Developing Single-Molecule TPM Experiments for Direct Observation of Successful RecA-Mediated Strand Exchange Reaction

RecA recombinases play a central role in homologous recombination. Once assembled on single-stranded (ss) DNA, RecA nucleoprotein filaments mediate the pairing of homologous DNA sequences and strand exchange processes. We have designed two experiments based on tethered particle motion (TPM) to investigate the fates of the invading and the outgoing strands during E. coli RecA-mediated pairing and strand exchange at the single-molecule level in the absence of force. TPM experiments measure the tethered bead Brownian motion indicative of the DNA tether length change resulting from RecA binding and dissociation. Experiments with beads labeled on either the invading strand or the outgoing strand showed that DNA pairing and strand exchange occurs successfully in the presence of either ATP or its non-hydrolyzable analog, ATPγS. The strand exchange rates and efficiencies are similar under both ATP and ATPγS conditions. In addition, the Brownian motion time-courses suggest that the strand exchange process progresses uni-directionally in the 5′-to-3′ fashion, using a synapse segment with a wide and continuous size distribution

Structural basis for inhibition of homologous recombination by the RecX protein

The RecA/RAD51 nucleoprotein filament is central to the reaction of homologous recombination (HR). Filament activity must be tightly regulated in vivo as unrestrained HR can cause genomic instability. Our mechanistic understanding of HR is restricted by lack of structural information about the regulatory proteins that control filament activity. Here, we describe a structural and functional analysis of the HR inhibitor protein RecX and its mode of interaction with the RecA filament. RecX is a modular protein assembled of repeated three-helix motifs. The relative arrangement of the repeats generates an elongated and curved shape that is well suited for binding within the helical groove of the RecA filament. Structure-based mutagenesis confirms that conserved basic residues on the concave side of RecX are important for repression of RecA activity. Analysis of RecA filament dynamics in the presence of RecX shows that RecX actively promotes filament disassembly. Collectively, our data support a model in which RecX binding to the helical groove of the filament causes local dissociation of RecA protomers, leading to filament destabilisation and HR inhibition

Energy-saving technologies in design and construction of residential buildings and industrial facilities in the far North

The article discusses the main problems of energy saving and special features of design, construction and operation of residential buildings and industrial structures in extreme climatic conditions of the Northern territories. The impact of permafrost, seasonal thawing of soils and low bearing capacity of thawed soils on the choice of ways of arrangement of cities and pipelines in the Northern regions of Russia have been analyzed. There have been assessed the effect of climate change and the associated natural and man-made risks in the Far North on the development of a system of measures to ensure the reliability of energy facilities and options for emergency response. The solutions allowing to increase energy-saving capabilities of buildings and to reduce expenditures at their operation have been designated. Necessity of using non-traditional and renewable energy sources has been considered

Variations in the origin of the medial and inferior calcaneal nerves

WOS: 000234017800002PubMed ID: 16333630Introduction: Entrapment of the medial heel region nerves is often mentioned as a possible cause of heel pain. Some authors have suggested that the medial and inferior calcaneal nerves may be involved in such heel pain, including plantar fasciitis, heel pain syndrome and fat pad disorders. The aim of this study was to give a detailed description of the medial heel that would determine the variability and pattern of the medial and inferior calcaneal nerves, as well as to relate these findings to the currently used incision line for tarsal tunnel, fixations of fractures with external nailing, medial displacement osteotomy and nerve blocks in podiatric medicine. Materials and methods: The origin, relationship, distribution, variability and innervation of medial and inferior calcaneal nerves were studied with the use of a 3.5 power loupe magnification for dissection of 25 adult male feet of formalin-fixed cadavers. The medial heel was found to be innervated by just one medial calcaneal nerve in 38% of the feet, by two medial calcaneal nerves in 46%, by three medial calcaneal nerves in 12% and by four medial calcaneal nerves in 4%. An origin for a medial calcaneal nerve from the medial plantar nerve was found in 46% of the feet. This nerve most often innervates the skin of the posteromedial arch. Results: In our dissection, the rate of occurrence of the medial and inferior calcaneal nerves in medial heel region was 100%. When compared with the inferior calcaneal nerve, the medial calcaneal nerve was posterior, superior and thicker. The inferior calcaneal nerve supplies deeper structures. In the majority of the cases, inferior calcaneal nerve aroused from the lateral plantar nerve, but it may also arise from the tibial nerve, sometimes in a common origin with the medial calcaneal nerve. Conclusions: Knowledge of fine anatomy of the calcaneal nerves is necessary to ensure safe surgical intervention in the medial heel region

RecFOR proteins are essential for Pol V-mediated translesion synthesis and mutagenesis

When the replication fork moves through the template DNA containing lesions, daughter-strand gaps are formed opposite lesion sites. These gaps are subsequently filled-in either by translesion synthesis (TLS) or by homologous recombination. RecA filaments formed within these gaps are key intermediates for both of the gap-filling pathways. For instance, Pol V, the major lesion bypass polymerase in Escherichia coli, requires a functional interaction with the tip of the RecA filament. Here, we show that all three recombination mediator proteins RecFOR are needed to build a functionally competent RecA filament that supports efficient Pol V-mediated TLS in the presence of ssDNA-binding protein (SSB). A positive contribution of RecF protein to Pol V lesion bypass is demonstrated. When Pol III and Pol V are both present, Pol III imparts a negative effect on Pol V-mediated lesion bypass that is counteracted by the combined action of RecFOR and SSB. Mutations in recF, recO or recR gene abolish induced mutagenesis in E. coli