A random mutation capture assay to detect genomic point mutations in mouse tissue

Herein, a detailed protocol for a random mutation capture (RMC) assay to measure nuclear point mutation frequency in mouse tissue is described. This protocol is a simplified version of the original method developed for human tissue that is easier to perform, yet retains a high sensitivity of detection. In contrast to assays relying on phenotypic selection of reporter genes in transgenic mice, the RMC assay allows direct detection of mutations in endogenous genes in any mouse strain. Measuring mutation frequency within an intron of a transcribed gene, we show this assay to be highly reproducible. We analyzed mutation frequencies from the liver tissue of animals with a mutation within the intrinsic exonuclease domains of the two major DNA polymerases, δ and ε. These mice exhibited significantly higher mutation frequencies than did wild-type animals. A comparison with a previous analysis of these genotypes in Big Blue mice revealed the RMC assay to be more sensitive than the Big Blue assay for this application. As RMC does not require analysis of a particular gene, simultaneous analysis of mutation frequency at multiple genetic loci is feasible. This assay provides a versatile alternative to transgenic mouse models for the study of mutagenesis in vivo

The metabolome regulates the epigenetic landscape during naive-to-primed human embryonic stem cell transition.

For nearly a century developmental biologists have recognized that cells from embryos can differ in their potential to differentiate into distinct cell types. Recently, it has been recognized that embryonic stem cells derived from both mice and humans exhibit two stable yet epigenetically distinct states of pluripotency: naive and primed. We now show that nicotinamide N-methyltransferase (NNMT) and the metabolic state regulate pluripotency in human embryonic stem cells (hESCs).  Specifically, in naive hESCs, NNMT and its enzymatic product 1-methylnicotinamide are highly upregulated, and NNMT is required for low S-adenosyl methionine (SAM) levels and the H3K27me3 repressive state. NNMT consumes SAM in naive cells, making it unavailable for histone methylation that represses Wnt and activates the HIF pathway in primed hESCs. These data support the hypothesis that the metabolome regulates the epigenetic landscape of the earliest steps in human development

Decreased Mitochondrial DNA Mutagenesis in Human Colorectal Cancer

Genome instability is regarded as a hallmark of cancer. Human tumors frequently carry clonally expanded mutations in their mitochondrial DNA (mtDNA), some of which may drive cancer progression and metastasis. The high prevalence of clonal mutations in tumor mtDNA has commonly led to the assumption that the mitochondrial genome in cancer is genetically unstable, yet this hypothesis has not been experimentally tested. In this study, we directly measured the frequency of non-clonal (random) de novo single base substitutions in the mtDNA of human colorectal cancers. Remarkably, tumor tissue exhibited a decreased prevalence of these mutations relative to adjacent non-tumor tissue. The difference in mutation burden was attributable to a reduction in C∶G to T∶A transitions, which are associated with oxidative damage. We demonstrate that the lower random mutation frequency in tumor tissue was also coupled with a shift in glucose metabolism from oxidative phosphorylation to anaerobic glycolysis, as compared to non-neoplastic colon. Together these findings raise the intriguing possibility that fidelity of mitochondrial genome is, in fact, increased in cancer as a result of a decrease in reactive oxygen species-mediated mtDNA damage

Roles of DNA polymerase I in leading and lagging-strand replication defined by a high-resolution mutation footprint of ColE1 plasmid replication

DNA polymerase I (pol I) processes RNA primers during lagging-strand synthesis and fills small gaps during DNA repair reactions. However, it is unclear how pol I and pol III work together during replication and repair or how extensive pol I processing of Okazaki fragments is in vivo. Here, we address these questions by analyzing pol I mutations generated through error-prone replication of ColE1 plasmids. The data were obtained by direct sequencing, allowing an accurate determination of the mutation spectrum and distribution. Pol I’s mutational footprint suggests: (i) during leading-strand replication pol I is gradually replaced by pol III over at least 1.3 kb; (ii) pol I processing of Okazaki fragments is limited to ∼20 nt and (iii) the size of Okazaki fragments is short (∼250 nt). While based on ColE1 plasmid replication, our findings are likely relevant to other pol I replicative processes such as chromosomal replication and DNA repair, which differ from ColE1 replication mostly at the recruitment steps. This mutation footprinting approach should help establish the role of other prokaryotic or eukaryotic polymerases in vivo, and provides a tool to investigate how sequence topology, DNA damage, or interactions with protein partners may affect the function of individual DNA polymerases

Quantification of random mutations in the mitochondrial genome

Mitochondrial DNA (mtDNA) mutations contribute to the pathology of a number of age-related disorders, including Parkinson disease [A. Bender et al., Nat. Genet. 38 (2006) 515,Y. Kraytsberg et al., Nat. Genet. 38 (2006) 518], muscle-wasting [J. Wanagat, Z. Cao, P. Pathare, J.M. Aiken, FASEB J. 15 (2001) 322], and the metastatic potential of cancers [K. Ishikawa et al., Science 320 (2008) 661]. The impact of mitochondrial DNA mutations on a wide variety of human diseases has made it increasingly important to understand the mechanisms that drive mitochondrial mutagenesis. In order to provide new insight into the etiology and natural history of mtDNA mutations, we have developed an assay that can detect mitochondrial mutations in a variety of tissues and experimental settings [M. Vermulst et al., Nat. Genet. 40 (2008) 4, M. Vermulst et al., Nat. Genet. 39 (2007) 540]. This methodology, termed the Random Mutation Capture assay, relies on single-molecule amplification to detect rare mutations among millions of wild-type bases [J.H. Bielas, L.A. Loeb, Nat. Methods 2 (2005) 285], and can be used to analyze mitochondrial mutagenesis to a single base pair level in mammals

Molecularly Evolved Thymidylate Synthase Inhibits 5-Fluorodeoxyuridine Toxicity in Human Hematopoietic Cells

Thymidylate synthase (TS) inhibitors, such as 5-fluorouracil (5-FU) and 5-fluorodeoxyuridine (5-FUdR), are amongst the most frequently used chemotherapeutic drugs available, although their efficacy is often limited by myelotoxicity. An emerging strategy for overcoming bone marrow toxicity involves ex vivo genetic transfer of drug resistance to autologous hematopoietic progenitor cells, followed by reimplantation of the transfected cells before chemotherapy. Here we establish that expression of mutant TS genes, selected from millions of engineered variants, renders human hematopoietic cells resistant to 5-FUdR, and identify the most efficacious variant for gene therapeutic rescue of drug-induced myelosuppression