Genome-wide hypermutation and structural instability

National Institute Of Environmental Health Sciences

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Abstract

Purpose or scope: A role for somatic mutations in carcinogenesis and genetic disease is well accepted, but the degree to which mutation rates influence cancer initiation and development is under continuous debate. Recently accumulated genomic data has revealed that thousands of tumor samples are riddled by hypermutation, broadening support that many cancers acquire a mutator phenotype. This major expansion of cancer mutation datasets has provided unprecedented statistical power for the analysis of mutation spectra, which has confirmed several classical sources of mutation in cancer, highlighted new prominent mutation sources and empowered the search for cancer drivers. In our work we combined mechanistic knowledge obtained through our experiments with yeast models to interrogate the large whole-genome datasets of cancer mutations in order to gain mechanistic insight for understanding the impact of mutations on cancer and genetic disease. Research subject: The optimal levels of genome instability needed to sustain fitness of an organism are maintained by a complex set of DNA metabolic functions and pathways. Understanding the interplay between the biological mechanisms maintaining a stable genome and the environmental factors promoting genome instability is important for improving policies pertaining to the impact of the environment on human health. My long-term interest is in understanding physiological mechanisms and environmental causes of extreme levels of genome instability that can give rise to diseases and may alter the life-span of organisms. During the reviewed period, me and my group addressed these questions by combining the following general approaches: (i) Gaining new mechanistic information through research in yeast models reporter based and whole-genome sequencing. This approach elucidates mechanisms of genome instability and defines their specific features. (ii) Using mechanistic knowledge acquired from small genome studies for designing analyses of publicly available large datasets of genome changes in human cancers. Knowledge acquired from mechanistic research in yeast allows to build stringent statistical hypotheses thereby increasing the statistical power in bioinformatic interrogation of the exponentially growing datasets of cancer genomics such as The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC). (iii) Assessing load and signatures of somatic genome changes in humans. Analytical pipeline and information about mutation signatures generated through interrogation of cancer genomics data sets are applied to whole-genome sequencing analyses of cells isolated from healthy individuals. The combination of approaches (i) and (iii) provides additional research opportunities by way of using new knowledge generated through bioinformatic analysis of large public datasets and through sequencing genomes of human subjects for developing the next level of mechanistic hypotheses testable via small genome systems. (iv) Changes in RNA sequences occur through the life of an organism and through generations. These variations are associated with differential exposure to endogenous and environmental damaging agents acting on DNA genomes of cellular organisms as well as on genomes of DNA and RNA viruses. Over the last several years my group was studying only DNA mutations. However, recent studies, including our own, revealed that changes in RNA genomes and in RNA-editing of non-replicating cellular RNAs can result from the same agents that act on DNAs. Considering importance of factors affecting stability of viral RNA genomes and generation of cellular RNA editome, Therefore we extended experimental and bioinformatics tools already developed by my group in DNA research to studying induced changes in RNA sequences. Accomplishments: Mutagens often prefer specific nucleotides or oligonucleotide motifs that can be revealed by studying the hypermutation spectra in single-stranded (ss) DNA. We utilized a yeast model to explore mutagenesis by glycidamide, a simple epoxide formed endogenously in humans from the environmental toxicant acrylamide. Glycidamide caused ssDNA hypermutation in yeast predominantly in cytosine and adenines. The most frequent mutations in adenines occurred in the nAtnGt trinucleotide motif. Base substitutions A to G in this motif relied on Rev1 translesion polymerase activity. Inactivating Rev1 did not alter the nAt trinucleotide preference, suggesting it may be an intrinsic specificity of the chemical reaction between glycidamide and adenine in the ssDNA. We found this mutational motif enriched in published sequencing data from glycidamide-treated mouse cells and ubiquitous in human cancers. In cancers, this motif was positively correlated with the single base substitution (SBS) smoking-associated SBS4 signature, with the clock-like signatures SBS1, SBS5, and was strongly correlated with smoking history and with age of tumor donors. Clock-like feature of the motif was also revealed in cells of human skin and brain. Given its pervasiveness, we propose that this mutational motif reflects mutagenic lesions to adenines in ssDNA from a potentially broad range of endogenous and exogenous agents. We found that: --Base specificity of glycidamide mutagenesis in ssDNA may reflect a broad class of small electrophiles reacting with DNA bases via SN2 mechanism --The nAt trinucleotide mutations preference reflects the specificity of glycidamide-induced lesions. --Transcription strand and replication timing biases of the aTn to aCn (nAt to nGt) motif in mammalian cells and in human cancers indicated the involvement of mutagenesis in transient ssDNA. --The mutation load associated with aTn to aCn (nAt to nGt) motif can serve as a cumulative record of mutagenesis caused by exogenous and endogenous DNA damage in an individual