Journal: bioRxiv

Article Title: ATR-I774Yfs*5 promotes genomic instability through micronuclei formation

doi: 10.1101/2022.03.10.483800

Figure Lengend Snippet: (A) Lollipop plot of ATR mutations across all cancer types; there is a mutational hotspot at the codon 774 which occurs at a far greater frequency compared to any other ATR mutation. (B) Distribution pattern of all identified ATR I774Yfs*5/Nfs*3 mutations from publicly available databases; cancers typically associated with microsatellite instability (colorectal, endometrial, gastric, and pancreatic cancers) account for about two thirds of all identified ATR-I774Yfs*5/Nfs*3 mutations. (C) Nucleotide context of these truncation mutations show they are frameshifts that occurs as the result of a single adenosine insertion (I774Nfs*3) or deletion (I774Yfs*5) in a poly-adenosine region of ATR producing a truncation mutation at codon 776 (TAG stop codon) or codon 778 (TAA stop codon) for the insertion or deletion events, respectively. While the insertion or deletion of a single A could theoretically occur at any nucleotide along the poly-adenosine sequence, the resulting amino acid change only begins at codon 774 (boxed). (D) Co-incidence between all observed ATR-I774Yfs*5/I774Nfs*3 mutations and mutations in genes in the MutSα /MutLα complexes. Some ATR mutants were co-incident with multiple MutSα/MutLα complex genes, while some had no co-incidence. (E) A map of the functional domains of ATR, showing the location of the mutation of interest, ATR-I774Yfs*5 (F) ATR-I774Yfs*5/I774Nfs*3 mutations generate a putative truncated ATR product which lacks numerous functional domains including the C-terminal kinase domain but still retains the RPA and ATRIP interaction domains as well as the nuclear localization sequence (NLS).

Article Snippet: ATR-I774Yfs*5-GFP is a modification of the ATR-WT-GFP PCDNA 3.1 plasmid, with an additional modification of a deletion of 1 adenosine in the 10-A polynucleotide sequence in exon 10 (codons 771-774) of the ATR cDNA sequence (Applied Biological Materials).

Techniques: Mutagenesis, Sequencing, Functional Assay

Journal: bioRxiv

Article Title: ATR-I774Yfs*5 promotes genomic instability through micronuclei formation

doi: 10.1101/2022.03.10.483800

Figure Lengend Snippet: (A) representative images of log growth phase HCT-116 ATR-WT and D15 (heterozygous ATR-I774Yfs*5) labeled with EdU to show proliferation in the absence of thymidine treatment as indicated by red staining (DAPI staining to indicate DNA). (B) EdU incorporation into HCT-116 ATR-WT and a representative ATR heterozygotic mutant HCT-116 cell clone (D15) are shown in cells treated with 2mM thymidine for 24 hours prior to EdU treatment. (C) Quantification of EdU incorporation into nuclei of HCT-116 ATR-WT and all three ATR heterozygotic mutant HCT-116 cell clones ± 2 μM thymidine. (D) Quantification of EdU incorporation into observed micronuclei in HCT-116 ATR-WT and all three ATR heterozygotic mutant HCT-116 cell clones ± 2 μM thymidine. (E) Quantification of micronuclear incidence in HCT-116 ATR-WT and all three ATR heterozygotic mutant HCT-116 cell clones ± 2 μM thymidine. For (C), (D) , and (E) , * denotes p <0.05 using Fisher’s Exact test). (F) A chart depicting the cell counts for each clone in the two treatment conditions, along with EdU positive cell counts, total number of observed micronuclei, and the number of EdU positive micronuclei. These values were used to determine statistical significances as shown in (C), (D), and (E).

Article Snippet: ATR-I774Yfs*5-GFP is a modification of the ATR-WT-GFP PCDNA 3.1 plasmid, with an additional modification of a deletion of 1 adenosine in the 10-A polynucleotide sequence in exon 10 (codons 771-774) of the ATR cDNA sequence (Applied Biological Materials).

Techniques: Labeling, Staining, Mutagenesis, Clone Assay