RecBCD is required to complete chromosomal replication: Implications for double-strand break frequencies and repair mechanisms

Several aspects of the mechanism of homologous double-strand break repair remain unclear. Although intensive efforts have focused on how recombination reactions initiate, far less is known about the molecular events that follow. Based upon biochemical studies, current models propose that RecBCD processes double-strand ends and loads RecA to initiate recombinational repair. However, recent studies have shown that RecBCD plays a critical role in completing replication events on the chromosome through a mechanism that does not involve RecA or recombination. Here, we examine several studies, both early and recent, that suggest RecBCD also operates late in the recombination process – after initiation, strand invasion, and crossover resolution have occurred. Similar to its role in completing replication, we propose a model in which RecBCD is required to resect and resolve the DNA synthesis associated with homologous recombination at the point where the missing sequences on the broken molecule have been restored. We explain how the impaired ability to complete chromosome replication in recBC and recD mutants is likely to account for the loss of viability and genome instability in these mutants, and conclude that spontaneous double-strand breaks and replication fork collapse occur far less frequently than previously speculated.     

Invasive plants may promote predator‐mediated feedback that inhibits further invasion

Understanding the impacts of invasive species requires placing invasion within a full community context. Plant invaders are often considered in the context of herbivores that may drive invasion by avoiding invaders while consuming natives (enemy escape), or inhibit invasion by consuming invaders (biotic resistance). However, predators that attack those herbivores are rarely considered as major players in invasion. Invasive plants often promote predators, generally by providing improved habitat. Here, we show that predator‐promoting invaders may initiate a negative feedback loop that inhibits invasion. By enabling top‐down control of herbivores, predator‐promoting invaders lose any advantage gained through enemy escape, indirectly favoring natives. In cases where palatable invaders encounter biotic resistance, predator promotion may allow an invader to persist, but not dominate. Overall, results indicate that placing invaders in a full community context may reveal reduced impacts of invaders compared to expectations based on simple plant–plant or plant–herbivore subsystems.     

Kinematics of primate midfoot flexibility

This study describes a unique assessment of primate intrinsic foot joint kinematics based upon bone pin rigid cluster tracking. It challenges the assumption that human evolution resulted in a reduction of midfoot flexibility, which has been identified in other primates as the “midtarsal break.” Rigid cluster pins were inserted into the foot bones of human, chimpanzee, baboon, and macaque cadavers. The positions of these bone pins were monitored during a plantarflexion‐dorsiflexion movement cycle. Analysis resolved flexion‐extension movement patterns and the associated orientation of rotational axes for the talonavicular, calcaneocuboid, and lateral cubometatarsal joints. Results show that midfoot flexibility occurs primarily at the talonavicular and cubometatarsal joints. The rotational magnitudes are roughly similar between humans and chimps. There is also a similarity among evaluated primates in the observed rotations of the lateral cubometatarsal joint, but there was much greater rotation observed for the talonavicular joint, which may serve to differentiate monkeys from the hominines. It appears that the capability for a midtarsal break is present within the human foot. A consideration of the joint axes shows that the medial and lateral joints have opposing orientations, which has been associated with a rigid locking mechanism in the human foot. However, the potential for this same mechanism also appears in the chimpanzee foot. These findings demonstrate a functional similarity within the midfoot of the hominines. Therefore, the kinematic capabilities and restrictions for the skeletal linkages of the human foot may not be as unique as has been previously suggested. Am J Phys Anthropol 155:610–620, 2014. © 2014 Wiley Periodicals, Inc.     

TALE nickase mediates high efficient targeted transgene integration at the human multi-copy ribosomal DNA locus

Although targeted gene addition could be stimulated strikingly by a DNA double strand break (DSB) created by either zinc finger nucleases (ZFNs) or TALE nucleases (TALENs), the DSBs are really mutagenic and toxic to human cells. As a compromised solution, DNA single-strand break (SSB) or nick has been reported to mediate high efficient gene addition but with marked reduction of random mutagenesis. We previously demonstrated effective targeted gene addition at the human multicopy ribosomal DNA (rDNA) locus, a genomic safe harbor for the transgene with therapeutic potential. To improve the transgene integration efficiency by using TALENs while lowering the cytotoxicity of DSBs, we created both TALENs and TALE nickases (TALENickases) targeting this multicopy locus. A targeting vector which could integrate a GFP cassette at the rDNA locus was constructed and co-transfected with TALENs or TALENickases. Although the fraction of GFP positive cells using TALENs was greater than that using TALENickases during the first few days after transfection, it reduced to a level less than that using TALENickases after continuous culture. Our findings showed that the TALENickases were more effective than their TALEN counterparts at the multi-copy rDNA locus, though earlier studies using ZFNs and ZFNickases targeting the single-copy loci showed the reverse. Besides, TALENickases mediated the targeted integration of a 5.4 kb fragment at a frequency of up to 0.62% in HT1080 cells after drug selection, suggesting their potential application in targeted gene modification not being limited at the rDNA locus.     

Edaphic,Nutritive, and Species Assemblage Differences between Hotspots and Matrix Vegetation: Two African Case Studies

Areas of locally intense and frequent grazing, or ‘hotspots’, are pervasive features in tropical grasslands and savannas. In some ecosystems, hotspot presence is clearly associated with edaphic factors (e.g., high clay content and elevated soil fertility), such as those that develop in abandoned cattle bomas. Studies in a range of other savanna ecosystems, however, have failed to find intrinsic soil differences between hotspots and the surrounding matrix. Also, it remains unclear to what extent hotspots are associated with specific assemblages of nutrient‐rich plant species, as opposed to being a manifestation of intraspecific variation in nutritive quality. We conducted simultaneous studies in Kruger (South Africa) and Serengeti (Tanzania) National Parks to re‐evaluate the role of edaphic correlates of hotspot occurrence and to test whether intraspecific variation in plant quality occurs across hotspot‐matrix boundaries. We sampled soils and plants in paired hotspot and matrix plots at multiple sites within each ecosystem to test our a priori hypothesis that hotspots would be associated with distinct species assemblages and differences in soil fertility. We found clear hotspot‐matrix differences in foliar N, particularly within species, despite finding no differences in any soil or plant–soil variables, including N mineralization potential and mycorrhizal inoculation levels. We found only weak differences in community composition across the boundary, suggesting that intraspecific variation in foliar N rather than species turnover is mainly responsible for the enhanced nutritive value of hotspot vegetation. We propose that grazer–plant interactions may be stronger drivers of hotspot maintenance in these systems than plant–soil interactions.     

Reverse genetics in eukaryotes

Reverse genetics consists in the modification of the activity of a target gene to analyse the phenotypic consequences. Four main approaches are used towards this goal and will be explained in this review. Two of them are centred on genome alterations. Mutations produced by random chemical or insertional mutagenesis can be screened to recover only mutants in a specific gene of interest. Alternatively, these alterations may be specifically targeted on a gene of interest by HR (homologous recombination). The other two approaches are centred on mRNA. RNA interference is a powerful method to reduce the level of gene products, while MO (morpholino) antisense oligonucleotides alter mRNA metabolism or translation. Some model species, such as Drosophila, are amenable to most of these approaches, whereas other model species are restricted to one of them. For example, in mice and yeasts, gene targeting by HR is prevalent, whereas in Xenopus and zebrafish MO oligonucleotides are mainly used. Genome‐wide collections of mutants or inactivated models obtained in several species by these approaches have been made and will help decipher gene functions in the post‐genomic era.     

Photosynthetic regulation of C4 desert plant Haloxylon ammodendron under drought stress

About 20-year-old desert plants of C₄ species, Haloxylon ammodendron, growing at the southern edge of the Badain Jaran Desert in China, were selected to study the photosynthetic characteristics and changes in chlorophyll fluorescence when plants were subject to a normal arid environment (AE), moist atmospheric conditions during post-rain (PR), and the artificial supplement of soil water (SW). Results showed that under high radiation, in the AE, the species down-regulated its net assimilation rate (A) and maximum photochemical efficiency of PS II (Fv/Fm), indicating photoinhibition. However, under the PR and SW environments, A was up-regulated, with a unimodal diurnal course of A and a small diurnal change in Fv/Fm, suggesting no photoinhibition. When the air humidity or SW content was increased, the light compensation points were reduced; light saturation points were enhanced; while light saturated rate of CO₂ assimilation (A _max) and apparent quantum yield of CO₂ assimilation (Φ_C) increased. Φ_C was higher while the A _max was reduced under PR relative to the SW treatment. It was concluded that under high-radiation conditions drought stress causes photoinhibition of H. ammodendron. Increasing air humidity or soil moisture content can reduce photoinhibition and increase the efficiency of solar energy use.     

Pol32 is required for Pol zeta-dependent translesion synthesis and prevents double-strand breaks at the replication fork

POL32 encodes a non-essential subunit of Polδ and plays a role in Polδ processivity and DNA repair. In order to understand how Pol32 is involved in these processes, we performed extensive genetic analysis and demonstrated that POL32 is required for Polζ-mediated translesion synthesis, but not for Polη-mediated activity. Unlike Polζ, inactivation of Pol32 does not result in decreased spontaneous mutagenesis, nor does it limit genome instability in the absence of the error-free postreplication repair pathway. In contrast, inactivation of Pol32 results in an increased rate of replication slippage and recombination. A genome-wide synthetic lethal screen revealed that in the absence of Pol32, homologous recombination repair and cell cycle checkpoints play crucial roles in maintaining cell survival and growth. These results are consistent with a model in which Pol32 functions as a coupling factor to facilitate a switch from replication to translesion synthesis when Polδ encounters replication-blocking lesions. When Pol32 is absent, the S-phase checkpoint complex Mrc1–Tof1 becomes crucial to stabilize the stalled replication fork and recruit Top3 and Sgs1. Lack of any of the above activities will cause double strand breaks at or near the replication fork that require recombination as well as Rad9 for cell survival.     

Goose-mediated nutrient enrichment and planktonic grazer control in arctic freshwater ponds

A dramatic increase in the breeding population of geese has occurred over the past few decades at Svalbard. This may strongly impact the fragile ecosystems of the Arctic tundra because many of the ultra-oligotrophic freshwater systems experience enrichment from goose feces. We surveyed 21 shallow tundra ponds along a gradient of nutrient enrichment based on exposure to geese. Concentrations of total phosphorus (P) and dissolved inorganic nitrogen (DIN) in the tundra ponds ranged from 2–76 to 2–23 μg l⁻¹ respectively, yet there was no significant increase in phytoplankton biomass (measured as chlorophyll a; range: 0.6–7.3 μg l⁻¹) along the nutrient gradient. This lack of response may be the result of the trophic structure of these ecosystems, which consists of only a two-trophic level food chain with high biomasses of the efficient zooplankton grazer Daphnia in the absence of fish and scarcity of invertebrate predators. Our results indicate that this may cause a highly efficient grazing control of phytoplankton in all ponds, supported by the fact that large fractions of the nutrient pools were bound in zooplankton biomass. The median percentage of Daphnia–N and Daphnia–P content to particulate (sestonic) N and P was 338 and 3009%, respectively, which is extremely high compared to temperate lakes. Our data suggest that Daphnia in shallow arctic ponds is heavily subsidized by major inputs of energy from other food sources (bacteria, benthic biofilm), which may be crucial to the persistence of strong top–down control of pelagic algae by Daphnia.