DMC1 and RAD51 bind FxxA and FxPP motifs of BRCA2 via two separate interfaces.

In vertebrates, the BRCA2 protein is essential for meiotic and somatic homologous recombination due to its interaction with the RAD51 and DMC1 recombinases through FxxA and FxPP motifs (here named A- and P-motifs, respectively). The A-motifs present in the eight BRC repeats of BRCA2 compete with the A-motif of RAD51, which is responsible for its self-oligomerization. BRCs thus disrupt RAD51 nucleoprotein filaments in vitro. The role of the P-motifs is less studied. We recently found that deletion of Brca2 exons 12-14 encoding one of them (the prototypical 'PhePP' motif), disrupts DMC1 but not RAD51 function in mouse meiosis. Here we provide a mechanistic explanation for this phenotype by solving the crystal structure of the complex between a BRCA2 fragment containing the PhePP motif and DMC1. Our structure reveals that, despite sharing a conserved phenylalanine, the A- and P-motifs bind to distinct sites on the ATPase domain of the recombinases. The P-motif interacts with a site that is accessible in DMC1 octamers and nucleoprotein filaments. Moreover, we show that this interaction also involves the adjacent protomer and thus increases the stability of the DMC1 nucleoprotein filaments. We extend our analysis to other P-motifs from RAD51AP1 and FIGNL1.

BRCA2-HSF2BP oligomeric ring disassembly by BRME1 promotes homologous recombination.

In meiotic homologous recombination (HR), BRCA2 facilitates loading of the recombinases RAD51 and DMC1 at the sites of double-strand breaks (DSBs). The HSF2BP-BRME1 complex interacts with BRCA2. Its absence causes a severe reduction in recombinase loading at meiotic DSB. We previously showed that, in somatic cancer cells ectopically producing HSF2BP, DNA damage can trigger HSF2BP-dependent degradation of BRCA2, which prevents HR. Here, we report that, upon binding to BRCA2, HSF2BP forms octameric rings that are able to interlock into a large ring-shaped 24-mer. Addition of BRME1 leads to dissociation of both of these ring structures and cancels the disruptive effect of HSF2BP on cancer cell resistance to DNA damage. It also prevents BRCA2 degradation during interstrand DNA crosslink repair in Xenopus egg extracts. We propose that, during meiosis, the control of HSF2BPBRCA2 oligomerization by BRME1 ensures timely assembly of the ring complex that concentrates BRCA2 and controls its turnover, thus promoting HR.

Conformational flexibility and oligomerization of BRCA2 regions induced by RAD51 interaction.

BRCA2 is a key breast cancer associated protein that is predicted to have interspersed regions of intrinsic disorder. Intrinsic disorder coupled with large size likely allows BRCA2 to sample a broad range of conformational space. We expect that the resulting dynamic arrangements of BRCA2 domains are a functionally important aspect of its role in homologous recombination DNA repair. To determine the architectural organization and the associated conformational landscape of BRCA2, we used scanning force microscopy based single molecule analyses to map the flexible regions of the protein and characterize which regions influence oligomerization. We show that the N- and the C-terminal regions are the main flexible regions. Both of these regions also influence BRCA2 oligomerization and interaction with RAD51. In the central Brc repeat region, Brc 1-4 and Brc 5-8 contribute synergistically to BRCA2 interaction with RAD51. We also analysed several single amino acid changes that are potentially clinically relevant and found one, the variant of F1524V, which disrupts key interactions and alters the conformational landscape of the protein. We describe the overall conformation spectrum of BRCA2, which suggests that dynamic structural transitions are key features of its biological function, maintaining genomic stability.

The Recombination Mediator BRCA2: Architectural Plasticity of Recombination Intermediates Revealed by Single-Molecule Imaging (SFM/TIRF).

Cellular functions are defined by dynamic assembly, rearrangement, and disassembly of biomolecules to achieve control and specificity. As an example, effective DNA repair is brought about by the concerted action of several DNA processing proteins. Both changes in the structure of individual proteins and in the arrangement of multiple proteins together (referred to here as architecture) are inherent to biological function. These dynamic changes are exemplified in the breast cancer susceptibility protein 2 (BRCA2). BRCA2 is a DNA repair protein that undergoes changes in its own structure and affects changes in molecular architecture with partners during homologous recombination (HR) repair of DNA double strand breaks. These challenging features of BRCA2 protein, its size and predicted stretches of intrinsically disordered regions, have made it difficult to determine the structural consequences and mechanistic importance of interactions between full-length BRCA2 with RAD51 and other HR proteins. In this chapter, we describe scanning force microscopy (SFM)-based approaches to study DNA-protein complexes involved in HR, the architectural plasticity of full-length BRCA2, and the dynamic reorganization of these molecular components associated with essential steps of HR.

Cohesin Releases DNA through Asymmetric ATPase-Driven Ring Opening.

Cohesin stably holds together the sister chromatids from S phase until mitosis. To do so, cohesin must be protected against its cellular antagonist Wapl. Eco1 acetylates cohesin's Smc3 subunit, which locks together the sister DNAs. We used yeast genetics to dissect how Wapl drives cohesin from chromatin and identified mutants of cohesin that are impaired in ATPase activity but remarkably confer robust cohesion that bypasses the need for the cohesin protectors Eco1 in yeast and Sororin in human cells. We uncover a functional asymmetry within the heart of cohesin's highly conserved ABC-like ATPase machinery and find that both ATPase sites contribute to DNA loading, whereas DNA release is controlled specifically by one site. We propose that Smc3 acetylation locks cohesin rings around the sister chromatids by counteracting an activity associated with one of cohesin's two ATPase sites.

A novel Fanconi anaemia subtype associated with a dominant-negative mutation in RAD51.

Fanconi anaemia (FA) is a hereditary disease featuring hypersensitivity to DNA cross-linker-induced chromosomal instability in association with developmental abnormalities, bone marrow failure and a strong predisposition to cancer. A total of 17 FA disease genes have been reported, all of which act in a recessive mode of inheritance. Here we report on a de novo g.41022153G>A; p.Ala293Thr (NM_002875) missense mutation in one allele of the homologous recombination DNA repair gene RAD51 in an FA-like patient. This heterozygous mutation causes a novel FA subtype, 'FA-R', which appears to be the first subtype of FA caused by a dominant-negative mutation. The patient, who features microcephaly and mental retardation, has reached adulthood without the typical bone marrow failure and paediatric cancers. Together with the recent reports on RAD51-associated congenital mirror movement disorders, our results point to an important role for RAD51-mediated homologous recombination in neurodevelopment, in addition to DNA repair and cancer susceptibility.

P2X7 receptor blockade reverses purinergic facilitation of neck muscle nociception in mice.

INTRODUCTION: Facilitation of neck muscle nociception mediated via purinergic signalling may play a role in the pathophysiology of tension-type headache (TTH). The present study addressed reversal of purinergic facilitation of brainstem nociception via P2X7 antagonist action in anaesthetized mice. METHODS: Following administration of α,ß-meATP (i.m. 20 µL/min, 20 µL each) into semispinal neck muscles, the impact of neck muscle nociceptive input on brainstem processing was monitored by the jaw-opening reflex in anaesthetized mice (n = 20). The hypothesized involvement of the P2X7 receptor in the α,ß-meATP effect was addressed with i.p. (systemic) and i.m. (semispinalis, 20 µL/min, 20 µL each) administration of P2X7 inhibitor A438079 during established facilitation; i.p. saline served as control. RESULTS: α,ß-meATP reliably induced jaw-opening reflex facilitation (256 ± 48% (mean ± SEM), n = 20). I.p. A438079 (150, 300 µmol/kg) completely reversed this α,ß-meATP effect dose-dependently. Neither saline nor intramuscular A438079 (100 µM) altered facilitated brainstem nociceptive processing. DISCUSSION: These data suggest that muscular structures are not directly involved in the P2X7 antagonist-mediated reversal of purinergic facilitation. Instead, involvement of neuronal structures, particularly of the central nervous system, seems more probable. The results from this animal experimental model may point to involvement of purinergic P2X7 receptors in TTH pathophysiology and may suggest potential future targets for its pharmacological treatment.

Impaired somatosensation in tongue mucosa of smokers.

Smoking has been indicated as a risk factor for oral diseases and can lead to altered sense of taste. So far, the effects of sensory changes on the tongue are not investigated. In this study, quantitative sensory testing was used to evaluate somatosensory function in the lingual region. Eighty healthy volunteers were investigated (20 smokers, 20 non-smokers). Subjects were bilaterally tested in innervation areas of lingual nerves. Thresholds of cold and warm detection, cold and heat pain, and mechanical detection were determined. As control for systemic, extraoral effects of smoking, tests were additionally performed in 40 volunteers (20 smokers, 20 non-smokers) on the skin of the chin innervated by the mental branch of the trigeminal nerve. Cold (p < 0.001), warm detection thresholds (p < 0.001), and thermal sensory limen (p < 0.001) showed higher sensitivity in non-smokers as compared to smokers. Heat pain and mechanical detection, as well as all tests in the skin of the chin, showed no significant differences. The impaired temperature perception in smokers indicates a reduction of somatosensory functions in the tongue, possibly caused by nerve degeneration associated with smoking. Possible systemic effects of smoking do not seem to affect extraoral trigeminal branches.

Acetylsalicylic acid inhibits α,ß-meATP-induced facilitation of neck muscle nociception in mice--implications for acute treatment of tension-type headache.

Infusion of α,ß-methylene ATP (α,ß-meATP) into murine neck muscle facilitates brainstem nociception. This animal experimental model is suggested to be appropriate for investigating pathophysiological mechanisms in tension-type headache. It was hypothesized that d-lysine acetylsalicylic acid (ASA, aspirin®) reverses this α,ß-meATP effect. Facilitation of neck muscle nociceptive processing was induced via bilateral infusion of α,ß-meATP into semispinal neck muscles (100 nM, 20 µl each) in 42 anesthetized mice. Brainstem nociception was monitored by the jaw-opening reflex elicited via electrical tongue stimulation. The hypothesis was addressed by subsequent (15, 30, 60 mg/kg) and preceding (60 mg/kg) intraperitoneal ASA injection. Saline served as control to ASA solution. Subsequent ASA dose-dependently reversed α,ß-meATP-induced reflex facilitation and was the most prominent with 60 mg/kg. Preceding 60 mg/kg ASA prevented reflex facilitation. Cyclooxygenases are involved in nociceptive transmission. Former experiments showed that unspecific inhibition of cyclooxygenases does not alter the α,ß-meATP effect. This suggests a specific mode of action of ASA. The concept is accepted that neck muscle nociception is involved in the pathophysiology of tension-type headache. Thus, objective proof of ASA effects in this experimental model may emphasize its major role in pharmacological treatment of tension-type headache attacks.

Bilateral Monteggia fracture in adults.

INTRODUCTION: In 1814 Giovanni Monteggia first described two cases of fractures of the proximal third of ulna with dislocation of the radial head. These fractures are more common in children than in adults, and mutual Monteggia fracture is a rare complication. This study presents a treatment course of a patient with bilateral Monteggia fracture. CASE REPORT: A 55-year-old patient was injured by falling in the yard. Radiography showed bilateral Monteggia fracture type II (by the Badon classification). Operative treatment of fracture was done by a compression plate on the right side and by the zuggurtung technique on the left one. Closed repositioning of the radial head was done on both sides. The patient was wearing a plaster splint for the upper arm for 21 days. After removing the fixation, the function of the elbow was determined by the Broberg Morrey score (BM) which was on the right side 45.5 and on the left side 47.5. After the proper physical therapy, four months after the surgery, BM score was 100 on the right side, and 93 on the left one. CONCLUSION: Surgical treatment and early rehabilitation is the key for the return of good function of both elbows.

Inhibition of nNOS prevents and inhibition of iNOS reverses α,ß-meATP-induced facilitation of neck muscle nociception in mice.

Infusion of α,ß-methylene ATP (α,ß-meATP) into murine neck muscle facilitates brainstem nociception. Unspecific nitric oxide synthase (NOS) inhibition prevents and reverses this sensitization. It is unclear whether neuronal (nNOS), inducible (iNOS) or endothelial NOS isoenzymes are involved in this α,ß-meATP effect. Hypothesized involvement of nNOS isoenzyme was addressed by preceding (0.5, 1, and 2 mg/kg) and subsequent (2 mg/kg) intraperitoneal injection of the nNOS-inhibitor NPLA. iNOS involvement was addressed by subsequent, intraperitoneal administration of the iNOS-inhibitor 1400 W (2 mg/kg). Brainstem nociception was monitored by the jaw-opening reflex elicited via electrical tongue stimulation in 45 anesthetized mice. Preceding NPLA dose-dependently prevented α,ß-meATP-induced reflex facilitation. Whereas subsequent inhibition of nNOS showed no effect, iNOS inhibition by 1400 W significantly reversed reflex facilitation. Data provide evidence that nNOS plays a major role in induction and iNOS in maintenance of facilitation in neck muscle nociception. Divergent roles of NOS isoenzymes may promote research on target specific treatment for headache and neck muscle pain.

Inter-subunit interactions that coordinate Rad51's activities.

Rad51 is the central catalyst of homologous recombination in eukaryotes and is thus critical for maintaining genomic integrity. Recent crystal structures of filaments formed by Rad51 and the closely related archeal RadA and eubacterial RecA proteins place the ATPase site at the protomeric interface. To test the relevance of this feature, we mutated conserved residues at this interface and examined their effects on key activities of Rad51: ssDNA-stimulated ATP hydrolysis, DNA binding, polymerization on DNA substrates and catalysis of strand-exchange reactions. Our results show that the interface seen in the crystal structures is very important for nucleoprotein filament formation. H352 and R357 of yeast Rad51 are essential for assembling the catalytically competent form of the enzyme on DNA substrates and coordinating its activities. However, contrary to some previous suggestions, neither of these residues is critical for ATP hydrolysis.

Human Rad51 filaments on double- and single-stranded DNA: correlating regular and irregular forms with recombination function.

Recombinase proteins assembled into helical filaments on DNA are believed to be the catalytic core of homologous recombination. The assembly, disassembly and dynamic rearrangements of this structure must drive the DNA strand exchange reactions of homologous recombination. The sensitivity of eukaryotic recombinase activity to reaction conditions in vitro suggests that the status of bound nucleotide cofactors is important for function and possibly for filament structure. We analyzed nucleoprotein filaments formed by the human recombinase Rad51 in a variety of conditions on double-stranded and single-stranded DNA by scanning force microscopy. Regular filaments with extended double-stranded DNA correlated with active in vitro recombination, possibly due to stabilizing the DNA products of these assays. Though filaments formed readily on single-stranded DNA, they were very rarely regular structures. The irregular structure of filaments on single-stranded DNA suggests that Rad51 monomers are dynamic in filaments and that regular filaments are transient. Indeed, single molecule force spectroscopy of Rad51 filament assembly and disassembly in magnetic tweezers revealed protein association and disassociation from many points along the DNA, with kinetics different from those of RecA. The dynamic rearrangements of proteins and DNA within Rad51 nucleoprotein filaments could be key events driving strand exchange in homologous recombination.