cancer-associated MUTYH variants

doi: 10.1093/nar/gkaf037

Figure Lengend Snippet: Defective MUTYH-APE1 Interactions Identified by the 8-oxoG•[O] reporter and co-IP experiments. ( A ) Schematic diagram of the fluorescent reporter for 8-oxoG•[O] repair. ( B ) Relative 8-oxoG•[O] repair values in living cells harboring various MUTYH mutants. Values calculated as described in . Bars represent the average of n = 3 biological replicates (circles show clone 1, triangles show clone 2, and squares show clone 3). Error bars represent the standard deviation of the three biological replicates. Data were analyzed with unpaired, one-tailed, parametric t -tests (in which the heterozygous or homozygous lines were compared to their wild-type counterparts), and P- values are marked as follows: ns = P ≥ .05, not significant; * P ≤ .05, ** P ≤ .01, and *** P ≤ 0.001 are significant. ( C ) Plotted are the ratios of the relative amount of APE1 protein to total MUTYH protein in MUTYH immunoprecipitated samples, normalized to that of untreated HEK293T cells (based on the co-IP experiments in ). Plotted for each experimental condition are n = 3 biological replicates containing homozygous genotypes (circles show clone 1, triangles show clone 2, and squares show clone 3). The D271G and L296L homozygous clones were normalized to the average of untreated HEK293T cells from the same blot (Un.1), and L111P and W131* homozygous clones were normalized to the average of untreated HEK293T cells on the same blot (Un.2). Error bars represent the standard deviation of the three biological replicates. Data were analyzed with unpaired, one-tailed, parametric t -tests, and P -values are marked as follows: ns = P ≥ .05, not significant; * P ≤ .05, ** P ≤ .01, and *** P ≤ .001 are significant.

Article Snippet: The membrane was then blocked with 5 ml of 5% nonfat dried milk in Tris-buffered saline with 0.1% Tween 20 (TBST; 20 mM Tris, 150 mM NaCl, and 0.1% Tween 20 from Thermo Fisher Scientific, #85114) for 1 h, and then incubated overnight at 4°C with the following primary antibodies diluted in 10 ml of TBST: MUTYH (C-6) (Santa Cruz Biotechnology #sc-374571, diluted 1:2500), APE1 (Thermo Fisher Scientific #PA5-29157, diluted 1:2500), and Normal Mouse IgG 3 (Cell Signaling #75952, diluted 1:3000).

Techniques: Co-Immunoprecipitation Assay, Standard Deviation, One-tailed Test, Immunoprecipitation, Clone Assay