Oxidative thymine dimer repair in the DNA helix

The metallointercalator Rh(phi)2DMB3+ (phi, 9,10-phenanthrenequinone diimine; DMB, 4,4'-dimethyl-2,2'-bipyridine) catalyzed the repair of a thymine dimer incorporated site-specifically in a 16-base pair DNA duplex by means of visible light. This repair could be accomplished with rhodium noncovalently bound to the duplex and at long range (16 to 26 angstroms), with the rhodium intercalator tethered to either end of the duplex assembly. This long-range repair was mediated by the DNA helix. Repair efficiency did not decrease with increasing distance between intercalated rhodium and the thymine dimer, but it diminished with disruption of the intervening pi-stack.     

The Bloom's syndrome helicase unwinds G4 DNA

BLM, the gene that is defective in Bloom's syndrome, encodes a protein homologous to RecQ subfamily helicases that functions as a 3'-5' DNA helicase in vitro. We now report that the BLM helicase can unwind G4 DNA. The BLM G4 DNA unwinding activity is ATP-dependent and requires a short 3' region of single-stranded DNA. Strikingly, G4 DNA is a preferred substrate of the BLM helicase, as measured both by efficiency of unwinding and by competition. These results suggest that G4 DNA may be a natural substrate of BLM in vivo and that the failure to unwind G4 DNA may cause the genomic instability and increased frequency of sister chromatid exchange characteristic of Bloom's syndrome.     

Solution-state structure of a DNA dodecamer duplex containing a Cis-syn thymine cyclobutane dimer, the major UV photoproduct of DNA

The solution structures of a duplex DNA dodecamer containing a cis-syn cyclobutane thymine dimer d(GCACGAAT[cs]TAAG).d(CTTAATTCG TGC) and its native parent sequence were determined using NMR data collected at 750 MHz. The dodecamer sequence corresponds to the section of a site-specific cis-syn dimer containing 49-mer that was found to be the binding site for the dimer-specific T4 denV endonuclease V repair enzyme by chemical and enzymatic footprinting experiments. Structures of both sequences were derived from NOE restrained molecular dynamics/simulated annealing calculations using a fixed outer layer of water and an inner dynamic layer of water with sodium counterions. The resulting structures reveal a subtle distortion to the phosphodiester backbone in the dimer-containing sequence which includes a BII phosphate at the T9pA10 junction immediately 3' to the dimer. The BII phosphate, established experimentally by analysis of the 31P chemical shifts and interpretation of the 3JP-H3' values using an optimized Karplus relationship, enables the DNA helix to accommodate the dimer by destacking the base 3' to the dimer. Furthermore, the structures provide explanations for the unusually shifted T8-N3H imino, A16-H2 and T8-Me proton resonances and T9pA10 (31)P NMR resonance and are consistent with bending, unwinding, and thermodynamic data. The implications of the structural data for the mechanism by which cis-syn dimers are recognized by repair enzymes and bypassed by DNA polymerases are also discussed.     

Chemical display of thymine residues flipped out by DNA methyltransferases

The DNA cytosine-C5 methyltransferase M. Hha I flips its target base out of the DNA helix during interaction with the substrate sequence GCGC. Binary and ternary complexes between M. Hha I and hemimethylated DNA duplexes were used to examine the suitability of four chemical methods to detect flipped-out bases in protein-DNA complexes. These methods probe the structural peculiarities of pyrimidine bases in DNA. We find that in cases when the target cytosine is replaced with thymine (GTGC), KMnO4proved an efficient probe for positive display of flipped-out thymines. The generality of this procedure was further verified by examining a DNA adenine-N6 methyltransferase, M. Taq I, in which case an enhanced reactivity of thymine replacing the target adenine (TCGT) in the recognition sequence TCGA was also observed. Our results support the proposed base-flipping mechanism for adenine methyltransferases, and offer a convenient laboratory tool for detection of flipped-out thymines in protein-DNA complexes.     

Gross efficiency of muscular work during step exercise at -15 degrees C and 21 degrees C

The systemic and regional hemodynamic effects of inhibition of endothelium-derived relaxing factor/nitric oxide (EDRF/NO) were studied in awake, indomethacin-treated rats. The radiolabeled microsphere method was used to determine the cardiac output, systemic vascular resistance (SVR), and regional blood flows and regional vascular resistances in 12 tissues before and after infusion of the EDRF/NO synthesis inhibitor, NG-monomethyl-L-arginine (NMMA, 100 mg/kg), and after reversal of NMMA by infusion of L-arginine (300 mg/kg). NMMA infusion resulted in increases in the blood pressure and SVR. After NMMA, blood flows were decreased to the cerebrum, heart, kidney, spleen, gastrointestinal tract, skin, ear, and white fat, whereas flow in the hepatic artery was increased. Vascular resistances were increased in every tissue studied except the hepatic artery, in which the resistance decreased after NMMA. L-arginine restored the vascular resistance to control values in 8 of the 12 tissues. The magnitude of the increase in the regional resistance was not uniform among the organs studied, and ranged from a maximum of 253% in brown fat to 22% in heart. These results indicate that EDRF/NO is an important mediator of regional hemodynamic control in numerous tissues of the intact rat. The marked heterogeneity in the magnitude of basal EDRF/NO-dependent tone suggests that the mechanisms mediating this cardiovascular control system are regulated locally.     

Efficient bypass of a thymine-thymine dimer by yeast DNA polymerase, Poleta

The RAD30 gene of the yeast Saccharomyces cerevisiae is required for the error-free postreplicational repair of DNA that has been damaged by ultraviolet irradiation. Here, RAD30 is shown to encode a DNA polymerase that can replicate efficiently past a thymine-thymine cis-syn cyclobutane dimer, a lesion that normally blocks DNA polymerases. When incubated in vitro with all four nucleotides, Rad30 incorporates two adenines opposite the thymine-thymine dimer. Rad30 is the seventh eukaryotic DNA polymerase to be described and hence is named DNA polymerase eta.     

Oxidative charge transfer To repair thymine dimers and damage guanine bases in DNA assemblies containing tethered metallointercalators

Potent oxidants which intercalate in DNA serve as tools to probe DNA-mediated electron-transfer reactions. A photoexcited rhodium intercalator, Rh(phi)2DMB3+ (phi = 9,10-phenanthrenequinone diimine and DMB = 4,4'-dimethyl-2,2'-bipyridine), tethered to DNA, promotes both oxidative damage to 5'-GG-3' doublets in DNA and the repair of thymine dimers from a remote site on the DNA duplex. DNA-mediated repair of a thymine dimer lesion by charge transfer from the tethered rhodium intercalator is quantitative, albeit with low photoefficiency, occurs in an intraduplex reaction over long range (36 A), and requires that the intervening bases be paired. When both oxidative reactions, repair and oxidative damage, are monitored on the same duplex, competition is evident; the presence of both a 5'-GG-3' site and the thymine dimer diminished the dimer repair efficiency by 20-40% and decreased damage at the 5'-GG-3' sites 2-fold compared to similar sequences lacking either the guanine doublet or thymine dimer, respectively. In addition to damage at the 5'-G of 5'-GG-3' sites, we also observe oxidation at the 3'-G of the 5'-GT<>TG-3' tetrad only in the presence of thymine dimer. Overall, the yield of repaired thymine strand was at least 10 times higher than the yield of oxidized guanine in the same sequences. While the 5-GG-3' may represent the thermodynamically favored site for oxidative reaction, repair of the thymine dimer appears to be kinetically more favorable. Dipyridophenanzine (dppz) complexes of ruthenium(III), less potent oxidants which intercalate in DNA, oxidize 5'-GG-3' doublets efficiently but cannot trigger the repair of the thymine dimer lesion. Oxidative damage to DNA from a distance, mediated by the DNA base pair stack, can, however, be utilized to probe the disruption in the base stack generated by the thymine dimer. The presence of the dimer does not diminish oxidation by a Ru(III) intercalator at a distal guanine doublet, suggesting that the disruption caused by the dimer does not block charge transfer through the DNA duplex. DNA-mediated electron-transfer reactions of metallointercalators therefore serve to illustrate important aspects of radical migration and its consequence with respect to reactions at a distance through the DNA base pair stack.     

Carboxyl-terminal domain dimer interface mutant 434 repressors have altered dimerization and DNA binding specificities

Strong dimerization of the repressor, mediated by the carboxyl (C)-terminal domain, is a prerequisite for forming a specific complex with DNA and cooperative DNA binding to form tetramers. We have generated a computer model of the C-terminal domain of the 434 repressor based on the crystal structure of the homologous UmuD' protein. This model predicts that residues in the primary sequence between 93 and 168 contribute to the dimer interface. We changed several amino acid residues located in this region. Gel filtration and crosslinking assays were used to characterize the strength and specificity of dimerization of the purified repressor C-terminal domain dimer interface mutants. These results indicate that amino acid residues K121, H139, D161 and N163 contribute to the strength and/or specificity of dimerization. The relative affinity of the bacteriophage 434 repressor for 434 operators is determined, in part, by the repressor's ability to detect sequence-dependent structural alterations in the non-contacted region at the center of an operator site. We find that the relative ability of C-terminal domain dimer interface mutant repressors to dimerize does not necessarily predict their relative abilities to bind DNA, and that these proteins are deficient in detecting non-contacted base-dependent differences in operator strength. Our results show that the structure of the DNA in complex with these mutant proteins differs from that found in wild-type repressor-operator complexes, even though the sites of these mutations lie in a separate domain from that which contacts the DNA. These observations demonstrate that the structural integrity of the C-terminal domain dimer interface is required to appropriately orient the DNA binding information contained within the DNA-contacting N-terminal domain.     

Reduction of thymine hydroperoxide by phospholipid hydroperoxide glutathione peroxidase and glutathione transferases

Thymine hydroperoxide (5-hydroperoxymethyluracil), a model compound representing products of oxidative damage to DNA, is a substrate for glutathione peroxidase and some isoforms of glutathione transferase. In this paper, we show that selenium-dependent human phospholipid hydroperoxide glutathione peroxidase (Se-PHGPx) exhibits about four orders of magnitude higher activity on thymine hydroperoxide than that of other human enzymes such as selenium-dependent glutathione peroxidase and various representatives of glutathione transferases. The results indicate that Se-PHGPx may be an important enzyme in repairing oxidatively damaged DNA.     

小儿拇长屈肌腱狭窄性腱鞘炎诊疗体会

目的:探讨手术治疗小儿拇长屈肌腱狭窄性腱鞘炎的临床疗效.方法:34例,其中9例为双侧共43指小儿先天性狭窄性腱鞘炎采用开放手术治疗,随访治疗效果.结果:29例患儿37指得到随访,随访时间9个月～2年,27例34指屈伸活动正常,2例3指关节略屈曲,但功能影响不大.结论:手术治疗狭窄性腱鞘炎是一种安全有效的方法.     

The effects of a temperature below 15 degrees C on the myocardial calcium and ultrastructure in donor heart preservation in a canine model

The effects of 6-h hypothermic cardioplegic arrest on myocardial biochemical, morphologic, and functional recovery were investigated in two groups of dogs. Group 1 (n = 6) was subjected to hypothermia of 15 degrees C and group 2 (n = 6) was subjected hypothermia of 5 degrees C. Although the myocardial calcium (Ca) concentration was significantly higher at the end of reperfusion in group 2 compared to group 1, the MB-fraction of creatine kinase, mitochondrial aspartate aminotransferase, recovery of left ventricular systolic function, and mitochondrial morphologic integrity were better in group 2 than in group 1. These findings suggest that hypothermia of 5 degrees C in 6-h cardioplegia is not necessarily coupled with interference in myocardial contractility, despite the Ca overload that occurs during reperfusion.     

Structures stabilizing the dimer interface on human 11 beta-hydroxysteroid dehydrogenase types 1 and 2 and human 15-hydroxyprostaglandin dehydrogenase and their homologs

Human 11 beta-hydroxysteroid dehydrogenase-types 1 and 2 and human 15-hydroxyprostaglandin dehydrogenase belong to a large family of oxidoreductases that includes human dihydropteridine reductase and Streptomyces hydrogenans 20 beta-hydroxysteroid dehydrogenase, for which 3D structures are available. Almost all of these enzymes are either dimers or tetramers. The dimer interface of rat dihydropteridine reductase consists of alpha-helices E and F from each monomer arranged in a four alpha-helix bundle [Varughese et al. (1992) Proc. Natl. Acad. Sci. USA 89, 6080-6084]. Alpha-helix F contains tyrosine-146 and lysine-150, residues that are highly conserved in this protein superfamily and have been proposed to be at the catalytic site. We have examined the dimer interface between alpha-helix F in human and rat dihydropteridine reductase and Streptomyces hydrogenans 20 beta-hydroxysteroid dehydrogenase as well as modeled 3D structures of steroid and prostaglandin dehydrogenases and homologs for stabilizing interactions. We find a site in the middle of alpha-helix F that stabilizes the dimer. This anchor is adjacent to conserved lysine on alpha-helix F. Our analysis suggests that sequence variation in the anchor may be important in substrate specificity.     

The evaluation of 15q proximal duplications by FISH

BACKGROUND: Preoperative cutaneous lymphoscintigraphy (LS) to identify sentinel (first-tier) lymph nodes was performed in 250 consecutive melanoma patients before wide local excision only or wide local excision with sentinel node biopsy. METHODS: The location of the sentinel nodes was marked on the overlying skin in all patients. Whether or not tracer was present in second-tier lymph nodes on the delayed scans was recorded for each patient and related to the lesion site at which the tracer had initially been injected. For 100 consecutive patients the rate of tracer movement through the lymphatic channels was compared to the incidence of second-tier drainage. RESULTS: Second-tier nodes were visualized in all patients with melanomas on the leg and thigh, and in almost all patients with melanomas on the forearm and hand, but were seen less often in patients with more centrally located melanomas. There was a significant correlation between the rate of lymph flow and the incidence of demonstrable second-tier drainage. CONCLUSION: The results suggest that the physiology of the lymphatic system varies depending on the origin of the lymphatic vessel. These findings have important implications for application of the sentinel node biopsy technique in individual patients.     

Enzymatic reaction with unnatural substrates: DNA photolyase (Escherichia coli) recognizes and reverses thymine [2+2] dimers in the DNA strand of a DNA/PNA hybrid duplex

Peptide nucleic acids (PNA) are mimics with normal bases connected to a pseudopeptide chain that obey Watson-Crick rules to form stable duplexes with itself and natural nucleic acids. This has focused attention on PNA as therapeutic or diagnostic reagents. Duplexes formed with PNA mirror some but not all properties of DNA. One fascinating aspect of PNA biochemistry is their reaction with enzymes. Here we show an enzyme reaction that operates effectively on a PNA/DNA hybrid duplex. A DNA oligonucleotide containing a cis, syn-thymine [2+2] dimer forms a stable duplex with PNA. The hybrid duplex is recognized by photolyase, and irradiation of the complex leads to the repair of the thymine dimer. This finding provides insight into the enzyme mechanism and provides a means for the selective repair of thymine photodimers.     

The int genes of bacteriophages P22 and lambda are regulated by different mechanisms

Bacteriophage P22 and lambda are related bacteriophages with similar gene organizations. In lambda the cll-dependent Pl promoter is responsible for lambda int gene expression. The only apparent counterpart to pl in P22 is oriented in the opposite direction, and cannot transcribe the P22 int gene. We show that this promoter, called P(al), is active both in vivo and in vitro, and is dependent upon the P22 cll-like gene, called c1. We have also determined the DNA sequence of a 3.3 kb segment that closes the gap between previously reported sequences to give a continuous sequence between the P22 pL promoter and the int gene. The newly determined sequence is densely packed with genes from the pL direction, and the proteins predicted by the sequence show excellent correlation with the proteins mapped by Youderian and Susskind in 1980. However, the sequence contains no apparent genes in the opposite (p(al)) direction, and no additional binding motifs for the P22 c1 protein. We conclude that int gene expression in P22 is regulated by a different mechanism than in lambda.     

The main-chain dynamics of the dynamin pleckstrin homology (PH) domain in solution: analysis of 15N relaxation with monomer/dimer equilibration

The backbone dynamics of the pleckstrin homology (PH) domain from dynamin were studied by 15N NMR relaxation (R1 and R2) and steady state heteronuclear 15N [1H] nuclear Overhauser effect measurements at 500 and 600 MHz, at protein concentrations of 1.7 mM and 300 microM, and by molecular dynamics (MD) simulations. The analysis was performed using the model-free approach. The method was extended in order to account for observed partial (equilibrium) dimerization of the protein at NMR concentrations. A model is developed that takes into account both rapid monomer-dimer exchange and anisotropy of the over-all rotation of the dimer. The data show complex dynamics of the dynamin PH domain. Internal motions in elements of the secondary structure are restricted, as inferred from the high value of the order parameter (S2 approximately 0.9) and from the local correlation time < 100 ps. Of the four extended loop regions that are disordered in the NMR-derived solution structure of the protein, loops beta 1/beta 2 and beta 5/beta 6 are involved in a large-amplitude (S2 down to 0.2 to 0.3) subnanosecond to nanosecond time-scale motion. Reorientation of the loops beta 3/beta 4 and beta 6/beta 7, in contrast, is restricted, characterized by the values of order parameter S2 approximately 0.9 more typical of the protein core. These loops, however, are involved in much slower processes of motion resulting in a conformational exchange on a microsecond to submillisecond time scale. The motions of the terminal regions (residues 1 to 10, 122 to 125) are practically unrestricted (S2 down to 0.05, characteristic times in nanosecond time scale), suggesting that these parts of the sequence do not participate in the protein fold. The analysis shows a larger sensitivity of the 15N relaxation data to protein microdynamic parameters (S2, tau loc) when protein molecular mass (tau c) increases. The use of negative values of the steady state 15N[1H] NOEs as an indicator of the residues not belonging to the folded structure is suggested. The amplitudes of local motion observed in the MD simulation are in a good-agreement with the NMR data for the amide NH groups located in the protein core.     

Flow Behavior and Hot Workability of Nb-15Si-22Ti-5Cr-3Al-2.5Hf Alloy

Constitutive models for flow behaviors of an arc-melted Nb-15Si-22Ti-5Cr-3Al-2.5Hf alloy at temperatures of 1350 °C to 1500 °C and strain rates of 0.001 to 0.1 s⁻¹ have been successfully established during work hardening and dynamic softening stages, respectively, and relatively small average absolute relative errors of the predicted flow stresses are reached (7.7 pct for the work hardening stage and 5.7 pct for the dynamic softening stage). The hot processing map has also been established successfully for this Nb-Si-based ultrahigh temperature alloy. The favorable conditions for hot working of this alloy have been determined as 1350 °C to 1380 °C/0.001 to 0.003 s⁻¹ and 1380 °C to 1440 °C/0.001 to 0.01 s⁻¹. The deformed microstructures under different conditions have been explored and the mechanisms for flow instability of this alloy have been revealed. Flow instability at relatively low temperatures and high strain rates (1350 °C and 1410 °C, 0.1 s⁻¹) is mainly derived from the cracking of Nb₅Si₃ and the debonding of Nbss/Nb₅Si₃ interfaces, while flow instability at higher temperatures (1500 °C) should primarily result from the development of cracks and holes within the Nbss phase because of excessive strain accumulation.