Explosive mutation accumulation triggered by heterozygous human Pol ε proofreading-deficiency is driven by suppression of mismatch repair
- Tulane University School of Medicine, United States
- The Hospital for Sick Children, Canada
- University of Toronto, Canada
Figures
Figure 1 with 2 supplements
Heterozygous inactivation of Pol ε proofreading causes an increase in specific base pair substitutions.
(A) Mutation rates were measured using the fluctuation assay at the HPRT1 locus by resistance to 6-thioguanine. Mutation rates and 95% confidence intervals were measured by fluctuation analysis as …
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Figure 1—source data 1
Pol ε rAAV targeting efficiencies in human HCT-116 cells.
HCT-116 cells (37.4 × 106) were transduced with Pol ε rAAV and grown in the presence of 10 μg/ml G418 to select for Neor clones. Targeted clones were identified by PCR analysis.
- https://doi.org/10.7554/eLife.32692.006
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Figure 1—source data 2
HPRT1 mutations sequenced from 6-thioguanine resistant Pol ε wt/exo- and Pol ε wt/wt HCT116 cells.
For each cell line, HPRT1 cDNA was made by RT-PCR, amplified and sequenced from independent 6-thioguanine resistant clones. Verified errors are indicated by type on the coding strand and position relative to the +1 start site. Insertion (ins) or deletion (Δ) of the indicated base(s) is denoted.
- https://doi.org/10.7554/eLife.32692.007
Figure 1—figure supplement 1
Generation of exonuclease-deficient Pol ε human cell lines by gene targeting.
(A) Gene targeting scheme to change the sequence coding for the exonuclease active site amino acid residues (DIE) at the endogenous human Pol ε locus (POLE) to DNA coding for exonuclease-inactive …
Figure 1—figure supplement 2
Southern blot of parental (HCT116) and knock-in clone (HCT116-Polεwt/exo-) after Cre-mediated excision.
Genomic DNA was digested with SacI and resolved on a 1% agarose gel in TBE. The DNA was transferred to Hybond N + membrane (Amersham) and blotted with probe against HA2 (shown in Figure 1—figure …
Figure 2 with 11 supplements
Whole-genome sequencing from defined population doubling Pol εwt/exo- mismatch repair-deficient cells.
(A) Whole genome sequencing (2.8 × 109 bp, average 30X coverage) was performed on Pol εwt/exo- cells lacking mismatch repair at two defined population doubling levels, P0 and P14, as described in …
Figure 2—figure supplement 1
Whole genome SNVs identified in Pol εwt/exo- (PDL = 14) in cells lacking functional mismatch repair identified.
Whole exome SNVs identified in HCT116 cells in the current study, as well as from HCT116 and HCC2998 cells previously by Abaan et al. were extracted and identified. All SNVs were then analyzed for …
Figure 2—figure supplement 2
POLE mutation signature extracted from POLE-mutant cell lines.
(A) Non negative matrix factorization (NMF) was used to extract six unique mutation signatures from POLE-mutant (n = 14) and POLE-wild type (n = 545) colorectal cancer patients, Pol ε-P286R HCC2998 …
Figure 2—figure supplement 3
Mutation counts in the indicated trinucleotide context (top) were plotted as a proportion of their occurrence (bottom) in WGS samples.
https://doi.org/10.7554/eLife.32692.011
Figure 2—figure supplement 4
Mutation counts in the indicated trinucleotide context (top) were plotted as a proportion of their occurrence (bottom) in WES samples.
https://doi.org/10.7554/eLife.32692.012
Figure 2—figure supplement 5
Relative contributions of Cosmic Mutation Signatures to individual patient mutation spectra were determined using deconstructSig.
The relative proportion of each signature is shown for several tumor types (bMMRD, blue; somatic endometrial, green; somatic colorectal, salmon) and for Pol ε (black) and Pol δ (gray) mutations.
Figure 2—figure supplement 6
Mean coverage was greater than 90x for each WES sample.
https://doi.org/10.7554/eLife.32692.014
Figure 2—figure supplement 7
Alignment rate to the reference genome exceeded 99% for each WES sample.
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Figure 2—figure supplement 8
Total reads exceeded 60 million for each WES sample.
https://doi.org/10.7554/eLife.32692.016
Figure 2—figure supplement 9
Greater than 90% of the bases in the WES genome exceeded 30x coverage.
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Figure 2—figure supplement 10
Greater than 85% of the bases in the WGS genome exceeded 20x coverage.
https://doi.org/10.7554/eLife.32692.018
Figure 2—figure supplement 11
Average alternate base quality to reference base quality of ~1.0.
https://doi.org/10.7554/eLife.32692.019
Figure 3
Mismatch repair suppresses exonuclease-deficient Pol ε-induced mutation rate increase.
(A) Lentivirus encoding human Mlh1 was generated and used to infect parental cells with wild type Pol ε and cells heterozygous for Pol ε exonuclease deficiency. Cell lysates were probed by Western …
Figure 4 with 1 supplement
Mismatch repair suppresses the majority of exonuclease-deficient Pol ε mutation specificity.
(A) Cells were continuously passaged and PDL was calculated using the following equation: PDL = [ln(Nt)-ln(N0*PE)]/ln2. Nt = Number of viable cells counted after passage; N0 = Number of cells seeded …
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Figure 4—source data 1
HPRT1 mutations sequenced from mismatch repair-proficient cells.
The HPRT1 ORF was sequenced from individual HPRT1-resistant clones at the indicated PDL as described (Figure 4A and Materials and methods). One clone was unable to be sequenced (n.d.).
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Figure 4—source data 2
Pol ε mutation spectra calculation of cosine similarity to cancer mutation spectra.
Cosine similarities were calculated between the six unique mutation signatures extracted from POLE tumors and Pol ε mutant cell lines (columns, from Figure 2—figure supplement 2A) and each of the 30 identified Cosmic mutation signatures (http://cancer.sanger.ac.uk/cancergenome/assets/signatures_probabilities.txt).
- https://doi.org/10.7554/eLife.32692.024
Figure 4—figure supplement 1
Whole exome SNVs identified in Pol εwt/exo- (PDL = 71) and Pol εwt/wt (PDL = 70) cells expressing Mlh1 were analyzed for their triplet nucleotide sequence context.
Occurrences of each of the 96 possible trinucleotide base pair substitutions was then plotted as a percentage of the total number of SNVs.
Figure 5 with 1 supplement
Model for Pol ε-dependent tumor mutation signature development.
Rapid, massive mutation accumulation and Pol ε mutation signature acquisition (blue circles) depends on both Pol ε exonuclease domain mutation and compromised mismatch repair function. In somatic …
Figure 5—figure supplement 1
Oncoprints were made using cBioPortal for colorectal (n = 8) and endometrial (n = 18) tumors from the TCGA studies containing Pol ε exonuclease domain mutations.
Also shown are mutations in canonical and associated mismatch repair genes. Missense (green), nonsense (black) and inframe deletion or insertion (tan) mutations are shown. Each column represents an …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| cell line (Homo sapiens, Male) | HCT116 cells | Other | RRID:CVCL_0291 | Prescott Deininger at Tulane Univeristy LCRC |
| cell line (H. sapiens, Male) | HCT116 + Mlh1 | This paper | NA | HCT116 cells stably expressing human Mlh1-ORF via lentivirus-mediated integration |
| cell line (H. sapiens, Male) | Exo-; Exonuclease-deficient HCT116 Cells | This paper | NA | HCT116 cells infected with rAAV containing D275A and E277A POLE mutations |
| cell line (H. sapiens, Male) | Exo-; Exonuclease-deficient HCT116 Cells + Mlh1 | This paper | NA | HCT116 cells stably expressing human Mlh1-ORF via lentivirus- mediated integration and infected with rAAV containing D275A and E277A POLE mutations |
| recombinant DNA reagent | ExoI-targeting rAAV vector | This paper | NA | Homology arms/SEPT Cassette/Exo- mutations |
| recombinant DNA reagent | pCMV-XL5-Mlh1 | Other | NA | Victoria Belancio at Tulane Univeristy LCRC |
| antibody | Mlh1 Antibody | Pharmingen | G168-728; RRID: AB_395227 | Rabbit monoclonal; (1:100) in Milk (1%) TBST (1X) x 1 hr at RT |
| chemical compound, drug | 6-Thioguanine; 6-TG | Sigma-Aldrich | A4882 | Used at 5 ug/mL final concentration |
| chemical compound, drug | Hypoxanthine-Aminopterin- Thymidine; HAT | Thermo Fisher Scientific | 21060017 | Used at 1X final concentration |
| chemical compound, drug | Geneticin; G418 | Thermo Fisher Scientific | 10131027 | Used at 400 ug/mL final concentration |
| other | Ad-CMV-Cre | Vector Biolabs | 1045 | Adenovirus expressing Cre recombinase for excision of SEPT cassette from ExoI-targeted cell lines |
| software, algorithm | BWA-MEM v0.7.8 | PMID: 19451168 | NA | Used to align reads to human reference |
| software, algorithm | Picard v1.108 | Broad Institute; https://broadinstitute.github.io/picard/. | NA | Identify duplicate reads |
| software, algorithm | The Genome Analysis Toolkit (GATK) v2.8.1 | PMCID: PMC2928508 | NA | locally realign reads to known indels and recalibrate base quality scores |
| software, algorithm | MuTect v1.1.4 | PMCID: PMC3833702 | NA | Identiy somatic point mutations between the tumour and matched normal |
| other | WES/WGS raw sequencing data | This paper | NCBI GEO Accession: PRJNA327240 | Raw FASTQ files for WES and WGS performed in this study |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.32692.027
