Evidence for a strong selenium-aromatic interaction derived from crystallographic data and ab initio quantum chemical calculations

Increasing attention is being paid to the role of selenium, both as an essential component required for the activity of many enzymes and in the context of selenium-based pharmaceutical agents. A wide range of therapeutics that include selenium are on the market and under development, such as antihypertensive, anticancerogenic, antiviral, and immunosuppressive agents. Computer-aided drug design (CADD) has proven to be an important tool for the development of new drugs. Many CADD techniques, including docking, molecular dynamics simulation, and other receptor-based approaches, require an accurate understanding of the nature of the intermolecular forces that act to stabilize protein-ligand complexes; moreover, a quantitative assessment of these interactions furthers our efforts to rationalize the drug design process. In this paper, we consider one class of interaction involving selenium, that between Se and aromatic rings. Prior work has shown that interactions between divalent sulfur and aromatic rings are observed much more frequently than would be expected on the basis of chance, both in protein structures and the crystal structures of organic compounds that include these moieties. Recent studies on the optimization of inhibitor-protein binding also suggest that sulfur-aromatic interactions are important in stabilizing these complexes and may be crucial focal point for CADD. Given that selenium and sulfur have similar chemistry, and that selenium is significantly more polarizable, we propose that Se-aromatic interactions may also play an important stabilizing role in the structure of folded proteins and in drug-protein complexes. We have tested this hypothesis against data from the Cambridge Crystallographic Database and ab initio quantum chemical calculations. We have found evidence that selenium does interact strongly with aromatic rings and may play a role analogous to sulfur in stabilizing protein folds. In addition, selenium should be considered along with sulfur in rational drug design strategies that seek to improve binding to target protein sites that include aromatic rings.     

Probing weakly polar interactions in cytochrome c.

Theoretical, statistical, and model studies suggest that proteins are stabilized by weakly polar attractions between sulfur atoms and properly oriented aromatic rings. The two sulfur-containing amino acids, methionine and cysteine, occur frequently among functional alleles in random mutant libraries of Saccharomyces cerevisiae iso-1-cytochrome c genes at positions that form a weakly polar aromatic-aromatic interaction, the wild-type protein. To determine if a weakly polar sulfur-aromatic interaction replaced the aromatic-aromatic interaction, the structure and stability of two variants were examined. Phenylalanine 10, which interacts with tyrosine 97, was replaced by methionine and cysteine. The cysteine was modified to form the methionine and cysteine analog, S-methyl cysteine (CysSMe). Proton NMR studies indicate that changing Phe 10 to Met or CysSMe affects only local structure and that the structures of sulfur-containing variants are nearly identical. Analysis of chemical shifts and nuclear Overhauser effect data indicates that both sulfur-containing side chains are in position to form a weakly polar interaction with Tyr 97. The F10M and F10CSMe variants are 2-3 kcal mol-1 less stable than iso-1-cytochrome c at 300 K. Comparison of the stabilities of the F10M and F10CSMe variants allows evaluation of the potential weakly polar interaction between the additional sulfur atom of F10CSMe and the aromatic moiety of Tyr 97. The F10CSMe;C102T variant is 0.7 +/- 0.3 kcal mol-1 more stable than the F10M;C102T protein. The increased stability is explained by the difference in hydrophobicity of the sulfur-containing side chains. We conclude that any weakly polar interaction between the additional sulfur and the aromatic ring is too weak to detect or is masked by destabilizing contributions to the free energy of denaturation.     

Structural influence of hydrophobic core residues on metal binding and specificity in carbonic anhydrase II

Aromatic residues in the hydrophobic core of human carbonic anhydrase II (CAII) influence metal ion binding in the active site. Residues F93, F95, and W97 are contained in a beta-strand that also contains two zinc ligands, H94 and H96. The aromatic amino acids contribute to the high zinc affinity and slow zinc dissociation rate constant of CAII [Hunt, J. A., and Fierke, C. A. (1997) J. Biol. Chem. 272, 20364-20372]. Substitution of these aromatic amino acids with smaller side chains enhances Cu(2+) affinity while decreasing Co(2+) and Zn(2+) affinity [Hunt, J. A., Mahiuddin, A., & Fierke, C. A. (1999) Biochemistry 38, 9054-9062]. Here, X-ray crystal structures of zinc-bound F93I/F95M/W97V and F93S/F95L/W97M CAIIs reveal the introduction of new cavities in the hydrophobic core, compensatory movements of surrounding side chains, and the incorporation of buried water molecules; nevertheless, the enzyme maintains tetrahedral zinc coordination geometry. However, a conformational change of direct metal ligand H94 as well as indirect (i.e., "second-shell") ligand Q92 accompanies metal release in both F93I/F95M/W97V and F93S/F95L/W97M CAIIs, thereby eliminating preorientation of the histidine ligands with tetrahedral geometry in the apoenzyme. Only one cobalt-bound variant, F93I/F95M/W97V CAII, maintains tetrahedral metal coordination geometry; F93S/F95L/W97M CAII binds Co(2+) with trigonal bipyramidal coordination geometry due to the addition of azide anion to the metal coordination polyhedron. The copper-bound variants exhibit either square pyramidal or trigonal bipyramidal metal coordination geometry due to the addition of a second solvent molecule to the metal coordination polyhedron. The key finding of this work is that aromatic core residues serve as anchors that help to preorient direct and second-shell ligands to optimize zinc binding geometry and destabilize alternative geometries. These geometrical constraints are likely a main determinant of the enhanced zinc/copper specificity of CAII as compared to small molecule chelators.     

Fetal Subjects, Feminist Positions

Fetal Subjects, Feminist Positions. Lynn M. Morgan and Meredith W. Michaels. eds. Philadelphia: University of Pennsylvania Press, 1999. vi +345 pp.     

CD and Fourier transform ir spectroscopic studies of peptides. II. Detection of β-turns in linear peptides

Comparative CD and Fourier transform ir (FTIR) spectroscopic data on N-Boc protected linear peptides with or without the (Pro-Gly) β-turn motif (e.g., Boc-Tyr-Pro-Gly-Phe-Leu-OH and Boc-Tyr-Gly-Pro-Phe-Leu-OH) are reported herein. The CD spectra, reflecting both backbone and aromatic contributions, were not found to be characteristic of the presence of β-turns. In the amide I region of the FTIR spectra, analyzed by self-deconvolution and curve-fitting methods, the β-turn band shewed up between 1639 and 1633 cm^?1 in trifluoroethanol (TFE) but only for models containing the (Pro-Gly) core. This band war-also present in the spectra in chloroform but absent in dimethylsulfoxide. These findings, in agreement with recent ir data on cyclic models and 3₁₀-helical polypeptides and protein in D₂O [see S. J. Prestrelski, D. M. Byler, and M. P. Thompson (1991), International Journal of Peptide and Protein Research, Vol. 37, pp. 508–512; H. H. Mantsch, A. Perczel. M. Hollósi, and G. D. Fasman (1992), FASEB Journal, Vol. 6, p. A341; H. H. Mantsch. A. Perczel, M. Hollósi, and G. Fasman (1992), Biopolymers. Vol. 33, pp. 201–207; S. M. Miick, G. V. Martinez, W. R. Fiori, A. P. Tedd, and G. L. Millhauser (1992). Nature, Vol. 359, pp. 653–655], suggest that the amide I band, with a major contribution from the acceptor C ? O of the 1 ← 4 intramolecular H bond of β-turns, appears near or below 1640 cm^?1, rather than above 1660 cm^?1. In TFE, bands between 1670 and 1660 cm^?1 are mainly due to “free” carbonyls, that is, C ? O's of amides that are solvated but not involved in the characteristic H bonds of periodic secondary structures or β-turns. © 1994 John Wiley & Sons, Inc.     

NMR conformational analysis of proadrenomedullin N-terminal 20 peptide, a proangiogenic factor involved in tumor growth

The preferred conformation of Proadrenomedullin N-Terminal 20 Peptide (PAMP; ARLDVASEFRKKWNKWALSR-amide) has been determined using 1H and 13C two-dimensional nuclear magnetic resonance (NMR) spectroscopy and molecular modeling. PAMP is a peptide that has various physiological functions, including its role as a proangiogenic factor in facilitating tumor growth and its inhibitory effect on catecholamine secretion at nicotinic receptors. The preferred conformation of PAMP was determined in a helix-inducing trifluoroethanol and water (TFE/H2O) solution, and in a membrane-mimetic sodium dodecylsulfate-d25 (SDS) micellar solution. The secondary structure consists of an alpha-helix for residues Arg2 to Arg20 in TFE/H2O solution and an alpha-helix for residues Arg2 to Ala17 in SDS solution. We postulate that the polar charged residues Arg2, Lys12, and Arg20 are responsible for the initial interaction of the peptide with the micelle, and that this is followed by the binding of the hydrophobic residues Leu3, Val5, Phe9, Trp13, and Trp16 to the micellar core. The three C-terminal amino acid residues adopt an extended structure in SDS, suggesting that they are important in receptor recognition and binding. This is supported by truncation studies done by Mahata et al. (Hypertension, 1998, Vol. 32, pp. 907-916), which show the importance of the C-terminal in physiological activity. Furthermore, Belloni et al. (Hypertension, 1999, Vol. 33, pp. 1185-1189), and Martinez et al. (Cancer Research, 2004, Vol. 64, pp. 6489-6494) suggested that the N-terminal was also important in PAMP activity. However, no differences in conformational preference of the N-terminal were observed between the two solvent systems.     

Antimicrobial Activity of a New Series of Fluorine-Containing Tertiary Alcohols

The in vitro antimicrobial activity of a new series of synthetic fluorine-substituted triaryl alcohols against the human pathogens Staphylococcus aureus, Escherichia coli and Candida albicans was studied with the aim of overcoming multiple drug resistance and improving the clinical usefulness of antimicrobial drugs. The nature and positions of substituents attached to aromatic rings, as well as their electronegativities and sizes, seem to affect the preferred molecular conformations and, hence, the binding of the compounds to the corresponding cell receptors.__________From Bioorganicheskaya Khimiya, Vol. 31, No. 4, 2005, pp. 441–444.Original English Text Copyright ¢ 2005 by Rute G. da Costa, Joao M. Curto, Olivia R. Furtado, Sonia Savluchinske Feio, Jose C. Roseiro.The text was submitted by the authors in English.     

Book review

Chronopharmacology. Cellular and Biochemical Interactions (Cellular Clocks Series/3), by Prof. Dr. Björn Lemmer, Center of Pharmacology, J.W. Goethe‐University, Frankfurt am Main, FRG. 1989. 744 pp., $150.00 (US and Canada), $180.00 (All other countries). ISBN 0–8247–8103–1 Published by Marcel Dekker, Inc. New York, USA.

Chronobiology and chronomedicine. Basic research and applications. Proceedings of the IVth Annual Meeting of the European Society for Chronobiology, Birmingham, July 1988, by E. Morgan. 1990, 407 pp., soft cover sFr. 79.‐ISBN 3–631–42301–2 Published by Verlag Peter Lang, Frankfurt a.M., Bern, New York, Paris.

Biological rhythms in clinical practice by J. Ahrendt, J.M. Waterhouse and D.S. Minors 1989. 299 pp.m, hard cover £ 48.‐ISBN 0–7236–0961–6     

Kinetics of the interaction of desAABB-fibrin monomer with immobilized fibrinogen

The soluble and stable fibrin monomer-fibrinogen complex (SF) is well known to be present in the circulating blood of healthy individuals and of patients with thrombotic diseases. However, its physiological role is not yet fully understood. To deepen our knowledge about this complex, a method for the quantitative analysis of interaction between soluble fibrin monomers and surface-immobilized fibrinogen has been established by means of resonant mirror (IAsys) and surface plasmon resonance (BIAcore) biosensors. The protocols have been optimized and validated by choosing appropriate immobilization procedures with regeneration steps and suitable fibrin concentrations. The highly specific binding of fibrin monomers to immobilized fibrin(ogen), or vice versa, was characterized by an affinity constant of approximately 10(-8)M, which accords better with the direct dissociation of fibrin triads (KD approximately 10(-8) -10(-9) M) (J. R. Shainoff and B. N. Dardik, Annals of the New York Academy of Science, 1983, Vol. 27, pp. 254-268) than with earlier estimations of the KD for the fibrin-fibrinogen complex (KD approximately 10(-6) M) (J. L. Usero, C. Izquierdo, F. J. Burguillo, M. G. Roig, A. del Arco, and M. A. Herraez, International Journal of Biochemistry, 1981, Vol. 13, pp. 1191-1196).     

Reviews of Publications

Book reviewed in this article:
Hair of West-European Mammals. Atlas and Identification Key , by B. J. Teerink.
Koobi Fora Research Project, Vol. 3, The Fossil Ungulates: Geology, Fossil Artiodactyls, and Palaeoenvironments , edited by J. M. Harris
Biomechanics in Evolution (Society for Experimental Biology Series No. 36) , edited by J. M. V. Rayner and R. J. Wooton.
Efficiency and Economy in Animal Physiology , edited by R. W. Blake.     

The role of stacking interactions in the mechanisms of clonidine binding

The stacking interactions of the clonidine aromatic ring with the aromatic rings of Phe or Tyr of alpha2-adrenoreceptor and Tyr aromatic ring of the pore of the tetradotoxin-resistant channel have been investigated. Ab initio quantum chemical calculations for a model system of two parallel aromatic rings were performed by GAMESS software with 6-31G** basis set in the framework of the Moller-Plesset second-order perturbation theory with full geometry optimization without any symmetry. It was shown that the parallel shifted conformation of two aromatic rings is energetically most favorable. The 2,6-chlorination of one of the benzene rings leads to the amplification of the stacking interaction, an increase in the relative shift of the rings and possible growth of both the hypotensive and analgetic functions of clonidine due to the increase in the binding energy. The 4-fluoridization of the clonidine benzene ring can amplify its analgetic function but practically excludes its hypotensive action.     

The Cambridge History of the Native Peoples of the Americas: Vol. 2: Mesoamerica, Part 1.; The Cambridge History of the Native Peoples of the Americas: Vol. 2: Mesoamerica, Part 2.

The Cambridge History of the Native Peoples of the Americas: Vol. 2: Mesoamerica, Part 1. Richard E. W. Adams and Murdo J. MacLeod. eds. Cambridge: Cambridge University Press, 2000. 571 pp.
The Cambridge History of the Native Peoples of the Americas: Vol. 2: Mesoamerica, Part 2. Richard E. W. Adams and Murdo J. MacLeod. eds. Cambridge: Cambridge University Press, 2000. 455 pp.     

Thermodynamic substantiation of water-bridged collagen structure.

A solution of the problem of topology of a hydrogen bond net in a triple helix of collagen is suggested on the basis of an analysis of thermodynamic data on denaturation of phylogenetically different collagen [T. V. Burjanadze (1982), Vol. 21, pp. 1489-1501; T. V. Burjanadze, E. I. Tiktopulo, and P. L. Privalov (1987), Dokl. Akad. Nauk. USSR, Vol. 293, pp. 720-724] as well as on the earlier evaluation of the energy of the OH group of the 4-hydroxyproline bond [A. R. Ward and P. Mason (1973), Journal of Molecular Biology, Vol. 29, pp. 431-435]. It is shown that only the water-bridged collagen structure [G. N. Ramachandran and R. Chandrasekharan (1968), Biopolymers, Vol. 6, pp. 1649-1661; G. N. Ramachandran, M. Bansal, and R. S. Bhatnagar (1973), Biochimica Biophysica Acta, Vol. 322, pp. 166-171; M. Bansal, C. Ramakrishnan, and G. N. Ramachandran (1975), Proceedings of the Indian Academy of Sciences, Vol. 82, pp. 152-164] can explain both the change of thermostability upon proline hydroxylation [J. Rosenbloom, M. Harsch, and S. Jimenez (1973), Archives of Biochemistry and Biophysics, Vol. 158, pp. 478-484] and its phylogenetic change [T. V. Burjanadze (1982)].     

REVIEWS

Book reviewed in this article:
Histological Technique. H. M. C arleton and R. A. B. D rury
Radioisotopes in Scientific Research. Vol. iv. Research with Isotopes in Plant Biology and Some General Problems. R. C. Extermann.
Chemical Aspects of Ecology in Relation to Agriculture. H ubert M artin .
Methods of Testing Chemicals on Insects. H. H. S hepard .
Beneficial Insects. B. D. M oreton .
The Grafter's Handbook (2nd edition). R. J. G arner .
Common Names of British Insect and other Pests. Compiled by I. T homas and H. W. J anson .     

A molecular thermodynamic approach to predict the secondary structure of homopolypeptides in aqueous systems.

Under physiological conditions, many polypeptide chains spontaneously fold into discrete and tightly packed three-dimensional structures. The folded polypeptide chain conformation is believed to represent a minimum Gibbs energy of the system, governed by the weak interactions that operate between the amino acid residues and between the residues and the solvent. A semiempirical molecular thermodynamic model is proposed to represent the Gibbs energy of folding of aqueous homopolypeptide systems. The model takes into consideration both the entropy contribution and the enthalpy contribution of folding homopolypeptide chains in aqueous solutions. The entropy contribution is derived from the Flory-Huggins expression for the entropy of mixing. It accounts for the entropy loss in folding a random-coiled polypeptide chain into a specific polypeptide conformation. The enthalpy contribution is derived from a molecular segment-based Non-Random Two Liquid (NRTL) local composition model [H. Renon and J. M. Prausnitz (1968) AIChE J., Vol. 14, pp. 135-142; C.-C. Chen and L. B. Evans (1986) AIChE J., Vol. 32, pp. 444-454], which takes into consideration of the residue-residue, residue-solvent, and solvent-solvent binary physical interactions along with the local compositions of amino acid residues in aqueous homopolypeptides. The UNIFAC group contribution method [A. Fredenslund, R. L. Jones, and J. M. Prausnitz (1975) AIChE J., 21, 1086-1099; A. Fredenslund, J. Gmehling, and P. Rasmussen (1977) Vapor-Liquid Equilibrium Using UNIFAC, Elsevier Scientific Publishing Company, Amsterdam], developed originally to estimate the excess Gibbs energy of solutions of small molecules, was used to estimate the NRTL binary interaction parameters. The model yields a hydrophobicity scale for the 20 amino acid side chains, which compares favorably with established scales [Y. Nozaki and C. Tanford (1971) Journal of Biological Chemistry, Vol. 46, pp. 2211-2217; E. B. Leodidis and T. A. Hatton (1990) Journal of Physical Chemistry, Vol. 94, pp. 6411-6420]. In addition, the model generates qualitatively correct thermodynamic constants and it accurately predicts thermodynamically favorable folding of a number of aqueous homopolypeptides from random-coiled states into alpha-helices. The model further facilitates estimation of the Zimm-Bragg helix growth parameter s and the nucleation parameter sigma for amino acid residues [B. H. Zimm and J. K. Bragg (1959) Journal of Chemical Physics, Vol. 31, pp. 526-535]. The calculated values of the two parameters fall into the ranges suggested by Zimm and Bragg.     

Book Reviews

Benzodiazepine Recognition Site Ligands: Biochemistry and Pharmacology (Advances in Biochemical Psychopharmacology, Vol. 38) edited by G. Biggio and E. Costa
Experimental Allergic Encephalomyelitis—A Useful Model for Multiple Sclerosis (Progress in Clinical and Biological Research, Vol. 146) edited by E. C. Alvoid, M. W. Kies, and A. J. Suckling. Alan R. Liss
Cellular Components of the Cerebral Cortex (Cerebral Cortex, Vol. 1) edited by A. Peters and E. G. Jones     

Book Reviews

M icrobial I nteractions and C ommunities Vol. 1. (1982). Edited by A.T. Bull & J.H. Slater. 567 pp. London, New York: Academic Press. £33.80, US $69.50.
B ioactive M icrobial P toducts : S earch and D iscovery (Special Publications of the Society for General Microbiology No. 6) (1982). Edited by J.D. Bu'Lock, L.J. Nisbet & O.J. Winstanley. 148 pp. London and New York: Academic Press. £10.00, US $20.50.
I solation and I dentification M ethods for F ood P oisoning O rganisms (Society for Applied Bacteriology Technical Series No. 17). (1982). Edited by Janet E.L. Corry, Diane Roberts & F.A. Skinner. 406 pp. London and New York: Acadmic Press. £30.00, US $55.50.
P lasmids (Study in Biology No. 142) (1982). M.J. Day. 52 pp. London: Edward Arnold. £2.50.
S mith's I ntroduction T o I ndustrial M ycology 7th Edition (1981). A.H.S. Onions, D. Allsopp & H.O.W. Eggins. 398 pp. London: Edward Arnold. £37.50.     

Book Reviews

Abstract: Book reviewed in this article:
Nutrition and the Brain , Vol. 8 by R. J. Wurtman and J. J. Wurtman.
Glossary of Biochemistry and Molecular Biology by D. M. Glick.
Antiepileptic Drugs (Third Edition) edited by R. H. Levy, F. E. Dreifuss, R. H. Mattson, B. S. Meldrum, and J. K. Penry.
Developmental Neurobiology (Nestlé Nutrition Workshop Series, Vol. 12) edited by P. Evrard and A. Minkowski.
Allosteric Modulation of Amino Acid Receptors: Therapeutic Implications (Fidia Research Foundation Symposium Series, Vol. 1) by E. A. Barnard and E. Costa.
Neurochemical Pharmacology—A Tribute to B. B. Brodie (Fidia Research Foundation Symposium Series, Vol. 2) edited by E. Costa.
Neuromethods Vol. 11: Carbohydrate and Energy Metabolism edited by A. A. Boulton, G. B. Baker, and R. F. Butterworth.
Adenosine and Adenosine Receptors edited by M. Williams.
Laughing Death: The Untold Story of Kuru by V. Zigas.
Psychoactive Drugs: Tolerance and Sensitization edited by A. J. Goudie and M. W. Emmett-Oglesby.
The NMDA Receptor edited by J. C. Watkins and G. L. Collingridge.
Basic and Clinical Aspects of Neuroscience (Vol. 3): The Role of Brain Dopamine edited by E. Flückiger, E. E. Müller, and M. O. Thorner.
Glycine Neurotransmission edited by O. P. Otterson and J. Storm-Mathisen.
The Neuropeptide Cholecystokinin (CCK): Anatomy and Biochemistry, Receptors, Pharmacology and Physiology edited by J. Hughes, G. Dockray, and G. Woodruff.     

Rad50S alleles of the Mre11 complex: questions answered and questions raised

We find that Rad50S mutations in yeast and mammals exhibit constitutive PIKK (PI3-kinase like kinase)-dependent signaling [T. Usui, H. Ogawa, J.H. Petrini, A DNA damage response pathway controlled by Tel1 and the Mre11 complex. Mol. Cell 7 (2001) 1255-1266.; M. Morales, J.W. Theunissen, C.F. Kim, R. Kitagawa, M.B. Kastan, J.H. Petrini, The Rad50S allele promotes ATM-dependent DNA damage responses and suppresses ATM deficiency: implications for the Mre11 complex as a DNA damage sensor. Genes Dev. 19 (2005) 3043-4354.]. The signaling depends on Mre11 complex functions, consistent with its role as a DNA damage sensor. Rad50S is distinct from hypomorphic mutations of Mre11 and Nbs1 in mammals [M. Morales, J.W. Theunissen, C.F. Kim, R. Kitagawa, M.B. Kastan, J.H. Petrini, The Rad50S allele promotes ATM-dependent DNA damage responses and suppresses ATM deficiency: implications for the Mre11 complex as a DNA damage sensor. Genes Dev. 19 (2005) 3043-3054.; J.P. Carney, R.S. Maser, H. Olivares, E.M. Davis, Le M. Beau, J.R. Yates, III, L. Hays, W.F. Morgan, J.H. Petrini, The hMre11/hRad50 protein complex and Nijmegen breakage syndrome: linkage of double-strand break repair to the cellular DNA damage response. Cell 93 (1998) 477-486.; G.S. Stewart, R.S. Maser, T. Stankovic, D.A. Bressan, M.I. Kaplan, N.G. Jaspers, A. Raams, P.J. Byrd, J.H. Petrini, A.M. Taylor, The DNA double-strand break repair gene hMRE11 is mutated in individuals with an ataxia-telangiectasia-like disorder. Cell 99 (1999) 577-587.; B.R. Williams, O.K. Mirzoeva, W.F. Morgan, J. Lin, W. Dunnick, J.H. Petrini, A murine model of nijmegen breakage syndrome. Curr. Biol. 12 (2002) 648-653.; J.W. Theunissen, M.I. Kaplan, P.A. Hunt, B.R. Williams, D.O. Ferguson, F.W. Alt, J.H. Petrini, Checkpoint failure and chromosomal instability without lymphomagenesis in Mre11(ATLD1/ATLD1) mice. Mol. Cell 12 (2003) 1511-1523.] and the Mre11 complex deficiency in yeast [T. Usui, H. Ogawa, J.H. Petrini, A DNA damage response pathway controlled by Tel1 and the Mre11 complex. Mol. Cell 7 (2001) 1255-1266.; D'D. Amours, S.P. Jackson, The yeast Xrs2 complex functions in S phase checkpoint regulation. Genes Dev. 15 (2001) 2238-49. ; M. Grenon, C. Gilbert, N.F. Lowndes, Checkpoint activation in response to double-strand breaks requires the Mre11/Rad50/Xrs2 complex. Nat. Cell Biol. 3 (2001) 844-847. ] where the signaling is compromised. Herein, we describe evidence for chronic signaling by Rad50S and discuss possible mechanisms.