Mismatch Repair Balances Leading and Lagging Strand DNA Replication Fidelity

The two DNA strands of the nuclear genome are replicated asymmetrically using three DNA polymerases, α, δ, and ε. Current evidence suggests that DNA polymerase ε (Pol ε) is the primary leading strand replicase, whereas Pols α and δ primarily perform lagging strand replication. The fact that these polymerases differ in fidelity and error specificity is interesting in light of the fact that the stability of the nuclear genome depends in part on the ability of mismatch repair (MMR) to correct different mismatches generated in different contexts during replication. Here we provide the first comparison, to our knowledge, of the efficiency of MMR of leading and lagging strand replication errors. We first use the strand-biased ribonucleotide incorporation propensity of a Pol ε mutator variant to confirm that Pol ε is the primary leading strand replicase in Saccharomyces cerevisiae. We then use polymerase-specific error signatures to show that MMR efficiency in vivo strongly depends on the polymerase, the mismatch composition, and the location of the mismatch. An extreme case of variation by location is a T-T mismatch that is refractory to MMR. This mismatch is flanked by an AT-rich triplet repeat sequence that, when interrupted, restores MMR to >95% efficiency. Thus this natural DNA sequence suppresses MMR, placing a nearby base pair at high risk of mutation due to leading strand replication infidelity. We find that, overall, MMR most efficiently corrects the most potentially deleterious errors (indels) and then the most common substitution mismatches. In combination with earlier studies, the results suggest that significant differences exist in the generation and repair of Pol α, δ, and ε replication errors, but in a generally complementary manner that results in high-fidelity replication of both DNA strands of the yeast nuclear genome.     

A Two-Antibody Pan-Ebolavirus Cocktail Confers Broad Therapeutic Protection in Ferrets and Nonhuman Primates

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Internalization and degradation of peptides of the bombesin family in Swiss 3T3 cells occurs without ligand-induced receptor down-regulation.

The binding of [125I]gastrin releasing peptide ([125I]GRP) to Swiss 3T3 cells at 37 degrees C increases rapidly, reaching a maximum after 30 min and decreasing afterwards. The decrease in cell-associated radioactivity at this temperature is accompanied by extensive degradation of the labelled peptide. At 4 degrees C equilibrium binding is achieved after 6 h and [125I]GRP degradation is markedly inhibited. Extraction of surface-bound ligand at low pH demonstrates that the iodinated peptide is internalized within minutes after addition to 3T3 cells at 37 degrees C. The rate of internalization is strikingly temperature-dependent and is virtually abolished at 4 degrees C. In addition, lysomotropic agents including chloroquine increase the cell-associated radioactivity in cells incubated with [125I]GRP. The binding of [125I]GRP to Swiss 3T3 cells was not affected by pretreatment for up to 24 h with either GRP or bombesin at mitogenic concentrations. Furthermore, pretreatment with GRP did not reduce the affinity labelling of a Mr 75,000-85,000 surface protein recently identified as a putative receptor for bombesin-like peptides. These results demonstrate that while peptides of the bombesin family are rapidly internalized and degraded by Swiss 3T3 cells, the cell surface receptors for these molecules are not down-regulated.     

Chronic social stress induces DNA methylation changes at an evolutionary conserved intergenic region in chromosome X

Chronic stress resulting from prolonged exposure to negative life events increases the risk of mood and anxiety disorders. Although chronic stress can change gene expression relevant for behavior, molecular regulators of this change have not been fully determined. One process that could play a role is DNA methylation, an epigenetic process whereby a methyl group is added onto nucleotides, predominantly cytosine in the CpG context, and which can be induced by chronic stress. It is unknown to what extent chronic social defeat, a model of human social stress, influences DNA methylation patterns across the genome. Our study addressed this question by using a targeted-capture approach called Methyl-Seq to investigate DNA methylation patterns of the dentate gyrus at putative regulatory regions across the mouse genome from mice exposed to 14 days of social defeat. Findings were replicated in independent cohorts by bisulfite-pyrosequencing. Two differentially methylated regions (DMRs) were identified. One DMR was located at intron 9 of Drosha, and it showed reduced methylation in stressed mice. This observation replicated in one of two independent cohorts. A second DMR was identified at an intergenic region of chromosome X, and methylation in this region was increased in stressed mice. This methylation difference replicated in two independent cohorts and in Major Depressive Disorder (MDD) postmortem brains. These results highlight a region not previously known to be differentially methylated by chronic social defeat stress and which may be involved in MDD.     

Disentangling the potential effects of land‐use and climate change on stream conditions

Land‐use and climate change are significantly affecting stream ecosystems, yet understanding of their long‐term impacts is hindered by the few studies that have simultaneously investigated their interaction and high variability among future projections. We modeled possible effects of a suite of 2030, 2060, and 2090 land‐use and climate scenarios on the condition of 70,772 small streams in the Chesapeake Bay watershed, United States. The Chesapeake Basin‐wide Index of Biotic Integrity, a benthic macroinvertebrate multimetric index, was used to represent stream condition. Land‐use scenarios included four Special Report on Emissions Scenarios (A1B, A2, B1, and B2) representing a range of potential landscape futures. Future climate scenarios included quartiles of future climate changes from downscaled Coupled Model Intercomparison Project ‐ Phase 5 (CMIP5) and a watershed‐wide uniform scenario (Lynch2016). We employed random forests analysis to model individual and combined effects of land‐use and climate change on stream conditions. Individual scenarios suggest that by 2090, watershed‐wide conditions may exhibit anywhere from large degradations (e.g., scenarios A1B, A2, and the CMIP5 25th percentile) to small degradations (e.g., scenarios B1, B2, and Lynch2016). Combined land‐use and climate change scenarios highlighted their interaction and predicted, by 2090, watershed‐wide degradation in 16.2% (A2 CMIP5 25th percentile) to 1.0% (B2 Lynch2016) of stream kilometers. A goal for the Chesapeake Bay watershed is to restore 10% of stream kilometers over a 2008 baseline; our results suggest meeting and sustaining this goal until 2090 may require improvement in 11.0%–26.2% of stream kilometers, dependent on land‐use and climate scenario. These results highlight inherent variability among scenarios and the resultant uncertainty of predicted conditions, which reinforces the need to incorporate multiple scenarios of both land‐use (e.g., development, agriculture, etc.) and climate change in future studies to encapsulate the range of potential future conditions.     

Neural substrates of sensory-guided locomotor decisions in the rat superior colliculus

Deciding in which direction to move is a ubiquitous feature of animal behavior, but the neural substrates of locomotor choices are not well understood. The superior colliculus (SC) is a midbrain structure known to be important for controlling the direction of gaze, particularly when guided by visual or auditory cues, but which may play a more general role in behavior involving spatial orienting. To test this idea, we recorded and manipulated activity in the SC of freely moving rats performing an odor-guided spatial choice task. In this context, not only did a substantial majority of SC neurons encode choice direction during goal-directed locomotion, but many also predicted the upcoming choice and maintained selectivity for it after movement completion. Unilateral inactivation of SC activity profoundly altered spatial choices. These results indicate that the SC processes information necessary for spatial locomotion, suggesting a broad role for this structure in sensory-guided orienting and navigation.     

Improving the limit of detection in portable luminescent assay readers through smart optical design

Critical biomarkers of disease are increasingly being detected by point‐of‐care assays. Chemiluminescence (CL) and electrochemiluminescence (ECL) are often used in such assays due to their convenience and that they do not require light sources or other components that could complicate or add cost to the system. Reports of these assays often include readers built on a cellphone platform or constructed from low‐cost components. However, the impact the optical design has on the limit of detection (LOD) in these systems remains unexamined. Here, we report a theoretical rubric to evaluate different optical designs in terms of maximizing the use of photons emitted from a CL or ECL assay to improve the LOD. We demonstrate that the majority of cellphone designs reported in the literature are not optimized, in part due to misunderstandings of the optical tradeoffs in collection systems, and in part due to limitations imposed on the designs arising from the use of a mobile phone with a very small lens aperture. Based on the theoretical rubric, we design a new portable reader built using off‐the‐shelf condenser optics, and demonstrate a nearly 10× performance enhancement compared to prior reports on an ECL assays running on a portable chip.