Mutational signatures of DNA mismatch repair deficiency in C. elegans and human cancers

Throughout their lifetime, cells are subject to extrinsic and intrinsic mutational processes leaving behind characteristic signatures in the genome. DNA mismatch repair (MMR) deficiency leads to hypermutation and is found in different cancer types. Although it is possible to associate mutational signatures extracted from human cancers with possible mutational processes, the exact causation is often unknown. Here, we use C. elegans genome sequencing of pms-2 and mlh-1 knockouts to reveal the mutational patterns linked to C. elegans MMR deficiency and their dependency on endogenous replication errors and errors caused by deletion of the polymerase epsilon subunit pole-4. Signature extraction from 215 human colorectal and 289 gastric adenocarcinomas revealed three MMR-associated signatures, one of which closely resembles the C. elegans MMR spectrum and strongly discriminates microsatellite stable and unstable tumors (AUC = 98%). A characteristic difference between human and C. elegans MMR deficiency is the lack of elevated levels of N (C) under barG > NTG mutations in C. elegans, likely caused by the absence of cytosine (CpG) methylation in worms. The other two human MMR signatures may reflect the interaction between MMR deficiency and other mutagenic processes, but their exact cause remains unknown. In summary, combining information from genetically defined models and cancer samples allows for better aligning mutational signatures to causal mutagenic processes

Protection of the C. elegans germ cell genome depends on diverse DNA repair pathways during normal proliferation.

Maintaining genome integrity is particularly important in germ cells to ensure faithful transmission of genetic information across generations. Here we systematically describe germ cell mutagenesis in wild-type and 61 DNA repair mutants cultivated over multiple generations. ~44% of the DNA repair mutants analysed showed a >2-fold increased mutagenesis with a broad spectrum of mutational outcomes. Nucleotide excision repair deficiency led to higher base substitution rates, whereas polh-1(Polη) and rev-3(Polζ) translesion synthesis polymerase mutants resulted in 50-400 bp deletions. Signatures associated with defective homologous recombination fall into two classes: 1) brc-1/BRCA1 and rad-51/RAD51 paralog mutants showed increased mutations across all mutation classes, 2) mus-81/MUS81 and slx-1/SLX1 nuclease, and him-6/BLM, helq-1/HELQ or rtel-1/RTEL1 helicase mutants primarily accumulated structural variants. Repetitive and G-quadruplex sequence-containing loci were more frequently mutated in specific DNA repair backgrounds. Tandem duplications embedded in inverted repeats were observed in helq-1 helicase mutants, and a unique pattern of 'translocations' involving homeologous sequences occurred in rip-1 recombination mutants. atm-1/ATM checkpoint mutants harboured structural variants specifically enriched in subtelomeric regions. Interestingly, locally clustered mutagenesis was only observed for combined brc-1 and cep-1/p53 deficiency. Our study provides a global view of how different DNA repair pathways contribute to prevent germ cell mutagenesis

Mutational signatures are jointly shaped by DNA damage and repair.

Funder: Biotechnology and Biological Sciences Research Council East of Scotland Bioscience Doctoral Training Partnership Ph.D. studentshipFunder: Korean Institute for Basic Science, IBS-R022-A1-2019Cells possess an armamentarium of DNA repair pathways to counter DNA damage and prevent mutation. Here we use C. elegans whole genome sequencing to systematically quantify the contributions of these factors to mutational signatures. We analyse 2,717 genomes from wild-type and 53 DNA repair defective backgrounds, exposed to 11 genotoxins, including UV-B and ionizing radiation, alkylating compounds, aristolochic acid, aflatoxin B1, and cisplatin. Combined genotoxic exposure and DNA repair deficiency alters mutation rates or signatures in 41% of experiments, revealing how different DNA alterations induced by the same genotoxin are mended by separate repair pathways. Error-prone translesion synthesis causes the majority of genotoxin-induced base substitutions, but averts larger deletions. Nucleotide excision repair prevents up to 99% of point mutations, almost uniformly across the mutation spectrum. Our data show that mutational signatures are joint products of DNA damage and repair and suggest that multiple factors underlie signatures observed in cancer genomes

Optimization of In Vivo Electroporation Conditions and Delivery of DNA Vaccine Encoding SARS-CoV-2 RBD Using the Determined Protocol

Vaccination against SARS-CoV-2 and other viral infections requires safe, effective, and inexpensive vaccines that can be rapidly developed. DNA vaccines are candidates that meet these criteria, but one of their drawbacks is their relatively weak immunogenicity. Electroporation (EP) is an effective way to enhance the immunogenicity of DNA vaccines, but because of the different configurations of the devices that are used for EP, it is necessary to carefully select the conditions of the procedure, including characteristics such as voltage, current strength, number of pulses, etc. In this study, we determined the optimal parameters for delivery DNA vaccine by electroporation using the BEX CO device. BALB/c mice were used as a model. Plasmid DNA phMGFP was intramuscular (I/M) injected into the quadriceps muscle of the left hind leg of animals using insulin syringes, followed by EP. As a result of the experiments, the following EP parameters were determined: direct and reverse polarity rectangular DC current in three pulses, 12 V voltage for 30 ms and 950 ms intervals, with a current limit of 45 mA. The selected protocol induced a low level of injury and provided a high level of GFP expression. The chosen protocol was used to evaluate the immunogenicity of the DNA vaccine encoding the receptor-binding domain (RBD) of the SARS-CoV-2 protein (pVAXrbd) injected by EP. It was shown that the delivery of pVAXrbd via EP significantly enhanced both specific humoral and cellular immune responses compared to the intramuscular injection of the DNA vaccine

Summary of the analysis for association between DNA methylation and gene expression, and between DNA methylation and phenotypes.

<p>Summary of the analysis for association between DNA methylation and gene expression, and between DNA methylation and phenotypes.</p

Associations between DNA methylation at cg06500161 (<i>ABCG1</i>) and different phenotypes, based on quintiles of methylation level.

<p>Associations between DNA methylation at cg06500161 (<i>ABCG1</i>) and different phenotypes, based on quintiles of methylation level.</p

Pathway analysis based on the top 1,000 CpG sites associated with fasting glucose (for results from model 2).

<p>Pathway analysis based on the top 1,000 CpG sites associated with fasting glucose (for results from model 2).</p

Regression models for the analysis of association between DNA methylation and measures of glucose metabolism.

<p>Regression models for the analysis of association between DNA methylation and measures of glucose metabolism.</p