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. 2020 Apr 24;11(1):1980.
doi: 10.1038/s41467-020-15901-w.

Transcription-coupled Repair and Mismatch Repair Contribute Towards Preserving Genome Integrity at Mononucleotide Repeat Tracts

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Free PMC article

Transcription-coupled Repair and Mismatch Repair Contribute Towards Preserving Genome Integrity at Mononucleotide Repeat Tracts

Ilias Georgakopoulos-Soares et al. Nat Commun. .
Free PMC article

Abstract

The mechanisms that underpin how insertions or deletions (indels) become fixed in DNA have primarily been ascribed to replication-related and/or double-strand break (DSB)-related processes. Here, we introduce a method to evaluate indels, orientating them relative to gene transcription. In so doing, we reveal a number of surprising findings: First, there is a transcriptional strand asymmetry in the distribution of mononucleotide repeat tracts in the reference human genome. Second, there is a strong transcriptional strand asymmetry of indels across 2,575 whole genome sequenced human cancers. We suggest that this is due to the activity of transcription-coupled nucleotide excision repair (TC-NER). Furthermore, TC-NER interacts with mismatch repair (MMR) under physiological conditions to produce strand bias. Finally, we show how insertions and deletions differ in their dependencies on these repair pathways. Our analytical approach reveals insights into the contribution of DNA repair towards indel mutagenesis in human cells.

Conflict of interest statement

S.N.Z. has patent applications with the UK IPO. S.N.Z. is also a consultant for Artios Pharma Ltd, Astra Zeneca and the Scottish Genomes Partnership. The other authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Indel characteristics across cancer types.
a The ratio of deletions to insertions for each tumour type. (Mann–Whitney U test, p value < 0.05 per cancer type). b Distribution of the size of insertions and of deletions for each tumour type. Deletions displayed greater size variance in comparison to insertions across cancer types (Levene’s test, p value < 0.05) and for individual cancer types (p value < 0.001 in breast, pancreas, liver, ovary, skin, lung, cervix, bone, head/neck, colorectal, p value < 0.05 in biliary and lymphoid cancers).
Fig. 2
Fig. 2. Strand asymmetries of polynucleotide (polyN) repeat tracts within transcribed regions.
a Enrichment of various polyN motifs across genes. Each gene is divided into ten bins, and two additional bins are added at either end of each gene. For any given bin, blue indicates relative enrichment in comparison to all other bins for that polyN, whereas red indicates relative depletion. b Scheme depicting the identification of polyN motifs on the template (blue) or non-template (orange) strands, dependent on the direction of the gene. RNA-polymerase II (RNAPII) binds to the template strand and mediates transcription. Thus, in the panel above, where the gene is on the (+) strand, the polyA tracts are on the non-template strand. In the panel below, where the gene is on the (−) strand, the polyA tracts are on the template strand. c Density of polyT and polyG motifs around the transcription start site (TSS). The gradient of pink to purple represents polyG tracts of 1-5 bp length, whereas the gradient of light blue to dark blue represents polyT tracts of 1–5 bp length. Error bars represent standard error from 1000-fold bootstrapping. d Density of polyT and polyG motifs around the transcription end site (TES). Error bars represent standard error from 1000-fold bootstrapping.
Fig. 3
Fig. 3. Transcriptional strand asymmetry of indels that occur at polyN tracts across multiple cancer types.
a Transcriptional strand asymmetries of indels occurring at polyT motifs. Average bias is shown with error bars showing standard deviation after 1000 bootstraps. Myeloid, cervix and thyroid cancers were excluded due to low numbers of total indels (Supplementary Table 1). T template, NT non-template. Strand bias was calculated as mutational density of non-template strand over total mutational density (of non-template and template strands). b Strand bias of MSI and MSS samples in stomach, biliary, uterus and colorectal tumours (Mann–Whitney U p value < 0.001 in all cases, Bonferroni corrected). c Transcriptional strand asymmetries of indels occurring at polyG motifs. Average bias is shown, with error bars showing standard deviation from bootstrapping. d Relationship between indel strand bias and gene expression levels in lung cancer (Mann–Whitney U p value < 0.001 for comparisons between low and medium expressed genes and between medium and highly expressed genes) according to length of polyG tracts (Kruskal–Wallis H-test with Bonferroni correction, p value < 0.001 for medium and high expression genes, p value > 0.05 for low expression genes). e Scheme depicting mechanism of indel mutagenesis at polyT tracts. DNA damage, shown as asterisks (*) that arise at T nucleotides of polyT tracts can occur on both template and non-template strands. The subsequent DNA repair, postulated to be TC-NER, results in preferential correction of DNA damage on the template strand, leaving T insertions (highlighted in as red T’s) and T deletions (shown as red −) on the non-template strand. f Schematic depicting mechanism of indel mutagenesis at polyG tracts in lung cancers from smokers. DNA damage in the form of adducted guanines (*) is asymmetrically repaired by TC-NER, with preferential repair of the template strand, thus accumulating more G indels on the non-template strand.
Fig. 4
Fig. 4. Transcriptional strand asymmetry at insertions and deletions.
a Transcriptional strand asymmetry of insertions and deletions at polyT tracts. Error bars represent standard deviation from bootstrapping with replacement. Both insertions and deletions displayed a strand asymmetry bias towards the non-template strand for polyT tracts across cancer types (binomial test with Bonferroni correction, p value < 0.001 for insertions and p value < 0.05 for deletions). b Transcriptional strand asymmetry occurring at polyT tracts according to level of gene expression for insertions. Mann–Whitney U with Bonferroni correction, p value < 0.001 when comparing low and high expression gene sets across all cancer types except skin, ovarian and lymphoid cancers (p value < 0.05) and CNS (p value > 0.05). c Transcriptional strand asymmetry occurring at polyT tracts according to level of gene expression for deletions. Mann–Whitney U with Bonferroni correction, p value < 0.001 when comparing low and high expression gene sets for skin and p value < 0.05 for stomach and pancreatic cancers. d Hierarchical clustering displaying transcriptional strand asymmetries for indels overlapping dinucleotide motifs. Dinucleotide repeat tracts of up to five repeated units are displayed. Purple represents asymmetry towards the non-template strand, whereas orange represents asymmetry towards the template strand. In the dendrogram of cancers, biliary, uterus, colorectal and stomach cancers are more distant from the other cancers, and contain MSI samples, while lung cancers are also separable from other cancer types, further reinforcing our observations regarding the DNA damage and repair processes that contribute to the observed asymmetries. Across cancer types a non-template strand asymmetry preference was observed for TG, TC and CT motifs (binomial test with Bonferroni correction, p value < 0.001) and for GT motifs (binomial test with Bonferroni correction, p value < 0.05) and a template strand asymmetry for CA, GA and AG motifs (binomial test with Bonferroni correction, p value < 0.001) and for AC motifs (binomial test with Bonferroni correction, p value < 0.05).

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