Structural Insights into the Mechanism of Base Excision by MBD4

DNA glycosylases remove damaged or modified nucleobases by cleaving the N-glycosyl bond and the correct nucleotide is restored through subsequent base excision repair. In addition to excising threatening lesions, DNA glycosylases contribute to epigenetic regulation by mediating DNA demethylation and perform other important functions. However, the catalytic mechanism remains poorly defined for many glycosylases, including MBD4 (methyl-CpG binding domain IV), a member of the helix-hairpin-helix (HhH) superfamily. MBD4 excises thymine from G·T mispairs, suppressing mutations caused by deamination of 5-methylcytosine, and it removes uracil and modified uracils (e.g., 5-hydroxymethyluracil) mispaired with guanine. To investigate the mechanism of MBD4 we solved high-resolution structures of enzyme-DNA complexes at three stages of catalysis. Using a non-cleavable substrate analog, 2′-deoxy-pseudouridine, we determined the first structure of an enzyme-substrate complex for wild-type MBD4, which confirms interactions that mediate lesion recognition and suggests that a catalytic Asp, highly conserved in HhH enzymes, binds the putative nucleophilic water molecule and stabilizes the transition state. Observation that mutating the Asp (to Gly) reduces activity by 2700-fold indicates an important role in catalysis, but probably not one as the nucleophile in a double-displacement reaction, as previously suggested. Consistent with direct-displacement hydrolysis, a structure of the enzyme-product complex indicates a reaction leading to inversion of configuration. A structure with DNA containing 1-azadeoxyribose models a potential oxacarbenium-ion intermediate and suggests the Asp could facilitate migration of the electrophile towards the nucleophilic water. Finally, the structures provide detailed snapshots of the HhH motif, informing how these ubiquitous metal-binding elements mediate DNA binding.     

Local grassland restoration affects insect communities

1. It is hypothesised that ecological restoration in grasslands can induce an alternative stable state shift in vegetation. The change in vegetation influences insect community assemblages and allows for greater functional redundancy in pollination and refuge for native insect species. 2. Insect community assemblages at eight coastal California grassland sites were evaluated. Half of these sites had undergone restoration through active revegetation of native grassland flora and half were non‐restored. Insects were collected from Lupinus bicolor (Fabaceae) within 2 × 2‐m² plots in spring 2017. Lupinus bicolor is a common native species that is used in California restoration projects, and home and state landscaping projects. 3. Ordination demonstrated that insect community assemblages were different between restored and non‐restored sites. These differences were seen in insect functional groups as well as taxa‐specific differences and were found to be driven by environmental characteristics such as non‐native forb cover. 4. Functional redundancy of herbivores decreased at restored sites, while pollinators became more redundant compared with non‐restored sites. The assemblages of the common species found at restoration sites contained more native insects than those found at non‐restored sites, including species such as Bombus vosnesenskii. 5. Local grassland restoration has the potential to induce an alternative stable state change and affect insect community assemblages. Additionally, it was found that grassland restoration can be a potential conservation tool to provide refugia for bumblebees (Bombus), but additional studies are required to fully understand its broader applicability.     

Regional differences in the carbon isotopic compositions of teak from two monsoonal regimes of India

Inter and intra-annual carbon isotope compositions (δ¹³C) of several annual growth rings of teak trees from two monsoonal regimes from India were studied and compared with the corresponding oxygen isotopic (δ¹⁸O) variations. In teak from both the regimes, amplitudes of intra-annual δ¹³C were ∼2-3 times lower than that observed in δ¹⁸O. Seasonal cycle with lower δ¹³C values at the middle and higher at ring boundaries was observed for teak from central India, dominated by the southwest monsoon. Positive correlations of intra-annual δ¹³C values with the corresponding δ¹⁸O values of the same rings and with relative humidity (RH) of the concurrent period suggest a dominant role of RH in controlling δ¹³C values of teak from central India. Intra-annual δ¹³C variations of teak from southern India, receiving both the southwest and northeast monsoons, revealed an initial decreasing trend followed by an increasing trend before culminating in depleted ¹³C values at the end of the growing season. No correlation was observed between intra-annual δ¹³C and δ¹⁸O variations of teak trees from southern India. Regional differences in the climatology of δ¹³C of atmospheric CO₂ or the lengths of growing season could be likely reasons for differing intra-annual δ¹³C variations of teak from the two climatic regimes.     

Tracking wind‐dispersed seeds using 15N‐isotope enrichment

Seed dispersal influences a wide range of ecological processes. However, measuring dispersal patterns, particularly long‐distance dispersal, has been a difficult task. Marking bird‐dispersed seeds with stable ¹⁵N isotopes has been shown to be a user‐friendly method to trace seed dispersal. In this study, we determined whether ¹⁵N urea solution could be used to enrich seeds of two common wind‐dispersed plants, Eupatorium glaucescens (Asteraceae) and Sericocarpus tortifolius (Asteraceae). We further tested if the water type (distilled versus tap) in ¹⁵N urea solutions influences the level and variability of enrichment of plant seeds, and if increasing spraying frequency per se increases enrichment. Because droughts may lower seed set or kill plants, we wanted to investigate if the additional use of an externally applied anti‐transpirant affects the intake of externally applied ¹⁵N into seeds. The results demonstrate that ¹⁵N enrichment of seeds can facilitate dispersal experiments with wind‐dispersed plants. The use of distilled water in ¹⁵N urea solutions did not increase ¹⁵N enrichment compared to tap water. Further, enrichment was more efficient at lower spray frequencies. Both the use of tap water and low frequencies could lower time, effort and project costs. The results suggest that species can be protected from drought using an anti‐transpirant without decreasing the incorporation of ¹⁵N into seeds.     

Evolutionarily stable seasonal timing for insects with competition for renewable resource

Summary I study the evolutionarily stable seasonal patterns of hatching and pupation for herbivorous insects that engage in exploitative competition for a renewable resource. A longer larval feeding period enhances female fecundity, but also causes a higher mortality by predation and parasitism. Previously, it was shown that the evolutionarily stable population exhibits asynchronous starting and ending of the larval feeding period in a model in which larval growth rate decreases with the total larval biomass in the population due presumably to interference competition. Here I study the case in which resource availability changes not only with environmental seasonality but with the depletion by the feeding of larvae. I find that if the impact of the herbivory is strong, both hatching and pupation should occur asynchronously in the evolutionarily stable population. And if the favourable season for the host plant is short the ESS population may include synchronous timing of pupation. If the timing of hatching and pupation occurs asynchronously, in the first day of each interval some fraction of the population hatch or pupate, respectively and the rest do so gradually over the interval. In addition, if the environmental variable changes as a symmetric function of time, the length of the period in which hatching occurs tends to be much shorter than the period in which pupation occurs.     

Stable isotopic niche predicts fitness of prey in a wolf–deer system

Interindividual variation in niche presents a potentially central object on which natural selection can act. This may have important evolutionary implications because habitat use governs a suite of selective forces encountered by foragers. In a free-living native black-tailed deer, Odocoileus hemionus , population from coastal British Columbia, we used stable isotope analysis to identify individual variation in foraging niche and investigated its relationship to fitness. Using an intragenerational comparison of surviving and nonsurviving O. hemionus over 2 years of predation by wolves, Canis lupus, we detected resource-specific fitness. Individuals with isotopic signatures that suggested they foraged primarily in cedar ( Thuja plicata )-dominated and low-elevation hemlock ( Tsuga heterophylla )-dominated forest stands were more likely to be killed by C. lupus . High-quality forage in T. plicata stands, as indexed by protein content, may be involved in maintaining this foraging phenotype. Moreover, nonsurvivors diverged more than survivors from median isotopic signatures, suggesting selection against foraging specialization. Stable isotope analysis provides a novel opportunity to integrate ecological and selective landscapes in order to identify underlying ecological mechanisms of selection and provide insight into the maintenance of variability.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 90 , 125–137.     

A stable carbon isotope study of dissolved inorganic carbon cycling in a softwater lake

The dissolved inorganic carbon (DIC) cycle in a softwater lake was studied using natural variations of the stable isotopes of carbon,¹²C and¹³C. During summer stratification there was a progressive decrease in epilimnion DIC concentration with a concomitant increase in ¹³C_DIC), due to preferential uptake of¹²C by phytoplankton and a change in the dominant CO₂ source from inflow andin situ oxidation to invasion from the atmosphere. There was an increase in hypolimnion DIC concentration throughout summer with a concomitant general decrease in ¹³C_DIC from oxidation of the isotopically light particulate organic carbon that sank down through the thermocline from the epilimnion.Mass balance calculations of DI¹²C and DI¹³C in the epilimnion for the summer (June 23–September 25) yield a mean rate of net conversion of DIC to organic carbon (C_org) of 430 ± 150 moles d^-1 (6.5 ± 1.8 m moles m^-2 d^-1. Net CO₂ invasion from the atmosphere was 420 ± 120 moles d^-1 (6.2 ± 1.8 m moles m^-2 d^-1) with an exchange coefficient of 0.6 ± 0.3m d^-1. These results imply that at least for the summer months the phytoplankton obtained about 90% of their carbon from atmosphere CO₂. About 50% of CO₂ invasion and conversion to C_org for the summer occurred during a two week interval in mid-summer.DIC concentration increased in the hypolimnion at a rate of 350 ± 70 moles DIC d^-1 during summer stratification. The amount of DIC added to the hypolimnion was equivalent to 75 ± 20% of net conversion of DIC to C_org in the euphotic zone over spring and summer implying rapid degradation of POC in the hypolimnion. The ¹³C of DIC added to the deep water (-22.) was too heavy to have been derived from oxidation of particulate organic carbon alone. About 20% of the added DIC must have diffused from hypolimnetic sediments where relatively heavy CO₂ (-7) was produced by a combination of POC oxidation and as a by-product of methanogenesis.     

Water use efficiency and carbon isotope composition of plants in a cold desert environment

Summary The effects of the availabilities of water and nitrogen on water use efficiency (WUE) of plants were investigated in a sagebrush steppe. The four species studied wereArtemisia tridentata (shrub),Ceratoides lanata (suffrutescent shrub),Elymus lanceolatus (rhizomatous grass), andElymus elymoides (tussock grass). Water and nitrogen levels were manipulated in a two-by-two factorial design resulting in four treatments: control (no additions), added water, added nitrogen, and added water and nitrogen. One instantaneous and two long-term indicators of WUE were used to testa priori predictions of the ranking of WUE among treatments. The short-term indicator was the instantaneous ratio of assimilation to transpiration (A/E). The long-term measures were 1) the slope of the relationship between conductance to water vapor and maximum assimilation and 2) the carbon isotope composition (¹³C) of plant material. Additional water decreased WUE, whereas additional nitrogen increased WUE. For both A/E and ¹³C, the mean for added nitrogen alone was significantly greater than the mean for added water alone, and means for the control and added water and nitrogen fell in between. This ranking of WUE supported the hypothesis that both water and nitrogen limit plant gas exchange in this semiarid environment. The short- and long-term indicators were in agreement, providing evidence in support of theoretical models concerning the water cost of carbon assimilation.