Cyclobutane Thymine Dimers with a Disrupted Phosphodiester Bond Are Refractory to T4 Endonuclease V Digestion but Have Increased Sensitivity to UvrABC Nuclease†

Biochemistry ◽  
1998 ◽  
Vol 37 (10) ◽  
pp. 3243-3249 ◽  
Author(s):  
Yi Zheng ◽  
Darel Hunting ◽  
Moon-shong Tang
Keyword(s):  
Phosphodiester Bond ◽  
Endonuclease V ◽  
Thymine Dimers ◽  
Increased Sensitivity

scholarly journals Endonuclease V (nfi) Mutant ofEscherichia coli K-12

1998 ◽  
Vol 180 (1) ◽  
pp. 46-51 ◽  
Author(s):  
Guangming Guo ◽  
Bernard Weiss
Keyword(s):  
Fold Increase ◽  
Induced Mutations ◽  
Base Pairs ◽  
Single Stranded Dna ◽  
Abasic Sites ◽  
Endonuclease V ◽  
Increased Sensitivity ◽  
K 12 ◽  
Insertion Mutants

ABSTRACT Endonuclease V (deoxyinosine 3′ endonuclease), the product of thenfi gene, has a specificity that encompasses DNAs containing dIMP, abasic sites, base mismatches, uracil, and even untreated single-stranded DNA. To determine its importance in DNA repair pathways, nfi insertion mutants and overproducers (strains bearing nfi plasmids) were constructed. The mutants displayed a twofold increase in spontaneous mutations for several markers and an increased sensitivity to killing by bleomycin and nitrofurantoin. An nfi mutation increased both cellular resistance to and mutability by nitrous acid. This agent should generate potential cleavage sites for the enzyme by deaminating dAMP and dCMP in DNA to dIMP and dUMP, respectively. Relative to that of a wild-type strain, an nfi mutant displayed a 12- to 1,000-fold increase in the frequency of nitrite-induced mutations to streptomycin resistance, which are known to occur in A · T base pairs. An nfi mutation also enhanced the lethality caused by a combined deficiency of exonuclease III and dUTPase, which has been attributed to unrepaired abasic sites. However, neither the deficiency nor the overproduction of endonuclease V affected the growth of the single-stranded DNA phages M13 or φX174 nor of Uracil-containing bacteriophage λ. These results suggest that endonuclease V has a significant role in the repair of deaminated deoxyadenosine (deoxyinosine) and abasic sites in DNA, but there was no evidence for its cleavage in vivo of single-stranded or uracil-containing DNA.

Structure of human endonuclease V as an inosine-specific ribonuclease

2014 ◽  
Vol 70 (9) ◽  
pp. 2286-2294 ◽  
Author(s):  
Zhemin Zhang ◽  
Zhitai Hao ◽  
Zhong Wang ◽  
Qing Li ◽  
Wei Xie
Keyword(s):  
Metal Binding ◽  
Binding Pocket ◽  
Rnase H ◽  
Structural Basis ◽  
Phosphodiester Bond ◽  
Substrate Preferences ◽  
Cysteine Motif ◽  
Endonuclease V ◽  
Cleavage Efficiency ◽  
Domains Of Life

The 6-aminopurine ring of adenosine (A) can be deaminated to form the 6-oxopurine of inosine (I). Endonuclease Vs (EndoVs) are inosine-specific nucleases that cleave at the second phosphodiester bond 3′ to inosine. EndoV proteins are highly conserved in all domains of life, but the bacterial and human enzymes seem to display distinct substrate preferences. While the bacterial enzymes exhibit high cleavage efficiency on various nucleic acid substrates, human EndoV (hEndoV) is most active towards ssRNA but is much less active towards other substrates. However, the structural basis of substrate recognition by hEndoV is not well understood. In this study, the 2.3 Å resolution crystal structure of hEndoV was determined and its unusual RNA-cleaving properties were investigated. The enzyme preserves the general `RNase H-like' structure, especially in the wedge motif, the metal-binding site and the hypoxanthine-binding pocket. hEndoV also features several extra insertions and a characteristic four-cysteine motif, in which Cys227 and Cys228, two cysteines that are highly conserved in higher eukaryotes, play important roles in catalysis. The structure presented here helps in understanding the substrate preference of hEndoV catalysis.

Detection of viruses by electron microscopy: Increased sensitivity by direct micro-ultracentrifugation of samples

1987 ◽  
Vol 45 ◽  
pp. 908-909
Author(s):  
Alain R. Trudel ◽  
M. Trudel
Keyword(s):  
Electron Microscopy ◽  
Fold Increase ◽  
Observation Time ◽  
Clinical Samples ◽  
Maximum Speed ◽  
Viral Particles ◽  
Structural Details ◽  
Latex Spheres ◽  
Increased Sensitivity ◽  
Size Of Particles

AirfugeR (Beckman) direct ultracentrifugation of viral samples on electron microscopy grids offers a rapid way to concentrate viral particles or subunits and facilitate their detection and study. Using the A-100 fixed angle rotor (30°) with a K factor of 19 at maximum speed (95 000 rpm), samples up to 240 μl can be prepared for electron microscopy observation in a few minutes: observation time is decreased and structural details are highlighted. Using latex spheres to calculate the increase in sensitivity compared to the inverted drop procedure, we obtained a 10 to 40 fold increase in sensitivity depending on the size of particles. This technique also permits quantification of viral particles in samples if an aliquot is mixed with latex spheres of known concentration.Direct ultracentrifugation for electron microscopy can be performed on laboratory samples such as gradient or column fractions, infected cell supernatant, or on clinical samples such as urine, tears, cephalo-rachidian liquid, etc..

Preplate neurons in embryonic mouse cortex: Ultrastructural observations using photoconversion of the fluorescent lipophilic dye dil

1992 ◽  
Vol 50 (1) ◽  
pp. 906-907
Author(s):  
Linda C. Hassinger ◽  
James E. Crandall
Keyword(s):  
Mouse Embryos ◽  
Embryonic Mouse ◽  
Cortical Afferents ◽  
Mouse Cortex ◽  
Carbocyanine Dye ◽  
Increased Sensitivity

We have begun to look directly at small numbers of afferent axons to early generated neurons that form the preplate in the developing mouse cortex. The carbocyanine dye Dil (1’1, dioctadecyl-3,3,3’3’-tetramethyl-indocarbocyanine) has proved especially useful for this goal. DiI labels axons and their terminals with greater sensitivity and without some of the disadvantages of axon filling with HRP. The increased sensitivity provided by labeling embryonic axons with DiI has given us new insights into the development of cortical afferents. For instance, we reported originally that afferents from the thalamus were present below the cortex as early as embryonic day 15 (E15) based on HRP injections into mouse embryos. By using DiI placements into the thalamus in aldehyde-fixed brains, we now know that thalamic fibers reach the cortex 24 hrs earlier.

Bayesian removal of noise for increased sensitivity in vector pattern recognition lattice imaging of interfaces

1993 ◽  
Vol 51 ◽  
pp. 994-995
Author(s):  
L. Fei ◽  
P. Fraundorf
Keyword(s):  
Pattern Recognition ◽  
Perfect Crystal ◽  
Unit Cell ◽  
Ion Milling ◽  
Beam Radiation ◽  
Major Interest ◽  
Unit Cells ◽  
Mapping Process ◽  
Increased Sensitivity ◽  
Electron Microscopes

Interface structure is of major interest in microscopy. With high resolution transmission electron microscopes (TEMs) and scanning probe microscopes, it is possible to reveal structure of interfaces in unit cells, in some cases with atomic resolution. A. Ourmazd et al. proposed quantifying such observations by using vector pattern recognition to map chemical composition changes across the interface in TEM images with unit cell resolution. The sensitivity of the mapping process, however, is limited by the repeatability of unit cell images of perfect crystal, and hence by the amount of delocalized noise, e.g. due to ion milling or beam radiation damage. Bayesian removal of noise, based on statistical inference, can be used to reduce the amount of non-periodic noise in images after acquisition. The basic principle of Bayesian phase-model background subtraction, according to our previous study, is that the optimum (rms error minimizing strategy) Fourier phases of the noise can be obtained provided the amplitudes of the noise is given, while the noise amplitude can often be estimated from the image itself.

Structures of c-di-GMP/cGAMP degrading phosphodiesterase VcEAL: identification of a novel conformational switch and its implication

2019 ◽  
Vol 476 (21) ◽  
pp. 3333-3353 ◽  
Author(s):  
Malti Yadav ◽  
Kamalendu Pal ◽  
Udayaditya Sen
Keyword(s):  
Active Site ◽  
Metal Binding ◽  
Metal Ion ◽  
Triosephosphate Isomerase ◽  
Catalytic Mechanism ◽  
Degradation Mechanism ◽  
Structural Basis ◽  
Conserved Residues ◽  
Phosphodiester Bond ◽  
Cyclic Dinucleotides

Cyclic dinucleotides (CDNs) have emerged as the central molecules that aid bacteria to adapt and thrive in changing environmental conditions. Therefore, tight regulation of intracellular CDN concentration by counteracting the action of dinucleotide cyclases and phosphodiesterases (PDEs) is critical. Here, we demonstrate that a putative stand-alone EAL domain PDE from Vibrio cholerae (VcEAL) is capable to degrade both the second messenger c-di-GMP and hybrid 3′3′-cyclic GMP–AMP (cGAMP). To unveil their degradation mechanism, we have determined high-resolution crystal structures of VcEAL with Ca2+, c-di-GMP-Ca2+, 5′-pGpG-Ca2+ and cGAMP-Ca2+, the latter provides the first structural basis of cGAMP hydrolysis. Structural studies reveal a typical triosephosphate isomerase barrel-fold with substrate c-di-GMP/cGAMP bound in an extended conformation. Highly conserved residues specifically bind the guanine base of c-di-GMP/cGAMP in the G2 site while the semi-conserved nature of residues at the G1 site could act as a specificity determinant. Two metal ions, co-ordinated with six stubbornly conserved residues and two non-bridging scissile phosphate oxygens of c-di-GMP/cGAMP, activate a water molecule for an in-line attack on the phosphodiester bond, supporting two-metal ion-based catalytic mechanism. PDE activity and biofilm assays of several prudently designed mutants collectively demonstrate that VcEAL active site is charge and size optimized. Intriguingly, in VcEAL-5′-pGpG-Ca2+ structure, β5–α5 loop adopts a novel conformation that along with conserved E131 creates a new metal-binding site. This novel conformation along with several subtle changes in the active site designate VcEAL-5′-pGpG-Ca2+ structure quite different from other 5′-pGpG bound structures reported earlier.

The Effect of Stapedectomy on the Loudness of One’s Own Voice

1965 ◽  
Vol 8 (3) ◽  
pp. 223-234 ◽  
Author(s):  
William Melnick
Keyword(s):  
Middle Ear ◽  
Normal Subjects ◽  
Sound Energy ◽  
Loudness Function ◽  
Before And After ◽  
Increased Sensitivity

Five subjects with normal middle ear mechanisms, and otosclerotic patients, before and after stapedectomy, matched the loudness of their voices to the loudness of a 125-cps-sawtooth noise. The results showed loudness matching functions with gradual slopes, less than 1.00, for the normal subjects and the patients prior to stapedectomy. Post-surgically, the loudness function for the patients increased in steepness to considerably more than 1.00. These results are explained, most logically, in terms of increased sensitivity of the altered middle ear to sound energy generated by the listener’s own voice.

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