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1.
Polyamines such as spermine can have interaction with protein. The aim of the present study was to investigate how spermine could influence the structure, thermal stability, and the activity of α-chymotrypsin. Kinetics, thermodynamics, molecular dynamics (MD), and docking simulations studies were conducted to investigate the effect of spermine on the activity and structure of α-Chymotrypsin (α-Chy) in 50 mM Tris–HCl buffer, with the pH 8, using different spectroscopic techniques as well as molecular docking and MD simulations. The stability and activity of α-Chy were increased in the presence of spermine. The results of the kinetic study showed that the activity of spermine was increased. Enzyme activation was accompanied by changes on the α-Chy conformation. Fluorescence intensity changes showed dynamic quenching during spermine binding. The fluorescence quenching of the α-Chy suggested the more polar location of Trp residues. Near-UV and Far-UV circular dichroism studies also demonstrated the transfer of Trp, Phe, and Tyr residues to a more flexible environment. The increase in the absorption of α-Chy in the presence of spermine was as a result of the formation of spermine–α-Chy complex. Molecular docking results revealed the presence of one binding site with a negative value for the Gibbs free energy of the binding of spermine to α-Chy. Docking study also revealed that van der Waals interactions and hydrogen bonds played a major role in stabilizing the complex.  相似文献   
2.
Mutations in the human ChlR1 (DDX11) gene are associated with a unique genetic disorder known as Warsaw breakage syndrome characterized by cellular defects in genome maintenance. The DNA triplex helix structures that form by Hoogsteen or reverse Hoogsteen hydrogen bonding are examples of alternate DNA structures that can be a source of genomic instability. In this study, we have examined the ability of human ChlR1 helicase to destabilize DNA triplexes. Biochemical studies demonstrated that ChlR1 efficiently melted both intermolecular and intramolecular DNA triplex substrates in an ATP-dependent manner. Compared with other substrates such as replication fork and G-quadruplex DNA, triplex DNA was a preferred substrate for ChlR1. Also, compared with FANCJ, a helicase of the same family, the triplex resolving activity of ChlR1 is unique. On the other hand, the mutant protein from a Warsaw breakage syndrome patient failed to unwind these triplexes. A previously characterized triplex DNA-specific antibody (Jel 466) bound triplex DNA structures and inhibited ChlR1 unwinding activity. Moreover, cellular assays demonstrated that there were increased triplex DNA content and double-stranded breaks in ChlR1-depleted cells, but not in FANCJ−/− cells, when cells were treated with a triplex stabilizing compound benzoquinoquinoxaline, suggesting that ChlR1 melting of triple-helix structures is distinctive and physiologically important to defend genome integrity. On the basis of our results, we conclude that the abundance of ChlR1 known to exist in vivo is likely to be a strong deterrent to the stability of triplexes that can potentially form in the human genome.  相似文献   
3.
The allosteric communication between the ATP- and DNA-binding sites of RecQ helicases enables efficient coupling of ATP hydrolysis to translocation along single-stranded DNA (ssDNA) and, in turn, the restructuring of multistranded DNA substrates during genome maintenance processes. In this study, we used the tryptophan fluorescence signal of Escherichia coli RecQ helicase to decipher the kinetic mechanism of the interaction of the enzyme with ssDNA. Rapid kinetic experiments revealed that ssDNA binding occurs in a two-step mechanism in which the initial binding step is followed by a structural transition of the DNA-bound helicase. We found that the nucleotide state of RecQ greatly influences the kinetics of the detected structural transition, which leads to a high affinity DNA-clamped state in the presence of the nucleotide analog ADP-AlF4. The DNA binding mechanism is largely independent of ssDNA length, indicating the independent binding of RecQ molecules to ssDNA and the lack of significant DNA end effects. The structural transition of DNA-bound RecQ was not detected when the ssDNA binding capability of the helicase-RNase D C-terminal domain was abolished or the domain was deleted. The results shed light on the nature of conformational changes leading to processive ssDNA translocation and multistranded DNA processing by RecQ helicases.  相似文献   
4.
ARTEMIS is a member of the metallo-β-lactamase protein family. ARTEMIS has endonuclease activity at DNA hairpins and at 5′- and 3′-DNA overhangs of duplex DNA, and this endonucleolytic activity is dependent upon DNA-PKcs. There has been uncertainty about whether ARTEMIS also has 5′-exonuclease activity on single-stranded DNA and 5′-overhangs, because this 5′-exonuclease is not dependent upon DNA-PKcs. Here, we show that the 5′-exonuclease and the endonuclease activities co-purify. Second, we show that a point mutant of ARTEMIS at a putative active site residue (H115A) markedly reduces both the endonuclease activity and the 5′-exonuclease activity. Third, divalent cation effects on the 5′-exonuclease and the endonuclease parallel one another. Fourth, both the endonuclease activity and 5′-exonuclease activity of ARTEMIS can be blocked in parallel by small molecule inhibitors, which do not block unrelated nucleases. We conclude that the 5′-exonuclease is intrinsic to ARTEMIS, making it relevant to the role of ARTEMIS in nonhomologous DNA end joining.  相似文献   
5.
8-Halogenated guanine (haloG), a major DNA adduct formed by reactive halogen species during inflammation, is a promutagenic lesion that promotes misincorporation of G opposite the lesion by various DNA polymerases. Currently, the structural basis for such misincorporation is unknown. To gain insights into the mechanism of misincorporation across haloG by polymerase, we determined seven x-ray structures of human DNA polymerase β (polβ) bound to DNA bearing 8-bromoguanine (BrG). We determined two pre-catalytic ternary complex structures of polβ with an incoming nonhydrolyzable dGTP or dCTP analog paired with templating BrG. We also determined five binary complex structures of polβ in complex with DNA containing BrG·C/T at post-insertion and post-extension sites. In the BrG·dGTP ternary structure, BrG adopts syn conformation and forms Hoogsteen base pairing with the incoming dGTP analog. In the BrG·dCTP ternary structure, BrG adopts anti conformation and forms Watson-Crick base pairing with the incoming dCTP analog. In addition, our polβ binary post-extension structures show Hoogsteen BrG·G base pair and Watson-Crick BrG·C base pair. Taken together, the first structures of haloG-containing DNA bound to a protein indicate that both BrG·G and BrG·C base pairs are accommodated in the active site of polβ. Our structures suggest that Hoogsteen-type base pairing between G and C8-modified G could be accommodated in the active site of a DNA polymerase, promoting G to C mutation.  相似文献   
6.
Injury and repair in biocide-treated spores of Bacillus subtilis   总被引:3,自引:0,他引:3  
Abstract Bacillus subtilis NCTC 8236 spores exposed to appropriate concentrations of test biocides (glutaraldehyde, two iodine and two chlorine preparations) were able to repair injury if subsequently held in nutrient broth at 37°C but not in broth at 22°C, sterile filtered water at 4, 22 or 37°C or germination medium at 37°C. Repair appeared to occur primarily during outgrowth and was initiated soonest for iodine-treated spores and latest for glutaraldehyde-treated ones.  相似文献   
7.
Musculoskeletal modeling allows for analysis of individual muscles in various situations. However, current techniques to realistically simulate muscle response when significant amounts of intentional coactivation is required are inadequate. This would include stiffening the neck or spine through muscle coactivation in preparation for perturbations or impacts. Muscle coactivation has been modeled previously in the neck and spine using optimization techniques that seek to maximize the joint stiffness by maximizing total muscle activation or muscle force. These approaches have not sought to replicate human response, but rather to explore the possible effects of active muscle. Coactivation remains a challenging feature to include in musculoskeletal models, and may be improved by extracting optimization objective functions from experimental data. However, the components of such an objective function must be known before fitting to experimental data. This study explores the effect of components in several objective functions, in order to recommend components to be used for fitting to experimental data. Four novel approaches to modeling coactivation through optimization techniques are presented, two of which produce greater levels of stiffness than previous techniques. Simulations were performed using OpenSim and MATLAB cooperatively. Results show that maximizing the moment generated by a particular muscle appears analogous to maximizing joint stiffness. The approach of optimizing for maximum moment generated by individual muscles may be a good candidate for developing objective functions that accurately simulate muscle coactivation in complex joints. This new approach will be the focus of future studies with human subjects.  相似文献   
8.
The HOP2 protein is required for efficient double-strand break repair which ensures the proper synapsis of homologous chromosomes and normal meiotic progression. We previously showed that in vitro HOP2 shows two distinctive activities: when it is incorporated into a HOP2-MND1 heterodimer, it stimulates DMC1 and RAD51 recombination activities, and the purified HOP2 alone is proficient in promoting strand invasion. The structural and biochemical basis of HOP2 action in recombination are poorly understood; therefore, they are the focus of this work. Herein, we present the solution structure of the amino-terminal portion of mouse HOP2, which contains a typical winged helix DNA-binding domain. Together with NMR spectral changes in the presence of double-stranded DNA, protein docking on DNA, and mutation analysis to identify the amino acids involved in DNA coordination, our results on the three-dimensional structure of HOP2 provide key information on the fundamental structural and biochemical requirements directing the interaction of HOP2 with DNA. These results, in combination with mutational experiments showing the role of a coiled-coil structural feature involved in HOP2 self-association, allow us to explain important aspects of the function of HOP2 in recombination.  相似文献   
9.
Summary Immunohistochemical properties of the terminal nerve network in the rat heart were assessed by use of the elution-restaining method. The colocalization of the enzymes involved in catecholamine synthesis (tyrosine hydroxylase — TH, dopamine--hydroxylase — DBH) as well as the respective distributions of the neuropeptides associated with the adrenergic nervous system (neuropeptide tyrosine — NPY, C-terminal flanking peptide of neuropeptide Y — C-PON) were studied in series of serial sections throughout the interatrial septum and the atrioventricular junction. Our data suggest that ganglion cells of sulcus terminalis as well as the epicardial ganglia enclosed between the superior vena cava and ascending aorta are VIP- and TH-negative, but neuropeptide Y- and DBH-immunoreactive. They give rise to three intraseptal nerves directed towards the specialised structures of the atrioventricular junction. These nerve fascicles contain abundant, thick TH-immunoreactive nerve fibres and scarce, thin NPY- and DBH-immunoreactive fibres. The cell bodies of the intramural ganglion cells localized between the right and left branches of the bundle of His (Moravec and Moravec 1984) are strongly TH- and DBH-immunoreactive. They are innervated by thick nerve fibres having the same immunohistochemical properties (NPY- and DBH-immunoreactivities) as those of a subpopulation of the epicardial ganglion cells and seem to supply some of the TH-immunoreactive nerve fibres directed via the intraseptal nerves to the epicardial ganglia. The existence of a multicomponent nerve network, characterized by a reciprocal innervation of the sinus node and atrioventricular node areas, is suggested by our immunohistochemical data.  相似文献   
10.
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