DNA damage recognition and repair pathway coordination revealed by the structural biochemistry of DNA repair enzymes.

Cells have evolved distinct mechanisms for both preventing and removing mutagenic and lethal DNA damage. Structural and biochemical characterization of key enzymes that function in DNA repair pathways are illuminating the biological and chemical mechanisms that govern initial lesion detection, recognition, and excision repair of damaged DNA. These results are beginning to reveal a higher level of DNA repair coordination that ensures the faithful repair of damaged DNA. Enzyme-induced DNA distortions allow for the specific recognition of distinct extrahelical lesions, as well as tight binding to cleaved products, which has implications for the ordered transfer of unstable DNA repair intermediates between enzymes during base excision repair.

Structure and mechanism of the RuvB Holliday junction branch migration motor.

The RuvB hexamer is the chemomechanical motor of the RuvAB complex that migrates Holliday junction branch-points in DNA recombination and the rescue of stalled DNA replication forks. The 1.6 A crystal structure of Thermotoga maritima RuvB together with five mutant structures reveal that RuvB is an ATPase-associated with diverse cellular activities (AAA+-class ATPase) with a winged-helix DNA-binding domain. The RuvB-ADP complex structure and mutagenesis suggest how AAA+-class ATPases couple nucleotide binding and hydrolysis to interdomain conformational changes and asymmetry within the RuvB hexamer implied by the crystallographic packing and small-angle X-ray scattering in solution. ATP-driven domain motion is positioned to move double-stranded DNA through the hexamer and drive conformational changes between subunits by altering the complementary hydrophilic protein- protein interfaces. Structural and biochemical analysis of five motifs in the protein suggest that ATP binding is a strained conformation recognized both by sensors and the Walker motifs and that intersubunit activation occurs by an arginine finger motif reminiscent of the GTPase-activating proteins. Taken together, these results provide insights into how RuvB functions as a motor for branch migration of Holliday junctions.

Poetry, physiology, and puerperal fever: understanding the young Oliver Wendell Holmes.

The 19th-century American physician Oliver Wendell Holmes (1809-1894) is known, internationally, more for his literary output than for his contributions to medical science. Yet a single paper he wrote in 1843--"The Contagiousness of Puerperal Fever"--has made him a hero in the eyes of many (especially in the United States) of the struggle against that scourge. Why that one article, written when Holmes was still in his thirties, should--even in its expanded 1855 version--so routinely be referred to as a "classic of medical literature", and why its author should have been raised on such a high pedestal that some grant him a position beside Ignác Semmelweis, are complicated questions. This present paper is an attempt to begin assessing what it is that makes someone a medical hero by looking at three different aspects of Holmes's early career. He was even as a young man a poet and a physiologist/anatomist as well as the author of this important essay. Whether and how those three features of Holmes's many-sides public persona are connected is discussed as a prelude to considering whether his work on puerperal fever legitimates his status as a medical hero.

Management outcomes for ruptured and unruptured aneurysms in the elderly.

OBJECTIVE: In a patient older than 70 years, the decision to treat an intracranial aneurysm remains difficult whether it is ruptured or unruptured. We sought to review our institutional risks of treatment of such lesions in the context of the risks of rupture and its associated morbidity and mortality in this age group. METHODS: One hundred twenty-nine consecutive patients aged 70 years or older, who were treated at a single institution for an intracranial aneurysm, were retrospectively reviewed. Forty patients were treated for unruptured aneurysms, and 89 patients presented after subarachnoid hemorrhage. Seven additional patients in this age group who had solely intracavernous lesions, as well as one patient with a dolichoectatic fusiform basilar lesion, were excluded. Management outcomes were assessed using a modification of the Glasgow Outcome Scale, and additional physical and functional disability was assessed using the Barthel index and the Reintegration to Normal Living index. RESULTS: Six-month outcomes for the unruptured group were: excellent, 70%; good, 15%; fair, 5%; poor, 7.5%; and death (2.5%). Outcomes for all patients with ruptured lesions (including those not offered aggressive therapy) were: excellent, 34%; good, 9%; fair, 5.6%; poor, 3.4%; and death, 45%. Long-term follow-up was performed by questionnaire to assess physical and functional disability. Although physical disability (Barthel index) was similar among survivors, the Reintegration to Normal Living index, a global assessment of function, was significantly higher in patients with unruptured aneurysms (84.8 versus 70.1; P = 0.05), which highlights the disabling effects of hemorrhage. CONCLUSION: On the basis of an individual treatment center's management risks, annual aneurysmal rupture rates can be estimated that justify treatment in this difficult patient population. Despite recent controversy regarding aneurysmal hemorrhage rates, we think that symptomatic unruptured aneurysms should be treated and good results can be achieved, even in older patients.

In-chair movement: validity, reliability and implications for measuring sitting discomfort.

Sitting discomfort is traditionally evaluated with subjective rating scales which are referenced to an objective correlate (e.g. sitting posture) measured on a static (i.e. non-continuous) basis. Since sitting discomfort is dynamic in nature, it requires continuous, objective measurement. We therefore adapted an interface pressure mat to continuously record in-chair movement (ICM) as an indirect measurement of sitting discomfort by tracking the center of pressure (COP) at the buttock-chair interface. Here we report on two phases in the development of the COP system: laboratory validity and field reliability. In the laboratory study we confirmed system validity by simultaneously tracking ICM with the mat and a force platform (r2 > 0.80) and by comparing subjects' COP movement with their gross trunk movements (r2 > 0.86). In our field study we used the intraclass correlation coefficient to establish a data sampling (i.e. selection) protocol that was reliable. We collected ICM data on seated telecommunications Directory Assistance operators during 2 h field tests. Results showed that using a minimum sampling time of 5 min and then averaging a series of 5 min samples of ICM was more reliable than single discrete samples. Using the averaging protocol, we also showed that ICM increased significantly over 2 h and that ICM did not differ between trials.

Lessons learned from structural results on uracil-DNA glycosylase.

Uracil-DNA glycosylase (UDG) functions as a sentry guarding against uracil in DNA. UDG initiates DNA base excision repair (BER) by hydrolyzing the uracil base from the deoxyribose. As one of the best studied DNA glycosylases, a coherent and complete functional mechanism is emerging that combines structural and biochemical results. This functional mechanism addresses the detection of uracil bases within a vast excess of normal DNA, the features of the enzyme that drive catalysis, and coordination of UDG with later steps of BER while preventing the release of toxic intermediates. Many of the solutions that UDG has evolved to overcome the challenges of policing the genome are shared by other DNA glycosylases and DNA repair enzymes, and thus appear to be general.

Active and inhibited human catalase structures: ligand and NADPH binding and catalytic mechanism.

Human catalase is an heme-containing peroxisomal enzyme that breaks down hydrogen peroxide to water and oxygen; it is implicated in ethanol metabolism, inflammation, apoptosis, aging and cancer. The 1. 5 A resolution human enzyme structure, both with and without bound NADPH, establishes the conserved features of mammalian catalase fold and assembly, implicates Tyr370 as the tyrosine radical, suggests the structural basis for redox-sensitive binding of cognate mRNA via the catalase NADPH binding site, and identifies an unexpectedly substantial number of water-mediated domain contacts. A molecular ruler mechanism based on observed water positions in the 25 A-long channel resolves problems for selecting hydrogen peroxide. Control of water-mediated hydrogen bonds by this ruler selects for the longer hydrogen peroxide and explains the paradoxical effects of mutations that increase active site access but lower catalytic rate. The heme active site is tuned without compromising peroxide binding through a Tyr-Arg-His-Asp charge relay, arginine residue to heme carboxylate group hydrogen bonding, and aromatic stacking. Structures of the non-specific cyanide and specific 3-amino-1,2, 4-triazole inhibitor complexes of human catalase identify their modes of inhibition and help reveal the catalytic mechanism of catalase. Taken together, these resting state and inhibited human catalase structures support specific, structure-based mechanisms for the catalase substrate recognition, reaction and inhibition and provide a molecular basis for understanding ethanol intoxication and the likely effects of human polymorphisms.

The food of sweet and bitter fancy.

The MAP30 ribosomal inactivating protein structure has been determined by NMR spectroscopy. This anti-HIV and anti-cancer protein is an RNA and DNA glycosylase as well as a DNA apurinic/apyrimidinic (AP) lyase.

Evolution and mechanism from structures of an ADP-ribosylating toxin and NAD complex.

A member of the Bacillus-produced vegetative insecticidal proteins (VIPs) possesses high specificity against the major insect pest, corn rootworms, and belongs to a class of binary toxins and regulators of biological pathways distinct from classical A-B toxins. The 1.5 A resolution crystal structure of the enzymatic ADP-ribosyltransferase component, VIP2, from Bacillus cereus reveals structurally homologous N- and C-terminal alpha/beta domains likely representing the entire class of binary toxins and implying evolutionary relationships between families of ADP-ribosylating toxins. The crystal structure of the kinetically trapped VIP2-NAD complex identifies the NAD binding cleft within the C-terminal enzymatic domain and provides a structural basis for understanding the targeting and catalysis of the medically and environmentally important binary toxins. These structures furthermore provide specific experimental results to help resolve paradoxes regarding the specific mechanism of ADP-ribosylation of actin by implicating ground state destabilization and nicotinamide product sequestration as the major driving forces for catalysis.

DNA repair mechanisms for the recognition and removal of damaged DNA bases.

Recent structural and biochemical studies have begun to illuminate how cells solve the problems of recognizing and removing damaged DNA bases. Bases damaged by environmental, chemical, or enzymatic mechanisms must be efficiently found within a large excess of undamaged DNA. Structural studies suggest that a rapid damage-scanning mechanism probes for both conformational deviations and local deformability of the DNA base stack. At susceptible lesions, enzyme-induced conformational changes lead to direct interactions with specific damaged bases. The diverse array of damaged DNA bases are processed through a two-stage pathway in which damage-specific enzymes recognize and remove the base lesion, creating a common abasic site intermediate that is processed by damage-general repair enzymes to restore the correct DNA sequence.

The contribution of inducible nitric oxide and cytomegalovirus to the stability of complex carotid plaque.

BACKGROUND: Although the association between inflammation and atherosclerosis is well established, the biologic events that trigger the local inflammatory response within plaque are not fully understood. Cytotoxic free radicals and infectious agents, both of which are associated with an inflammatory response, have previously been implicated in the initiation and progression of atherosclerosis. In this study, we analyzed carotid plaque for evidence of oxidative vascular injury by determining the presence and distribution of inducible nitric oxide synthase (iNOS) expression and nitrotyrosine formation and for evidence of infection with cytomegalovirus. METHODS: Carotid plaque from 51 patients who underwent endarterectomy for either primary (n = 37) or recurrent (n = 14) stenosis were examined histologically (hematoxylin-eosin staining and Masson's trichrome staining) and with immunohistochemistry with specific antibodies to alpha-smooth muscle actin, macrophages (CD68), T-lymphocytes (CD3), and T-cell activation (human leukocyte antigen-DR). Twenty-eight specimens from patients with primary (n = 15) and recurrent (n = 13) stenosis were examined for the presence of iNOS and nitrotyrosine with immunohistochemistry and in situ hybridization (iNOS). Twenty-three additional specimens (22 primary, and 1 recurrent) were analyzed with antibodies to p53, cytomegalovirus, and the polymerase chain reaction (cytomegalovirus, n = 8). RESULTS: Primary atherosclerotic lesions were either complex heterogenous cellular plaques (n = 29) or relatively acellular fibrous plaques (n = 8). Ten of 14 recurrent plaques were either complex or fibrous lesions, and the remaining four were typical of myointimal thickening. CD68-positive staining cells were detected in all specimens regardless of their structural morphology. CD3-positive cells were interspersed between macrophages in all heterogeneous cellular plaques and only infrequently noted in fibrous plaques. iNOS and nitrotyrosine immunoreactivity were detected in macrophages and smooth muscle cells in all complex and fibrous plaques and in two of four myointimal plaques. The presence of iNOS and nitrotyrosine in plaque correlated with the existence of symptoms in 80% of primary and 62% of recurrent lesions. Cytomegalovirus was detected in only two of 23 carotid specimens (9%). CONCLUSION: The association between ischemic cerebrovascular symptoms and iNOS and nitrotyrosine immunoreactivity in complex primary and recurrent carotid plaque and the infrequent occurrence of cytomegalovirus in primary carotid lesions suggests that ongoing free radical oxidative damage rather than viral infection may contribute to plaque instability in patients with complex and fibrous carotid plaques.

RAD53, DUN1 and PDS1 define two parallel G2/M checkpoint pathways in budding yeast.

Eukaryotic checkpoint genes regulate multiple cellular responses to DNA damage. In this report, we examine the roles of budding yeast genes involved in G2/M arrest and tolerance to UV exposure. A current model posits three gene classes: those encoding proteins acting on damaged DNA (e.g. RAD9 and RAD24), those transducing a signal (MEC1, RAD53 and DUN1) or those participating more directly in arrest (PDS1). Here, we define important features of the pathways subserved by those genes. MEC1, which we find is required for both establishment and maintenance of G2/M arrest, mediates this arrest through two parallel pathways. One pathway requires RAD53 and DUN1 (the 'RAD53 pathway'); the other pathway requires PDS1. Each pathway independently contributes approximately 50% to G2/M arrest, effects demonstrable after cdc13-induced damage or a double-stranded break inflicted by the HO endonuclease. Similarly, both pathways contribute independently to tolerance of UV irradiation. How the parallel pathways might interact ultimately to achieve arrest is not yet understood, but we do provide evidence that neither the RAD53 nor the PDS1 pathway appears to maintain arrest by inhibiting adaptation. Instead, we think it likely that both pathways contribute to establishing and maintaining arrest.

Mutation of an active site residue in Escherichia coli uracil-DNA glycosylase: effect on DNA binding, uracil inhibition and catalysis.

The role of the conserved histidine-187 located in the leucine intercalation loop of Escherichia coli uracil-DNA glycosylase (Ung) was investigated. Using site-directed mutagenesis, an Ung H187D mutant protein was created, overproduced, purified to apparent homogeneity, and characterized in comparison to wild-type Ung. The properties of Ung H187D differed from Ung with respect to specific activity, substrate specificity, DNA binding, pH optimum, and inhibition by uracil analogues. Ung H187D exhibited a 55000-fold lower specific activity and a shift in pH optimum from pH 8.0 to 7.0. Under reaction conditions optimal for wild-type Ung (pH 8.0), the substrate preference of Ung H187D on defined single- and double-stranded oligonucleotides (25-mers) containing a site-specific uracil target was U/G-25-mer > U-25-mer > U/A-25-mer. However, Ung H187D processed these same DNA substrates at comparable rates at pH 7.0 and the activity was stimulated approximately 3-fold relative to the U-25-mer substrate. Ung H187D was less susceptible than Ung to inhibition by uracil, 6-amino uracil, and 5-fluorouracil. Using UV-catalyzed protein/DNA cross-linking to measure DNA binding affinity, the efficiency of Ung H187D binding to thymine-, uracil-, and apyrimidinic-site-containing DNA was (dT20) = (dT19-U) >/= (dT19-AP). Comparative analysis of the biochemical properties and the X-ray crystallographic structures of Ung and Ung H187D [Putnam, C. D., Shroyer, M. J. N., Lundquist, A. J., Mol, C. D., Arvai, A. S., Mosbaugh, D. W., and Tainer, J. A. (1999) J. Mol. Biol. 287, 331-346] provided insight regarding the role of His-187 in the catalytic mechanism of glycosylic bond cleavage. A novel mechanism is proposed wherein the developing negative charge on the uracil ring and concomitant polarization of the N1-C1' bond is sustained by resonance effects and hydrogen bonding involving the imidazole side chain of His-187.

Protein mimicry of DNA from crystal structures of the uracil-DNA glycosylase inhibitor protein and its complex with Escherichia coli uracil-DNA glycosylase.

Uracil-DNA glycosylase (UDG), which is a critical enzyme in DNA base-excision repair that recognizes and removes uracil from DNA, is specifically and irreversably inhibited by the thermostable uracil-DNA glycosylase inhibitor protein (Ugi). A paradox for the highly specific Ugi inhibition of UDG is how Ugi can successfully mimic DNA backbone interactions for UDG without resulting in significant cross-reactivity with numerous other enzymes that possess DNA backbone binding affinity. High-resolution X-ray crystal structures of Ugi both free and in complex with wild-type and the functionally defective His187Asp mutant Escherichia coli UDGs reveal the detailed molecular basis for duplex DNA backbone mimicry by Ugi. The overall shape and charge distribution of Ugi most closely resembles a midpoint in a trajectory between B-form DNA and the kinked DNA observed in UDG:DNA product complexes. Thus, Ugi targets the mechanism of uracil flipping by UDG and appears to be a transition-state mimic for UDG-flipping of uracil nucleotides from DNA. Essentially all the exquisite shape, electrostatic and hydrophobic complementarity for the high-affinity UDG-Ugi interaction is pre-existing, except for a key flip of the Ugi Gln19 carbonyl group and Glu20 side-chain, which is triggered by the formation of the complex. Conformational changes between unbound Ugi and Ugi complexed with UDG involve the beta-zipper structural motif, which we have named for the reversible pairing observed between intramolecular beta-strands. A similar beta-zipper is observed in the conversion between the open and closed forms of UDG. The combination of extremely high levels of pre-existing structural complementarity to DNA binding features specific to UDG with key local conformational changes in Ugi resolves the UDG-Ugi paradox and suggests a potentially general structural solution to the formation of very high affinity DNA enzyme-inhibitor complexes that avoid cross- reactivity.

Rational design of a functional metalloenzyme: introduction of a site for manganese binding and oxidation into a heme peroxidase.

The design of a series of functionally active models for manganese peroxidase (MnP) is described. Artificial metal binding sites were created near the heme of cytochrome c peroxidase (CCP) such that one of the heme propionates could serve as a metal ligand. At least two of these designs, MP6.1 and MP6.8, bind Mn2+ with Kd congruent with 0.2 mM, react with H2O2 to form stable ferryl heme species, and catalyze the steady-state oxidation of Mn2+ at enhanced rates relative to WT CCP. The kinetic parameters for this activity vary considerably in the presence of various dicarboxylic acid chelators, suggesting that the similar features displayed by native MnP are largely intrinsic to the manganese oxidation reaction rather than due to a specific interaction between the chelator and enzyme. Analysis of pre-steady-state data shows that electron transfer from Mn2+ to both the Trp-191 radical and the ferryl heme center of compound ES is enhanced by the metal site mutations, with transfer to the ferryl center showing the greatest stimulation. These properties are perplexingly similar to those reported for an alternate model for this site (1), despite rather distinct features of the two designs. Finally, we have determined the crystal structure at 1.9 A of one of our designs, MP6.8, in the presence of MnSO4. A weakly occupied metal at the designed site appears to coordinate two of the proposed ligands, Asp-45 and the heme 7-propionate. Paramagnetic nuclear magnetic resonance spectra also suggest that Mn2+ is interacting with the heme 7-propionate in MP6.8. The structure provides a basis for understanding the similar results of Yeung et al. (1), and suggests improvements for future designs.

Vitamin E levels in human atherosclerotic plaque: the influence of risk factors.

Recent studies suggest that vitamin E may be an important preventative factor in the development and progression of atherosclerosis. In order to more clearly define the role of vitamin E in atherosclerosis, we measured vitamin E, conjugated diens, and lipid flurochromes, as well as cholesterol, triglycerides and phospholipid in arterial and venous tissue of 83 patients. Serum cholesterol and triglyceride levels were significantly higher (P < 0.05) in patients with aortic occlusive (AIOD) and aneurysmal (AAA) disease than in control organ donors (OD). Tissue cholesterol concentrations were significantly elevated in AAA tissue when compared to OD and tissue from patients with peripheral occlusive disease (POD). Tissue from patients with AIOD contained greater concentrations of phospholipid (PL) than were measured in patients with POD and in OD. Vitamin E concentrations were highest in POD tissue and approximately 3.0, 2.0, and 1.6 fold greater than OD, AIOD and AAA tissue respectively. Diene conjugates and lipid flurochromes, measures of early and intermediate products of lipid peroxidation, were markedly elevated in all diseased arterial tissue compared to controls. There were no significant differences in tissue or serum lipid levels between saphenous vein (SVBG) and diseased vein grafts (DVG). However, conjugated diene concentrations were elevated in DVG compared to SVBG. Vitamin E levels were significantly elevated in diseased arterial and venous tissue (AIOD, AAA, POD, DVG) removed from patients with diabetes (P = 0.013) and hypertension (P = 0.049) compared to those without these risk factors. Diabetes was the only risk factor associated with significantly increased (P = 0.005) levels of vitamin E when only data from atherosclerotic arterial tissue (AAA, POD, AIOD) were analyzed. These preliminary data provide additional evidence of altered vitamin E metabolism and free radical processes in the tissues of patients with various manifestations of atherosclerosis.

Incidence and histologic characteristics of blebs in patients with abdominal aortic aneurysms.

PURPOSE: Aortic blebs-focal outpouchings within aortic aneurysms-may contribute to their eventual rupture. In this study we determine the incidence of aortic blebs and describe their microscopic features. METHODS: Computed tomographic scans of the abdominal aorta were obtained in 188 patients with aortic diameters measuring > or = 3 cm and were independently evaluated by a radiologist. The number and location of blebs were recorded, and each was measured with calipers. Sixteen blebs, with an adjacent uninvolved aneurysmal segment of aorta, and tissue from two patients with ruptured aneurysms were examined by light microscopy and immunohistochemical analysis. Specimens from six blebs and five aneurysms were examined for alpha 1 (I) procollagen messenger RNA by in situ hybridization. RESULTS: Twenty blebs, ranging in size from 5 to 30 mm (mean, 12 +/- 7 mm), were detected in 11% (20 of 188) of computed tomographic scans. Blebs were observed in 10% (11 of 111) of patients with aortic diameters between 3.0 and 4.9 cm, 10% (6 of 61) of patients with aneurysms between 5.0 and 6.9 cm, and 19% (3 of 16) of patients with aortic diameters > or = 7 cm. Histologically, the major difference between the aneurysmal aortic wall and blebs was found in the media. In aneurysmal aortas, the media consisted of multiple layers of fragmented elastic lamellae, whereas the number of elastic tissue elements along the circumference of the blebs progressively decreased; only a few isolated fragments of elastic tissue were present at the apices. Histologic evidence of rupture was evident in two specimens. A chronic inflammatory cell infiltrate composed of T and B lymphocytes, plasma cells, and macrophages, common to both the aneurysmal and the blebs, was most prominent in the adventitia of aneurysmal tissue, but involved both the media and adventitia of the blebs. In situ hybridization demonstrated the presence of alpha 1 (I) procollagen messenger RNA in four of the five aneurysm segments that were evaluated, compared with only one of six blebs. CONCLUSIONS: Blebs were discovered in aneurysms of all sizes; their frequency appeared to be unrelated to aneurysm size. The presence of inflammatory cell infiltrates and absence of alpha 1 (I) procollagen messenger RNA in five of six blebs suggest that a local imbalance of matrix degradation and repair plays a role in the cause of these lesions. Attenuation of the aortic wall accompanying the formation of blebs may predispose these sites to rupture.