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.

Publisher Correction: Molecular basis of microhomology-mediated end-joining by purified full-length Polθ.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Molecular basis of microhomology-mediated end-joining by purified full-length Polθ.

DNA polymerase θ (Polθ) is a unique polymerase-helicase fusion protein that promotes microhomology-mediated end-joining (MMEJ) of DNA double-strand breaks (DSBs). How full-length human Polθ performs MMEJ at the molecular level remains unknown. Using a biochemical approach, we find that the helicase is essential for Polθ MMEJ of long ssDNA overhangs which model resected DSBs. Remarkably, Polθ MMEJ of ssDNA overhangs requires polymerase-helicase attachment, but not the disordered central domain, and occurs independently of helicase ATPase activity. Using single-particle microscopy and biophysical methods, we find that polymerase-helicase attachment promotes multimeric gel-like Polθ complexes that facilitate DNA accumulation, DNA synapsis, and MMEJ. We further find that the central domain regulates Polθ multimerization and governs its DNA substrate requirements for MMEJ. These studies identify unexpected functions for the helicase and central domain and demonstrate the importance of polymerase-helicase tethering in MMEJ and the structural organization of Polθ.

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.

Imaging of DNA and Protein by SFM and Combined SFM-TIRF Microscopy.

Direct imaging is invaluable for understanding the mechanism of complex genome transactions where proteins work together to organize, transcribe, replicate and repair DNA. Scanning (or atomic) force microscopy is an ideal tool for this, providing 3D information on molecular structure at nm resolution from defined components. This is a convenient and practical addition to in vitro studies as readily obtainable amounts of purified proteins and DNA are required. The images reveal structural details on the size and location of DNA bound proteins as well as protein-induced arrangement of the DNA, which are directly correlated in the same complexes. In addition, even from static images, the different forms observed and their relative distributions can be used to deduce the variety and stability of different complexes that are necessarily involved in dynamic processes. Recently available instruments that combine fluorescence with topographic imaging allow the identification of specific molecular components in complex assemblies, which broadens the applications and increases the information obtained from direct imaging of molecular complexes. We describe here basic methods for preparing samples of proteins, DNA and complexes of the two for topographic imaging and quantitative analysis. We also describe special considerations for combined fluorescence and topographic imaging of molecular complexes.

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.

Innocuous peripheral nerve stimulation shifts stimulus-response function of painful laser stimulation in man.

OBJECTIVES: Electrical peripheral nerve stimulation (PNS) is discussed as an effective neuromodulatory treatment in chronic pain. This human experimental study hypothesized a rightward shift of stimulus-response function as a marker of antinociceptive and analgesic PNS effects. MATERIALS AND METHODS: Innocuous electrical PNS of the left superficial radial nerve trunk evoked paresthesia on the left hand dorsum in 29 healthy volunteers. In this innervation area, laser stimulation was performed before, during, and after PNS. Ten different laser intensities ranging between perception and tolerance thresholds were applied. Cortical laser-evoked potentials (LEP) were recorded, and perceptual ratings were documented. Data were analyzed in low, medium, and high laser intensity categories. Stimulus-response functions were calculated. Laser detection and pain thresholds were interpolated. RESULTS: Interpolated laser thresholds after logarithmic regression were not different from measured thresholds. Laser pain threshold increased during and after PNS. LEP amplitude decreased at medium and high intensities under PNS. Ratings transiently decreased during PNS at medium and high laser intensities. CONCLUSIONS: Modulation of laser pain threshold, perceptual ratings, and LEP indicates a rightward shift of stimulus-response function under PNS. These data emphasize antinociceptive and analgesic effects of PNS in an experimental human model and support its clinical neuromodulative relevance.

The Ku heterodimer and the metabolism of single-ended DNA double-strand breaks.

Single-ended double-strand breaks (DSBs) are a common form of spontaneous DNA break, generated when the replisome encounters a discontinuity in the DNA template. Given their prevalence, understanding the mechanisms governing the fate(s) of single-ended DSBs is important. We describe the influence of the Ku heterodimer and Mre11 nuclease activity on processing of single-ended DSBs. Separation-of-function alleles of yku70 were derived that phenocopy Ku deficiency with respect to single-ended DSBs but remain proficient for NHEJ. The Ku mutants fail to regulate Exo1 activity, and bypass the requirement for Mre11 nuclease activity in the repair of camptothecin-induced single-ended DSBs. Ku mutants exhibited reduced affinity for DNA ends, manifest as both reduced end engagement and enhanced probability of diffusing inward on linear DNA. This study reveals an interplay between Ku and Mre11 in the metabolism of single-ended DSBs that is distinct from repair pathway choice at double-ended DSBs.

Caffeine suppresses homologous recombination through interference with RAD51-mediated joint molecule formation.

Caffeine is a widely used inhibitor of the protein kinases that play a central role in the DNA damage response. We used chemical inhibitors and genetically deficient mouse embryonic stem cell lines to study the role of DNA damage response in stable integration of the transfected DNA and found that caffeine rapidly, efficiently and reversibly inhibited homologous integration of the transfected DNA as measured by several homologous recombination-mediated gene-targeting assays. Biochemical and structural biology experiments revealed that caffeine interfered with a pivotal step in homologous recombination, homologous joint molecule formation, through increasing interactions of the RAD51 nucleoprotein filament with non-homologous DNA. Our results suggest that recombination pathways dependent on extensive homology search are caffeine-sensitive and stress the importance of considering direct checkpoint-independent mechanisms in the interpretation of the effects of caffeine on DNA repair.

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.

A human XRCC4-XLF complex bridges DNA.

DNA double-strand breaks pose a significant threat to cell survival and must be repaired. In higher eukaryotes, such damage is repaired efficiently by non-homologous end joining (NHEJ). Within this pathway, XRCC4 and XLF fulfill key roles required for end joining. Using DNA-binding and -bridging assays, combined with direct visualization, we present evidence for how XRCC4-XLF complexes robustly bridge DNA molecules. This unanticipated, DNA Ligase IV-independent bridging activity by XRCC4-XLF suggests an early role for this complex during end joining, in addition to its more well-established later functions. Mutational analysis of the XRCC4-XLF C-terminal tail regions further identifies specialized functions in complex formation and interaction with DNA and DNA Ligase IV. Based on these data and the crystal structure of an extended protein filament of XRCC4-XLF at 3.94 Å, a model for XRCC4-XLF complex function in NHEJ is presented.

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.

Sample preparation for SFM imaging of DNA, proteins, and DNA-protein complexes.

Direct imaging is invaluable for understanding the mechanism of complex genome transactions where proteins work together to organize, transcribe, replicate, and repair DNA. Scanning (or atomic) force microscopy is an ideal tool for this, providing 3D information on molecular structure at nanometer resolution from defined components. This is a convenient and practical addition to in vitro studies as readily obtainable amounts of purified proteins and DNA are required. The images reveal structural details on the size and location of DNA-bound proteins as well as protein-induced arrangement of the DNA, which are directly correlated in the same complexes. In addition, even from static images, the different forms observed and their relative distributions can be used to deduce the variety and stability of different complexes that are necessarily involved in dynamic processes. Recently available instruments that combine fluorescence with topographic imaging allow the identification of specific molecular components in complex assemblies, which broadens the applications and increases the information obtained from direct imaging of molecular complexes. We describe here basic methods for preparing samples of proteins, DNA, and complexes of the two for topographic imaging and quantitative analysis. We also describe special considerations for combined fluorescence and topographic imaging of molecular complexes.

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.

[Length of hospitalization of patients with ankle fracture].

INTRODUCTION: Fractures of ankle are one of the most frequent interacticular fractures that require operative treatment. During the work, the influence of some particular factors (the length of the preoperative period, the complications of the operative period and the application of antibiotics) to the length of the postoperative intrahospital stay, are scrutinised. METHOD: The patients with ankle fracture treated by operation were comprised by the retrospective study in the Traumatologic department in the CHC Zemun in period of 2003 to 2006, and they were divided in three groups depending on the length of postoperative stay. RESULTS: The period of time before the operation (Chi = 0.405, p < 0.01), the appearance of complications (Chi = 0.465, p < 0.01), as well as the length of the period of antibiotic application (Chi = 0.580, p < .01), significantly influence to the length of the postoperative intrahospital stay. The everage length of intrahospital stay for the patients with registered complications was 19 days, while for the patients without registered complications was 10 days. There is statistically significant difference in the length of intrahospital recovery, depending on various complications (logrank = 35.74; df = 5; p < 0.01). CONCLUSION: It is necessary to treat these fractures as soon as possible, for this way of medical treatment results with less number of complications, shorter stay of patients in hospital and thereby reduced treatment costs.

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.