DNA synthesis determines the binding mode of the human mitochondrial single-stranded DNA-binding protein

[EN] Single-stranded DNA-binding proteins (SSBs) play a key role in genome maintenance, binding and organizing single-stranded DNA (ssDNA) intermediates. Multimeric SSBs, such as the human mitochondrial SSB (HmtSSB), present multiple sites to interact with ssDNA, which has been shown in vitro to enable them to bind a variable number of single-stranded nucleotides depending on the salt and protein concentration. It has long been suggested that different binding modes might be used selectively for different functions. To study this possibility, we used optical tweezers to determine and compare the structure and energetics of long, individual HmtSSB¿DNA complexes assembled on preformed ssDNA and on ssDNA generated gradually during `in situ¿ DNA synthesis. We show that HmtSSB binds to preformed ss-DNA in two major modes, depending on salt and protein concentration. However, when protein binding was coupled to strand-displacement DNA synthesis, only one of the two binding modes was observed under all experimental conditions. Our results reveal a key role for the gradual generation of ssDNA in modulating the binding mode of a multimeric SSB protein and consequently, in generating the appropriate nucleoprotein structure for DNA synthetic reactions required for genome maintenance.We are grateful to Prof. M. Salas laboratory (CBMSO-CSIC) for generously providing the Phi29 DNA polymerase and to Juan P. García Villaluenga (UCM) for useful discussions. Spanish Ministry of Economy and Competitiveness [MAT2015-71806-R to J.R.A-G, FIS2010-17440, FIS2015-67765-R to F.J.C., BFU2012-31825, BFU2015-63714-R to B.I.]; Spanish Ministry of Education, Culture and Sport [FPU13/02934 to J.J., FPU13/02826 to E.B-H.]; National Institutes of Health [GM45925 to L.S.K.]; University of Tampere (to G.L.C.); Programa de Financiacion Universidad Complutense de Madrid-Santander Universidades [CT45/15-CT46/15 to F.C.]. Funding for open access charge: Spanish Ministry of Economy and Competitiveness [BFU2015-63714-R].Morin, J.; Cerrón, F.; Jarillo, J.; Beltran-Heredia, E.; Ciesielski, G.; Arias-Gonzalez, JR.; Kaguni, L.... (2017). DNA synthesis determines the binding mode of the human mitochondrial single-stranded DNA-binding protein. Nucleic Acids Research. 45(12):7237-7248. https://doi.org/10.1093/nar/gkx395S723772484512Shereda, R. D., Kozlov, A. G., Lohman, T. M., Cox, M. M., & Keck, J. L. (2008). SSB as an Organizer/Mobilizer of Genome Maintenance Complexes. Critical Reviews in Biochemistry and Molecular Biology, 43(5), 289-318. doi:10.1080/10409230802341296Flynn, R. L., & Zou, L. (2010). Oligonucleotide/oligosaccharide-binding fold proteins: a growing family of genome guardians. Critical Reviews in Biochemistry and Molecular Biology, 45(4), 266-275. doi:10.3109/10409238.2010.488216Murzin, A. G. (1993). OB(oligonucleotide/oligosaccharide binding)-fold: common structural and functional solution for non-homologous sequences. The EMBO Journal, 12(3), 861-867. doi:10.1002/j.1460-2075.1993.tb05726.xKozlov, A. G., Weiland, E., Mittal, A., Waldman, V., Antony, E., Fazio, N., … Lohman, T. M. (2015). Intrinsically Disordered C-Terminal Tails of E. coli Single-Stranded DNA Binding Protein Regulate Cooperative Binding to Single-Stranded DNA. Journal of Molecular Biology, 427(4), 763-774. doi:10.1016/j.jmb.2014.12.020Kuznetsov, S. V., Kozlov, A. G., Lohman, T. M., & Ansari, A. (2006). Microsecond Dynamics of Protein–DNA Interactions: Direct Observation of the Wrapping/Unwrapping Kinetics of Single-stranded DNA around the E.coli SSB Tetramer. Journal of Molecular Biology, 359(1), 55-65. doi:10.1016/j.jmb.2006.02.070Lohman, T. M., & Ferrari, M. E. (1994). Escherichia Coli Single-Stranded DNA-Binding Protein: Multiple DNA-Binding Modes and Cooperativities. Annual Review of Biochemistry, 63(1), 527-570. doi:10.1146/annurev.bi.63.070194.002523Maier, D., Farr, C. L., Poeck, B., Alahari, A., Vogel, M., Fischer, S., … Schneuwly, S. (2001). Mitochondrial Single-stranded DNA-binding Protein Is Required for Mitochondrial DNA Replication and Development in Drosophila melanogaster. Molecular Biology of the Cell, 12(4), 821-830. doi:10.1091/mbc.12.4.821Ruhanen, H., Borrie, S., Szabadkai, G., Tyynismaa, H., Jones, A. W. E., Kang, D., … Yasukawa, T. (2010). Mitochondrial single-stranded DNA binding protein is required for maintenance of mitochondrial DNA and 7S DNA but is not required for mitochondrial nucleoid organisation. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1803(8), 931-939. doi:10.1016/j.bbamcr.2010.04.008Farr, C. L., Matsushima, Y., Lagina, A. T., Luo, N., & Kaguni, L. S. (2004). Physiological and Biochemical Defects in Functional Interactions of Mitochondrial DNA Polymerase and DNA-binding Mutants of Single-stranded DNA-binding Protein. Journal of Biological Chemistry, 279(17), 17047-17053. doi:10.1074/jbc.m400283200Van Tuyle, G. C., & Pavco, P. A. (1985). The rat liver mitochondrial DNA-protein complex: displaced single strands of replicative intermediates are protein coated. The Journal of Cell Biology, 100(1), 251-257. doi:10.1083/jcb.100.1.251Clayton, D. A. (1982). Replication of animal mitochondrial DNA. Cell, 28(4), 693-705. doi:10.1016/0092-8674(82)90049-6Farr, C. L., Wang, Y., & Kaguni, L. S. (1999). Functional Interactions of Mitochondrial DNA Polymerase and Single-stranded DNA-binding Protein. Journal of Biological Chemistry, 274(21), 14779-14785. doi:10.1074/jbc.274.21.14779Korhonen, J. A., Gaspari, M., & Falkenberg, M. (2003). TWINKLE Has 5′ → 3′ DNA Helicase Activity and Is Specifically Stimulated by Mitochondrial Single-stranded DNA-binding Protein. Journal of Biological Chemistry, 278(49), 48627-48632. doi:10.1074/jbc.m306981200Miralles Fusté, J., Shi, Y., Wanrooij, S., Zhu, X., Jemt, E., Persson, Ö., … Falkenberg, M. (2014). In Vivo Occupancy of Mitochondrial Single-Stranded DNA Binding Protein Supports the Strand Displacement Mode of DNA Replication. PLoS Genetics, 10(12), e1004832. doi:10.1371/journal.pgen.1004832Oliveira, M. T., & Kaguni, L. S. (2011). Reduced Stimulation of Recombinant DNA Polymerase γ and Mitochondrial DNA (mtDNA) Helicase by Variants of Mitochondrial Single-stranded DNA-binding Protein (mtSSB) Correlates with Defects in mtDNA Replication in Animal Cells. Journal of Biological Chemistry, 286(47), 40649-40658. doi:10.1074/jbc.m111.289983Williams, A. J., & Kaguni, L. S. (1995). Stimulation ofDrosophilaMitochondrial DNA Polymerase by Single-stranded DNA-binding Protein. Journal of Biological Chemistry, 270(2), 860-865. doi:10.1074/jbc.270.2.860Bogenhagen, D. F., Wang, Y., Shen, E. L., & Kobayashi, R. (2003). Protein Components of Mitochondrial DNA Nucleoids in Higher Eukaryotes. Molecular & Cellular Proteomics, 2(11), 1205-1216. doi:10.1074/mcp.m300035-mcp200BARAT-GUERIDE, M., DUFRESNE, C., & RICKWOOD, D. (1989). Effect of DNA conformation on the transcription of mitochondrial DNA. European Journal of Biochemistry, 183(2), 297-302. doi:10.1111/j.1432-1033.1989.tb14928.xYang, C., Curth, U., Urbanke, C., & Kang, C. (1997). Crystal structure of human mitochondrial single-stranded DNA binding protein at 2.4 Å resolution. Nature Structural Biology, 4(2), 153-157. doi:10.1038/nsb0297-153Raghunathan, S., Ricard, C. S., Lohman, T. M., & Waksman, G. (1997). Crystal structure of the homo-tetrameric DNA binding domain of Escherichia coli single-stranded DNA-binding protein determined by multiwavelength x-ray diffraction on the selenomethionyl protein at 2.9-A resolution. Proceedings of the National Academy of Sciences, 94(13), 6652-6657. doi:10.1073/pnas.94.13.6652CURTH, U., URBANKE, C., GREIPEL, J., GERBERDING, H., TIRANTI, V., & ZEVIANI, M. (1994). Single-stranded-DNA-binding proteins from human mitochondria and Escherichia coli have analogous physicochemical properties. European Journal of Biochemistry, 221(1), 435-443. doi:10.1111/j.1432-1033.1994.tb18756.xOverman, L. B., & Lohman, T. M. (1994). Linkage of pH, Anion and Cation Effects in Protein-Nucleic Acid Equilibria. Journal of Molecular Biology, 236(1), 165-178. doi:10.1006/jmbi.1994.1126Bhattacharyya, B., George, N. P., Thurmes, T. M., Zhou, R., Jani, N., Wessel, S. R., … Keck, J. L. (2013). Structural mechanisms of PriA-mediated DNA replication restart. Proceedings of the National Academy of Sciences, 111(4), 1373-1378. doi:10.1073/pnas.1318001111Carlini, L. E., Porter, R. D., Curth, U., & Urbanke, C. (1993). Viability and preliminary in vivo characterization of site directed mutants of Escherichia coli single-stranded DNA-binding protein. Molecular Microbiology, 10(5), 1067-1075. doi:10.1111/j.1365-2958.1993.tb00977.xGriffith, J. D., Harris, L. D., & Register, J. (1984). Visualization of SSB-ssDNA Complexes Active in the Assembly of Stable RecA-DNA Filaments. Cold Spring Harbor Symposia on Quantitative Biology, 49(0), 553-559. doi:10.1101/sqb.1984.049.01.062Morrical, S. W., & Cox, M. M. (1990). Stabilization of recA protein-ssDNA complexes by the single-stranded DNA binding protein of Escherichia coli. Biochemistry, 29(3), 837-843. doi:10.1021/bi00455a034Muniyappa, K., Williams, K., Chase, J. W., & Radding, C. M. (1990). Active nucleoprotein filaments of single-stranded binding protein and recA protein on single-stranded DNA have a regular repeating structure. Nucleic Acids Research, 18(13), 3967-3973. doi:10.1093/nar/18.13.3967Wessel, S. R., Marceau, A. H., Massoni, S. C., Zhou, R., Ha, T., Sandler, S. J., & Keck, J. L. (2013). PriC-mediated DNA Replication Restart Requires PriC Complex Formation with the Single-stranded DNA-binding Protein. Journal of Biological Chemistry, 288(24), 17569-17578. doi:10.1074/jbc.m113.478156Bell, J. C., Liu, B., & Kowalczykowski, S. C. (2015). Imaging and energetics of single SSB-ssDNA molecules reveal intramolecular condensation and insight into RecOR function. eLife, 4. doi:10.7554/elife.08646Suksombat, S., Khafizov, R., Kozlov, A. G., Lohman, T. M., & Chemla, Y. R. (2015). Structural dynamics of E. coli single-stranded DNA binding protein reveal DNA wrapping and unwrapping pathways. eLife, 4. doi:10.7554/elife.08193Zhou, R., Kozlov, A. G., Roy, R., Zhang, J., Korolev, S., Lohman, T. M., & Ha, T. (2011). SSB Functions as a Sliding Platform that Migrates on DNA via Reptation. Cell, 146(2), 222-232. doi:10.1016/j.cell.2011.06.036Pant, K., Karpel, R. L., Rouzina, I., & Williams, M. C. (2004). Mechanical Measurement of Single-molecule Binding Rates: Kinetics of DNA Helix-destabilization by T4 Gene 32 Protein. Journal of Molecular Biology, 336(4), 851-870. doi:10.1016/j.jmb.2003.12.025Pant, K., Karpel, R. L., Rouzina, I., & Williams, M. C. (2005). Salt Dependent Binding of T4 Gene 32 Protein to Single and Double-stranded DNA: Single Molecule Force Spectroscopy Measurements. Journal of Molecular Biology, 349(2), 317-330. doi:10.1016/j.jmb.2005.03.065Robberson, D. L., & Clayton, D. A. (1972). Replication of Mitochondrial DNA in Mouse L Cells and Their Thymidine Kinase- Derivatives: Displacement Replication on a Covalently-Closed Circular Template. Proceedings of the National Academy of Sciences, 69(12), 3810-3814. doi:10.1073/pnas.69.12.3810Ciesielski, G. L., Bermek, O., Rosado-Ruiz, F. A., Hovde, S. L., Neitzke, O. J., Griffith, J. D., & Kaguni, L. S. (2015). Mitochondrial Single-stranded DNA-binding Proteins Stimulate the Activity of DNA Polymerase γ by Organization of the Template DNA. Journal of Biological Chemistry, 290(48), 28697-28707. doi:10.1074/jbc.m115.673707Lázaro, J. M., Blanco, L., & Salas, M. (1995). [5] Purification of bacteriophage φ29 DNA polymerase. DNA Replication, 42-49. doi:10.1016/0076-6879(95)62007-9Ibarra, B., Chemla, Y. R., Plyasunov, S., Smith, S. B., Lázaro, J. M., Salas, M., & Bustamante, C. (2009). Proofreading dynamics of a processive DNA polymerase. The EMBO Journal, 28(18), 2794-2802. doi:10.1038/emboj.2009.219Morin, J. A., Cao, F. J., Lazaro, J. M., Arias-Gonzalez, J. R., Valpuesta, J. M., Carrascosa, J. L., … Ibarra, B. (2012). Active DNA unwinding dynamics during processive DNA replication. Proceedings of the National Academy of Sciences, 109(21), 8115-8120. doi:10.1073/pnas.1204759109Smith, S. B., Cui, Y., & Bustamante, C. (2003). [7] Optical-trap force transducer that operates by direct measurement of light momentum. Biophotonics, Part B, 134-162. doi:10.1016/s0076-6879(03)61009-8Bosco, A., Camunas-Soler, J., & Ritort, F. (2013). Elastic properties and secondary structure formation of single-stranded DNA at monovalent and divalent salt conditions. Nucleic Acids Research, 42(3), 2064-2074. doi:10.1093/nar/gkt1089Smith, S., Finzi, L., & Bustamante, C. (1992). Direct mechanical measurements of the elasticity of single DNA molecules by using magnetic beads. Science, 258(5085), 1122-1126. doi:10.1126/science.1439819Longley, M. J., Smith, L. A., & Copeland, W. C. (2009). Preparation of Human Mitochondrial Single-Stranded DNA-Binding Protein. Mitochondrial DNA, 73-85. doi:10.1007/978-1-59745-521-3_5Li, K., & Williams, R. S. (1997). Tetramerization and Single-stranded DNA Binding Properties of Native and Mutated Forms of Murine Mitochondrial Single-stranded DNA-binding Proteins. Journal of Biological Chemistry, 272(13), 8686-8694. doi:10.1074/jbc.272.13.8686Jarillo, J., Morín, J. A., Beltrán-Heredia, E., Villaluenga, J. P. G., Ibarra, B., & Cao, F. J. (2017). Mechanics, thermodynamics, and kinetics of ligand binding to biopolymers. PLOS ONE, 12(4), e0174830. doi:10.1371/journal.pone.0174830Bujalowski, W., & Lohman, T. M. (1986). Escherichia coli single-strand binding protein forms multiple, distinct complexes with single-stranded DNA. Biochemistry, 25(24), 7799-7802. doi:10.1021/bi00372a003Thömmes, P., Farr, C. L., Marton, R. F., Kaguni, L. S., & Cotterill, S. (1995). Mitochondrial Single-stranded DNA-binding Protein fromDrosophilaEmbryos. Journal of Biological Chemistry, 270(36), 21137-21143. doi:10.1074/jbc.270.36.21137Rodriguez, I., Lazaro, J. M., Blanco, L., Kamtekar, S., Berman, A. J., Wang, J., … de Vega, M. (2005). A specific subdomain in  29 DNA polymerase confers both processivity and strand-displacement capacity. Proceedings of the National Academy of Sciences, 102(18), 6407-6412. doi:10.1073/pnas.0500597102Kamtekar, S., Berman, A. J., Wang, J., Lázaro, J. M., de Vega, M., Blanco, L., … Steitz, T. A. (2004). Insights into Strand Displacement and Processivity from the Crystal Structure of the Protein-Primed DNA Polymerase of Bacteriophage φ29. Molecular Cell, 16(4), 609-618. doi:10.1016/j.molcel.2004.10.019Chrysogelos, S., & Griffith, J. (1982). Escherichia coli single-strand binding protein organizes single-stranded DNA in nucleosome-like units. Proceedings of the National Academy of Sciences, 79(19), 5803-5807. doi:10.1073/pnas.79.19.5803Hamon, L., Pastre, D., Dupaigne, P., Breton, C. L., Cam, E. L., & Pietrement, O. (2007). High-resolution AFM imaging of single-stranded DNA-binding (SSB) protein--DNA complexes. Nucleic Acids Research, 35(8), e58-e58. doi:10.1093/nar/gkm147Takamatsu, C., Umeda, S., Ohsato, T., Ohno, T., Abe, Y., Fukuoh, A., … Kang, D. (2002). Regulation of mitochondrial D‐loops by transcription factor A and single‐stranded DNA‐binding protein. EMBO reports, 3(5), 451-456. doi:10.1093/embo-reports/kvf099Wang, Y., & Bogenhagen, D. F. (2006). Human Mitochondrial DNA Nucleoids Are Linked to Protein Folding Machinery and Metabolic Enzymes at the Mitochondrial Inner Membrane. Journal of Biological Chemistry, 281(35), 25791-25802. doi:10.1074/jbc.m604501200Brown, T. A. (2005). Replication of mitochondrial DNA occurs by strand displacement with alternative light-strand origins, not via a strand-coupled mechanism. Genes & Development, 19(20), 2466-2476. doi:10.1101/gad.135210

Minimal lepton flavor violating realizations of minimal seesaw models

We study the implications of the global U(1)R symmetry present in minimal lepton flavor violating implementations of the seesaw mechanism for neutrino masses. In the context of minimal type I seesaw scenarios with a slightly broken U(1)R, we show that, depending on the R-charge assignments, two classes of generic models can be identified. Models where the right-handed neutrino masses and the lepton number breaking scale are decoupled, and models where the parameters that slightly break the U(1)R induce a suppression in the light neutrino mass matrix. We show that within the first class of models, contributions of right-handed neutrinos to charged lepton flavor violating processes are severely suppressed. Within the second class of models we study the charged lepton flavor violating phenomenology in detail, focusing on mu to e gamma, mu to 3e and mu to e conversion in nuclei. We show that sizable contributions to these processes are naturally obtained for right-handed neutrino masses at the TeV scale. We then discuss the interplay with the effects of the right-handed neutrino interactions on primordial B - L asymmetries, finding that sizable right-handed neutrino contributions to charged lepton flavor violating processes are incompatible with the requirement of generating (or even preserving preexisting) B - L asymmetries consistent with the observed baryon asymmetry of the Universe.Comment: 21 pages, 4 figures; version 2: Discussion on possible generic models extended, typos corrected, references added. Version matches publication in JHE

Calibration and in orbit performance of the reflection grating spectrometer onboard XMM-Newton

Context: XMM-Newton was launched on 10 December 1999 and has been operational since early 2000. One of the instruments onboard XMM-Newton is the reflection grating spectrometer (RGS). Two identical RGS instruments are available, with each RGS combining a reflection grating assembly (RGA) and a camera with CCDs to record the spectra. Aims: We describe the calibration and in-orbit performance of the RGS instrument. By combining the preflight calibration with appropriate inflight calibration data including the changes in detector performance over time, we aim at profound knowledge about the accuracy in the calibration. This will be crucial for any correct scientific interpretation of spectral features for a wide variety of objects. Methods: Ground calibrations alone are not able to fully characterize the instrument. Dedicated inflight measurements and constant monitoring are essential for a full understanding of the instrument and the variations of the instrument response over time. Physical models of the instrument are tuned to agree with calibration measurements and are the basis from which the actual instrument response can be interpolated over the full parameter space. Results: Uncertainties in the instrument response have been reduced to < 10% for the effective area and < 6 mA for the wavelength scale (in the range from 8 A to 34 A. The remaining systematic uncertainty in the detection of weak absorption features has been estimated to be 1.5%. Conclusions: Based on a large set of inflight calibration data and comparison with other instruments onboard XMM-Newton, the calibration accuracy of the RGS instrument has been improved considerably over the preflight calibrations.Comment: Accepted for publication in Astronomy and Astrophysics, Astronomical instrumentation sectio

Dynamical R-parity Breaking at the LHC

In a class of extensions of the minimal supersymmetric standard model with (B-L)/left-right symmetry that explains the neutrino masses, breaking R-parity symmetry is an essential and dynamical requirement for successful gauge symmetry breaking. Two consequences of these models are: (i) a new kind of R-parity breaking interaction that protects proton stability but adds new contributions to neutrinoless double beta decay and (ii) an upper bound on the extra gauge and parity symmetry breaking scale which is within the large hadron collider (LHC) energy range. We point out that an important prediction of such theories is a potentially large mixing between the right-handed charged lepton ($e^c$) and the superpartner of the right-handed gauge boson ($\widetilde W_R^+$), which leads to a brand new class of R-parity violating interactions of type $\widetilde{\mu^c}^\dagger\nu_\mu^c e^c$ and \widetilde{d^c}^\dagger\u^c e^c. We analyze the relevant constraints on the sparticle mass spectrum and the LHC signatures for the case with smuon/stau NLSP and gravitino LSP. We note the "smoking gun" signals for such models to be lepton flavor/number violating processes: $pp\to \mu^\pm\mu^\pm e^+e^-jj$ (or $\tau^\pm\tau^\pm e^+e^-jj$) and $pp\to\mu^\pm e^\pm b \bar{b} jj$ (or $\tau^\pm e^\pm b \bar{b} jj$) without significant missing energy. The predicted multi-lepton final states and the flavor structure make the model be distinguishable even in the early running of the LHC.Comment: 30 pages, 13 figures, 6 tables, reference adde

Muon conversion to electron in nuclei in type-I seesaw models

We compute the muon to electron conversion in the type-I seesaw model, as a function of the right-handed neutrino mixings and masses. The results are compared with previous computations in the literature. We determine the definite predictions resulting for the ratios between the muon to electron conversion rate for a given nucleus and the rate of two other processes which also involve a mu-e flavour transition: mu -> e gamma and mu -> eee. For a quasi-degenerate mass spectrum of right-handed neutrino masses -which is the most natural scenario leading to observable rates- those ratios depend only on the seesaw mass scale, offering a quite interesting testing ground. In the case of sterile neutrinos heavier than the electroweak scale, these ratios vanish typically for a mass scale of order a few TeV. Furthermore, the analysis performed here is also valid down to very light masses. It turns out that planned mu -> e conversion experiments would be sensitive to masses as low as 2 MeV. Taking into account other experimental constraints, we show that future mu -> e conversion experiments will be fully relevant to detect or constrain sterile neutrino scenarios in the 2 GeV-1000 TeV mass range.Comment: 32 pages 14 figures, references added and some minor precisions; results unchange

Reconstructing Seesaws

We explore some aspects of "reconstructing" the heavy singlet sector of supersymmetric type I seesaw models, for two, three or four singlets. We work in the limit where one light neutrino is massless. In an ideal world, where selected coefficients of the TeV-scale effective Lagrangian could be measured with arbitrary accuracy, the two-singlet case can be reconstructed, two three or more singlets can be differentiated, and an inverse seesaw with four singlets can be reconstructed. In a more realistic world, we estimate \ell_\a \to \ell_\b \gamma expectations with a "Minimal-Flavour-Violation-like" ansatz, which gives a relation between ratios of the three branching ratios. The two singlet model predicts a discrete set of ratios.Comment: 14 page

Rejection of the hypothesis that Markarian 501 TeV photons are pure Bose-Einstein condensates

The energy spectrum of the Blazar type galaxy Markarian 501 (Mrk 501) as measured by the High-Energy-Gamma-Ray Astronomy (HEGRA) air Cerenkov telescopes extends beyond 16 TeV and constitutes the most energetic photons observed from an extragalactic object. A fraction of the emitted spectrum is possibly absorbed in interactions with low energy photons of the diffuse extragalactic infrared radiation, which in turn offers the unique possibility to measure the diffuse infrared radiation density by TeV spectroscopy. The upper limit on the density of the extragalactic infrared radiation derived from the TeV observations imposes constraints on models of galaxy formation and stellar evolution. One of the recently published ideas to overcome severe absorption of TeV photons is based upon the assumption that sources like Mrk 501 could produce Bose-Einstein condensates of coherent photons. The condensates would have a higher survival probability during the transport in the diffuse radiation field and could mimic TeV air shower events. The powerful stereoscopic technique of the HEGRA air Cerenkov telescopes allows to test this hypothesis by reconstructing the penetration depths of TeV air shower events: Air showers initiated by Bose-Einstein condensates are expected to reach the maximum of the shower development in the atmosphere earlier than single photon events. By comparing the energy-dependent penetration depths of TeV photons from Mrk 501 with those from the TeV standard-candle Crab Nebula and simulated air shower events, we can reject the hypothesis that TeV photons from Mrk 501 are pure Bose-Einstein condensates.Comment: 9 pages, 2 figures, published by ApJ Letters, revised version (simulation results added

The Energy Spectrum of TeV Gamma-Rays from the Crab Nebula as measured by the HEGRA system of imaging air Cherenkov telescopes

The Crab Nebula has been observed by the HEGRA (High-Energy Gamma-Ray Astronomy) stereoscopic system of imaging air Cherenkov telescopes (IACTs) for a total of about 200 hrs during two observational campaigns: from September 1997 to March 1998 and from August 1998 to April 1999. The recent detailed studies of system performance give an energy threshold and an energy resolution for gamma-rays of 500 GeV and ~ 18%, respectively. The Crab energy spectrum was measured with the HEGRA IACT system in a very broad energy range up to 20 TeV, using observations at zenith angles up to 65 degrees. The Crab data can be fitted in the energy range from 1 to 20 TeV by a simple power-law, which yields dJg/dE = (2.79+/-0.02 +/- 0.5) 10^{-7} E^{-2.59 +/- 0.03 +/- 0.05}, ph m^{-2} s^{-1} TeV^{-1} The Crab Nebula energy spectrum, as measured with the HEGRA IACT system, agrees within 15% in the absolute scale and within 0.1 units in the power law index with the latest measurements by the Whipple, CANGAROO and CAT groups, consistent within the statistical and systematic errors quoted by the experiments. The pure power-law spectrum of TeV gamma-rays from the Crab Nebula constrains the physics parameters of the nebula environment as well as the models of photon emission.Comment: to appear in ApJ, 29 pages, 6 figure