Knowledge of the rate and nature of spontaneous mutation is fundamental to understanding evolutionary and molecular processes. In this report, we analyze spontaneous mutations accumulated over ...thousands of generations by wild-type Escherichia coli and a derivative defective in mismatch repair (MMR), the primary pathway for correcting replication errors. The major conclusions are (i) the mutation rate of a wild-type E. coli strain is ∼1 × 10 ⁻³ per genome per generation; (ii) mutations in the wild-type strain have the expected mutational bias for G:C > A:T mutations, but the bias changes to A:T > G:C mutations in the absence of MMR; (iii) during replication, A:T > G:C transitions preferentially occur with A templating the lagging strand and T templating the leading strand, whereas G:C > A:T transitions preferentially occur with C templating the lagging strand and G templating the leading strand; (iv) there is a strong bias for transition mutations to occur at 5′ApC3′/3′TpG5′ sites (where bases 5′A and 3′T are mutated) and, to a lesser extent, at 5′GpC3′/3′CpG5′ sites (where bases 5′G and 3′C are mutated); (v) although the rate of small (≤4 nt) insertions and deletions is high at repeat sequences, these events occur at only 1/10th the genomic rate of base-pair substitutions. MMR activity is genetically regulated, and bacteria isolated from nature often lack MMR capacity, suggesting that modulation of MMR can be adaptive. Thus, comparing results from the wild-type and MMR-defective strains may lead to a deeper understanding of factors that determine mutation rates and spectra, how these factors may differ among organisms, and how they may be shaped by environmental conditions.
This Special Issue addresses the intersections of outdoor recreation, nature-based tourism, and sustainability. Outdoor recreation and nature-based tourism provide essential benefits to individuals, ...communities, and society and thereby contribute to sustainability. Equitable provision of opportunities, cultural variations in desired experiences, barriers to outdoor recreation, and diverse perceptions of both nature and recreation add to the complexity in outdoor recreation and nature-based tourism service delivery. Outdoor recreation and nature-based tourism occur within a socioecological system with feedback loops to changing social, economic, technological, and ecological conditions. On a global scale, climate change and other disturbance factors are impacting ecosystems and opportunities, increasing the importance of adaptation strategies for longer-term planning. Population growth and regional shifts in demographics and distribution (e.g., urbanization), as well as socioeconomic trends, affect who engages in outdoor recreation and nature-based tourism, opportunities sought, nature access, and governance of outdoor services. Overall the complexity of sustainable outdoor recreation and tourism may suggest a need for different approaches to service delivery, culture change among service providers and managers of natural spaces, and novel approaches to inclusive governance and shared stewardship. Given the clear importance of outdoor recreation and nature-based tourism to society, we invite you to consider this initial introduction to our assembled collection, which is meant to advance our understanding of the intersections of outdoor recreation, nature-based tourism, and sustainability.
It would not be an exaggeration to say that among molecular technologies, it is PCR (polymerase chain reaction) that underpins the discipline of molecular ecology as we know it today. With PCR, it ...has been possible to target the amplification of particular fragments of DNA, which can then be analysed in a multitude of ways. The capability of PCR to amplify DNA from a mere handful of copies further means that conservationists and ecologists are able to sample DNA unobtrusively and with minimal disturbance to the environment and the organisms of interest. However, a key disadvantage of PCR‐based methods has been the necessity for a generally non‐portable, laboratory setting to undertake the time‐consuming thermocycling protocols. LAMP (loop‐mediated isothermal amplification) offers a logistically simpler protocol: a relatively rapid DNA amplification reaction occurs at one temperature, and the products are visualized with a colour change within the reaction tubes. In the first field application of LAMP for an ecological study, Centeno‐Cuadros et al. () demonstrates how LAMP can be used to determine the sex of three raptor species. By enabling DNA amplification in situ and in ‘real‐time’, LAMP promises to revolutionize how molecular ecology is practised in the field.
Changes in telomere length are associated with degenerative diseases and cancer. Oxidative stress and DNA damage have been linked to both positive and negative alterations in telomere length and ...integrity. Here we examined how the common oxidative lesion 8-oxo-7,8-dihydro-2'-deoxyguanine (8-oxoG) regulates telomere elongation by human telomerase. When 8-oxoG is present in the dNTP pool as 8-oxodGTP, telomerase utilization of the oxidized nucleotide during telomere extension is mutagenic and terminates further elongation. Depletion of MTH1, the enzyme that removes oxidized dNTPs, increases telomere dysfunction and cell death in telomerase-positive cancer cells with shortened telomeres. In contrast, a preexisting 8-oxoG within the telomeric DNA sequence promotes telomerase activity by destabilizing the G-quadruplex DNA structure. We show that the mechanism by which 8-oxoG arises in telomeres, either by insertion of oxidized nucleotides or by direct reaction with free radicals, dictates whether telomerase is inhibited or stimulated and thereby mediates the biological outcome.
A complete understanding of evolutionary processes requires that factors determining spontaneous mutation rates and spectra be identified and characterized. Using mutation accumulation followed by ...whole-genome sequencing, we found that the mutation rates of three widely diverged commensal Escherichia coli strains differ only by about 50%, suggesting that a rate of 1-2 × 10(-3) mutations per generation per genome is common for this bacterium. Four major forces are postulated to contribute to spontaneous mutations: intrinsic DNA polymerase errors, endogenously induced DNA damage, DNA damage caused by exogenous agents, and the activities of error-prone polymerases. To determine the relative importance of these factors, we studied 11 strains, each defective for a major DNA repair pathway. The striking result was that only loss of the ability to prevent or repair oxidative DNA damage significantly impacted mutation rates or spectra. These results suggest that, with the exception of oxidative damage, endogenously induced DNA damage does not perturb the overall accuracy of DNA replication in normally growing cells and that repair pathways may exist primarily to defend against exogenously induced DNA damage. The thousands of mutations caused by oxidative damage recovered across the entire genome revealed strong local-sequence biases of these mutations. Specifically, we found that the identity of the 3' base can affect the mutability of a purine by oxidative damage by as much as eightfold.
Cancer cells rely on the activation of telomerase or the alternative lengthening of telomeres (ALT) pathways for telomere maintenance and survival. ALT involves homologous recombination ...(HR)-dependent exchange and/or HR-associated synthesis of telomeric DNA. Utilizing proximity-dependent biotinylation (BioID), we sought to determine the proteome of telomeres in cancer cells that employ these distinct telomere elongation mechanisms. Our analysis reveals that multiple DNA repair networks converge at ALT telomeres. These include the specialized translesion DNA synthesis (TLS) proteins FANCJ-RAD18-PCNA and, most notably, DNA polymerase eta (Polη). We observe that the depletion of Polη leads to increased ALT activity and late DNA polymerase δ (Polδ)-dependent synthesis of telomeric DNA in mitosis. We propose that Polη fulfills an important role in managing replicative stress at ALT telomeres, maintaining telomere recombination at tolerable levels and stimulating DNA synthesis by Polδ.
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•Utilize BioID to profile the composition of telomeres in cancer cells•DNA polymerase eta (Polη) localizes specifically to ALT telomeres•Loss of Polη enhances ALT and promotes mitotic DNA synthesis at telomeres
Garcia-Exposito et al. use BioID to determine the composition of telomeres in human cancer cells that rely on distinct telomere elongation mechanisms to survive. They suggest that the translesion polymerase DNA polymerase eta (Polη) regulates the recombination-associated alternative lengthening of telomeres (ALT) mechanism.
Carney-Stratakis syndrome, an inherited condition predisposing affected individuals to gastrointestinal stromal tumor (GIST) and paraganglioma, is caused by germline mutations in succinate ...dehydrogenase (SDH) subunits B, C, or D, leading to dysfunction of complex II of the electron transport chain. We evaluated the role of defective cellular respiration in sporadic GIST lacking mutations in KIT or PDGFRA (WT). Thirty-four patients with WT GIST without a personal or family history of paraganglioma were tested for SDH germline mutations. WT GISTs lacking demonstrable SDH genetic inactivation were evaluated for SDHB expression by immunohistochemistry and Western blotting and for complex II activity. For comparison, SDHB expression was also determined in KIT mutant and neurofibromatosis-1-associated GIST, and complex II activity was also measured in SDH-deficient paraganglioma and KIT mutant GIST; 4 of 34 patients (12%) with WT GIST without a personal or family history of paraganglioma had germline mutations in SDHB or SDHC. WT GISTs lacking somatic mutations or deletions in SDH subunits had either complete loss of or substantial reduction in SDHB protein expression, whereas most KIT mutant GISTs had strong SDHB expression. Complex II activity was substantially decreased in WT GISTs. WT GISTs, particularly those in younger patients, have defects in SDH mitochondrial complex II, and in a subset of these patients, GIST seems to arise from germline-inactivating SDH mutations. Testing for germline mutations in SDH is recommended in patients with WT GIST. These findings highlight a potential central role of SDH dysregulation in WT GIST oncogenesis.
The rate of cytosine deamination is much higher in single-stranded DNA (ssDNA) than in double-stranded DNA, and copying the resulting uracils causes C to T mutations. To study this phenomenon, the ...catalytic domain of APOBEC3G (A3G-CTD), an ssDNA-specific cytosine deaminase, was expressed in an Escherichia coli strain defective in uracil repair (ung mutant), and the mutations that accumulated over thousands of generations were determined by whole-genome sequencing. C:G to T:A transitions dominated, with significantly more cytosines mutated to thymine in the lagging-strand template (LGST) than in the leading-strand template (LDST). This strand bias was present in both repair-defective and repair-proficient cells and was strongest and highly significant in cells expressing A3G-CTD. These results show that the LGST is accessible to cellular cytosine deaminating agents, explains the well-known GC skew in microbial genomes, and suggests the APOBEC3 family of mutators may target the LGST in the human genome.
Evaluation of tear film break-up pattern (TFBUP) is the main diagnostic method for tear film-oriented therapy (TFOT) of dry eye. This prospective, observational study examined TFBUPs in 154 ...eyes/sides from 78 patients with thyroid eye disease (TED) who met the diagnostic criteria for dry eye in Japan. TFBUPs were classified as area, line, spot, dimple, and random breaks. Results for the status of TED and dry eye were compared between the TFBUPs. Consequently, line, spot, dimple, and random breaks were observed in 80 (51.9%), 29 (18.8%), 10 (6.5%), and 35 eyes (22.7%) while no eyes showed area breaks. The random break group had the highest incidence of lid lag/Graefe sign and superior limbic keratoconjunctivitis (SLK) (P < 0.050). Although the incidence of each TFBUP is almost equal in patients with simple dry eye without TED, line breaks were more frequently observed in TED. In addition, while random breaks in simple dry eye are usually only associated with minor ocular surface damages, those in TED were associated with a higher incidence of concomitant SLK. These results will be helpful for understanding the etiology of dry eye in TED and for TFOT in TED.
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