Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Restriction endonucleases are remarkably resilient to alterations in their DNA binding specificity. To understand the basis of this immutability, we have determined the crystal structure of endonuclease BglII bound to its recognition sequence (AGATCT), at 1. 5 A resolution. We compare the structure of BglII to endonuclease BamHI, which recognizes a closely related DNA site (GGATCC). We show that both enzymes share a similar alpha/beta core, but in BglII, the core is augmented by a beta-sandwich domain that encircles the DNA to provide extra specificity. Remarkably, the DNA is contorted differently in the two structures, leading to different protein-DNA contacts for even the common base pairs. Furthermore, the BglII active site contains a glutamine in place of the glutamate at the general base position in BamHI, and only a single metal is found coordinated to the putative nucleophilic water and the phosphate oxygens. This surprising diversity in structures shows that different strategies can be successful in achieving site-specific recognition and catalysis in restriction endonucleases.
...
PMID:Understanding the immutability of restriction enzymes: crystal structure of BglII and its DNA substrate at 1.5 A resolution. 1065 3

TaqI is a metal-dependent endonuclease that recognizes T(downward arrow)CGA, with the arrow indicating the cleavage site. Mutations at K158 render the enzyme inactive and mutations at K157 significantly reduce DNA cleavage activity (W. Cao and F. Barany (1998) J. Biol. Chem. 273, 33002-33010). Aspartate, glutamate, and histidine substitutions were made at K158 in the wild-type and K157S mutant TaqI endonuclease to understand the functional organization of the active site. None of the mutants was active with Mg(2+), but the DNA cleavage activities were partly rescued by Mn2+ for K157S-K158E and K157S-K158H mutants. The rescuing effects were observed with Mn2+ but not with other divalent cations. K157S-K158E required higher Mn2+ concentrations than the wild-type enzyme for DNA cleavage activity, suggesting that a Mn2+ ion is weakly bound at the 158 position. The need to neutralize K157 to recover the catalytic activity of K158E and K158H indicates that K158 and K157 may interact functionally. In analogy with EcoRV, Ca2+ stimulated Mn2+-mediated cleavage for the wild-type TaqI, suggesting the existence of at least two metal ions at the catalytic center. A catalytic mechanism involving two metal ions and the K157-K158 pair is proposed for TaqI endonuclease.
...
PMID:Exploring the catalytic center of TaqI endonuclease: rescuing catalytic activity by double mutations and Mn2+. 1125 28

We determined whether neural responses to inflammation and hyperalgesia involve activation of kainate receptors, a subgroup of glutamate receptors. Inflammation was introduced into the hind paw by intraplantar injection of complete Freund's adjuvant. The inflammation-induced thermal hyperalgesia was attenuated by intrathecal administration of a non-selective alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptor antagonist, 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide disodium (NBQX), as well as by selective kainate receptor antagonists, 6,7,8,9-tetrohydro-5-nitro-1H-benz[g]indole-2,3-dione 3-oxime (NS-102) and 3S,4aR,6S,8aR-6-(4-carboxyphenyl)methyl-1,2,3,4,4a,5,6,7,8,8a-deca-hydroisoquinoline-3-carboxylic acid (LY382884). Reverse transcription-polymerase chain reaction (RT-PCR) indicated that the GluR5 and GluR6, but not the GluR7, KA1 and KA2 subunits, exhibited increased mRNA expression at 2 h to 3 days following inflammation (P<0.05). Western blot showed an increase in GluR6 protein levels (P<0.01) with a time course consistent with the changes in its mRNA levels. cDNA sequence and BbvI endonuclease digestion of the GluR6 PCR product revealed that the upregulated GluR6 mRNAs were predominantly the unedited form (Q). These results suggest that a selective upregulation of kainate receptor subunit expression contributes to inflammatory hyperalgesia.
...
PMID:Activation of spinal kainate receptors after inflammation: behavioral hyperalgesia and subunit gene expression. 1235 72

Adeno-associated virus type 2 Rep endonuclease activity is necessary for both viral DNA replication and site-specific integration of the viral genome into human chromosome 19. The biochemical activities required for site-specific endonuclease activity (namely specific DNA binding and transesterification activity) have been mapped to the amino-terminal domain of the AAV2 Rep protein. The amino-terminal 208 amino acids are alone sufficient for site-specific endonuclease activity, and nicking by this domain is metal-dependent. To identify this metal-binding site, we have employed a cysteine mutagenesis approach that targets conserved acidic amino acids. By using this technique, we provide functional biochemical data supporting a role for glutamate 83 in the coordination of metal ions in the context of Rep endonuclease activity. In addition, our biochemical data suggest that glutamate 164, although not involved in the coordination of metal ions, is closely associated with the active site. Thus, in lieu of a crystal structure for the AAV type 2 amino-terminal domain, our data corroborate the recently published structural studies of the AAV type 5 endonuclease and suggest that although the two enzymes are not highly conserved with respect to the AAV family, their active sites are highly conserved.
...
PMID:Identification of active site residues of the adeno-associated virus type 2 Rep endonuclease. 1248 Sep 38

NaeI endonuclease contains a 10-amino acid region with sequence similarity to the active site KXDG motif of DNA ligase except for leucine (Leu-43) in NaeI ((43)LXDG(46)). Changing Leu-43 to lysine abolishes the NaeI endonuclease activity and replaces it with topoisomerase and recombinase activities. Here we report the results of substituting Leu-43 with alanine, arginine, asparagine, glutamate, and histidine. Quantitating specific activities and DNA binding values for the mutant proteins determined the range of amino acids at position 43 that alter NaeI mechanism. Substituting alanine, asparagine, glutamate, and histidine for Leu-43 maintained endonuclease activity, but at a lower level. On the other hand, substituting positively charged arginine, like lysine at position 43, converted NaeI to a topoisomerase with no observable double-strand cleavage activity. The specific activities of NaeI-43K and NaeI-43R and their relative sensitivities to salt, the topoisomerase-inhibiting drug N-[4-(9-acridinylamino)-3-methoxyphenyl]methane-sulfonamide (amsacrine) and single-stranded DNA showed that the two activities are similar. The effect of placing a positive charge at position 43 on NaeI structure was determined by measuring (for NaeI and NaeI-43K) relative susceptibilities to proteolysis, UV, circular dichroism spectra, and temperature melting transitions. The results provide evidence that a positive charge at position 43 induces dramatic changes in NaeI structure that affect both the Endo and Topo domains of NaeI. The identification of four putative DNA ligase motifs in NaeI leads us to speculate that structural changes that superimpose these motifs on the ligase structure may account for the changes in activity.
...
PMID:Amino acid substitutions at position 43 of NaeI endonuclease. Evidence for changes in NaeI structure. 1251 52

The Saccharomyces cerevisiae Rad50-Mre11-Xrs2 complex plays a central role in the cellular response to DNA double strand breaks. Rad50 has a globular ATPase head domain with a long coiled-coil tail. DNA binding by Rad50 is ATP-dependent and the Rad50-Mre11-Xrs2 complex possesses DNA unwinding and endonuclease activities that are regulated by ATP. Here we have examined the role of the Rad50 Walker type A ATP binding motif in DNA double strand break repair by a combination of genetic and biochemical approaches. Replacement of the conserved lysine residue within the Walker A motif with alanine, glutamate, or arginine results in the same DNA damage sensitivity and homologous recombination defect as the rad50 deletion mutation. The Walker A mutations also cause a deficiency in non-homologous end-joining. As expected, complexes containing the rad50 Walker A mutant proteins are defective in ATPase, ATP-dependent DNA unwinding, and ATP-stimulated endonuclease activities. Although the DNA end-bridging activity of the Rad50-Mre11-Xrs2 complex is ATP-independent, the end-bridging activity of complexes containing the rad50 Walker A mutant proteins is salt-sensitive. These results provide a molecular explanation for the observed in vivo defects of the rad50 Walker mutant strains and reveal a novel ATP-independent function for Rad50 in DNA end-bridging.
...
PMID:Effect of amino acid substitutions in the rad50 ATP binding domain on DNA double strand break repair in yeast. 1554 77

Status epilepticus (SE)-induced neuronal death is morphologically necrotic and is initiated by excessive glutamate release, which activates postsynaptic N-methyl-D-aspartate (NMDA) receptors and triggers receptor-mediated calcium influx (excitotoxicity). This results in activation of intracellular proteases and neuronal nitric oxide synthase, with generation of free radicals, and damage to cellular membranes, structural proteins, and essential enzymes. Programmed cell death mechanisms, such as p53 activation, activation of cell death-promoting Bcl-2 family members, and endonuclease-induced DNA laddering, occur in SE-induced neuronal death. Caspase-independent excitotoxic mechanisms, such as NMDA-induced calpain I activation, with activation and translocation of the cell death-promoting Bcl-2 family member Bid from cytoplasm to mitochondria, and subsequent translocation of apoptosis-inducing factor and endonuclease G to nuclei (which cause large-scale and internucleosomal DNA cleavage, respectively), may be triggered by SE. Poly(ADP-ribose) polymerase-1 (PARP-1) activation and cysteinyl cathepsin and DNase II release from lysosomes may occur following SE as well, but these events await future investigation. In the future, rational combinations of central nervous system-penetrable neuroprotective agents, based on our knowledge of excitotoxic mechanisms, may be useful in refractory human SE.
...
PMID:Prolonged seizures and cellular injury: understanding the connection. 1627 99

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated protein genes (cas genes) have been suggested to act as an immune system in archaea and bacteria mimicking the eukaryotic RNA interference (RNAi) system. We have investigated the properties of the protein SSO2001 from Sulfolobus solfataricus (Sso) P2, which is part of the cas gene cluster. This study shows that SSO2001 is an endonuclease specifically digesting double-stranded oligonucleotides and preferably cleaving at G:C pairs. Point mutations identify both highly conserved aspartate and glutamate residues as being crucial for the nuclease activity. The catalytic activity shows an optimum at neutral pH and pH 3.
...
PMID:Characterization of the endonuclease SSO2001 from Sulfolobus solfataricus P2. 1917 59

Saccharomyces cerevisiae MutLalpha is a heterodimer of Mlh1 and Pms1 that participates in DNA mismatch repair (MMR). Both proteins have weakly conserved C-terminal regions (CTDs), with the CTD of Pms1 harboring an essential endonuclease activity. These proteins also have conserved N-terminal domains (NTDs) that bind and hydrolyze ATP and bind to DNA. To better understand Pms1 functions and potential interactions with DNA and/or other proteins, we solved the 2.5A crystal structure of yeast Pms1 (yPms1) NTD. The structure is similar to the homologous NTDs of Escherichia coli MutL and human PMS2, including the site involved in ATP binding and hydrolysis. The structure reveals a number of conserved, positively charged surface residues that do not interact with other residues in the NTD and are therefore candidates for interactions with DNA, with the CTD and/or with other proteins. When these were replaced with glutamate, several replacements resulted in yeast strains with elevated mutation rates. Two replacements also resulted in NTDs with decreased DNA binding affinity in vitro, suggesting that these residues contribute to DNA binding that is important for mismatch repair. Elevated mutation rates also resulted from surface residue replacements that did not affect DNA binding, suggesting that these conserved residues serve other functions, possibly involving interactions with other MMR proteins.
...
PMID:Functional residues on the surface of the N-terminal domain of yeast Pms1. 2013 91

Ribonuclease H (RNase H) belongs to the nucleotidyl-transferase superfamily and hydrolyzes the phosphodiester linkage on the RNA strand of a DNA/RNA hybrid duplex. Due to its activity in HIV reverse transcription, it represents a promising target for anti-HIV drug design. While crystallographic data have located two ions in the catalytic site, there is ongoing debate concerning just how many metal ions bound at the active site are optimal for catalysis. Indeed, experiments have shown a dependency of the catalytic activity on the Mg(2+) concentration. Moreover, in RNase H, the glutamate residue E188 has been shown to be essential for full enzymatic activation, regardless of the Mg(2+) concentration. The catalytic center is known to contain two Mg(2+) ions, and E188 is not one of the primary metal ligands. Herein, classical molecular dynamics (MD) simulations are employed to study the metal-ligand coordination in RNase H at different concentration of Mg(2+). Importantly, the presence of a third Mg(2+) ion, bound to the second-shell ligand E188, is a persistent feature of the MD simulations. Free energy calculations have identified two distinct conformations, depending on the concentration of Mg(2+). At standard concentration, a third Mg(2+) is found in the catalytic pocket, but it does not perturb the optimal RNase H active conformation. However, at higher concentration, the third Mg(2+) ion heavily perturbs the nucleophilic water and thereby influences the catalytic efficiency of RNase H. In addition, the E188A mutant shows no ability to engage additional Mg(2+) ions near the catalytic pocket. This finding likely explains the decrease in catalytic activity of E188A and also supports the key role of E188 in localizing the third Mg(2+) ion at the active site. Glutamate residues are commonly found surrounding the metal center in the endonuclease family, which suggests that this structural motif may be an important feature to enhance catalytic activity. The present MD calculations support the hypothesis that RNase H can accommodate three divalent metal ions in its catalytic pocket and provide an in-depth understanding of their dynamic role for catalysis.
...
PMID:Understanding the effect of magnesium ion concentration on the catalytic activity of ribonuclease H through computation: does a third metal binding site modulate endonuclease catalysis? 2073 47


<< Previous 1 2 3 4 Next >>

---