Nucleotidyl transferases

TUNEL is the name given to the in-situ DNA endlabelling technique which serves as a marker of apoptotic cells. This method is based on the specific binding of terminal de-oxy nucleotidyl transferases to the 3-hydroxy ends of DNA. The technique is normally used for examination under the light microscope but can also be adapted for examination under the electron microscope. 

TCDD causes bone marrow hypocellularity, with specific decreases in the total number of hematopoietic stem cells (HSC) and lymphocyte precursors.42 16 Exposure to TCDD also diminishes mRNA levels of recombination activating gene-1 and terminal deoxy-nucleotidyl transferase in bone marrow cells.47 The best characterized effect of TCDD on bone marrow is the impaired maturation of B cells. A single dose of TCDD causes a transient, time- and dose-dependent, and developmental stage-specific impairment in B cell maturation, with mature B cells affected first, followed by depletion of B cell progenitors.45-46... 

Yet another super family, the nucleotidyl-transferase family, also utilizes two-metal-ion-dependent catalysis the members include transposases, retrovirus integrases and Holliday junction resolvases4. Whereas in the nucleases, the Mg2+ ions are asymmetrically coordinated, and play distinct roles, in activating the nucleophile and stabilizing the transition state, respectively, in the transposases, they are symmetrically coordinated and exchange roles to alternatively activate a water molecule and a 3 -OH for successive strand cleavage and transfer. 

The enzymes that synthesise RNA and DNA are known as nucleic acid polymerases. They are classified as nucleotidyl transferases (Chapter 3). The basic reaction can be represented as follows ... 

StrD/BlmD 355 16736 dTDP-D-glucose synthase/nucleotidyl-transferase... 

A metal-nucleotide complex that exhibits low rates of ligand exchange as a result of substituting higher oxidation state metal ions with ionic radii nearly equal to the naturally bound metal ion. Such compounds can be prepared with chromium(III), cobalt(III), and rhodi-um(III) in place of magnesium or calcium ion. Because these exchange-inert complexes can be resolved into their various optically active isomers, they have proven to be powerful mechanistic probes, particularly for kinases, NTPases, and nucleotidyl transferases. In the case of Cr(III) coordination complexes with the two phosphates of ATP or ADP, the second phosphate becomes chiral, and the screw sense must be specified to describe the three-dimensional configuration of atoms. 

The systematic name for these enzymes is nucleoside triphosphate a-D-glycosyl phosphate-nucleotidyl transferases (E.C. 2.7.7 group). 

After synthesis, the pre-tRNA molecule folds up into the characteristic stem-loops structures (Fig. 1) and non-tRNA sequence is cleaved from the 5 and 3 ends by ribonucleases. In prokaryotes, the CCA sequence at the 3 end of the tRNA (which is the site of bonding to the amino acid) is enclosed by the tRNA gene but this is not the case in eukaryotes. Instead, the CCA is added to the 3 end after the trimming reactions by tRNA nucleotidyl transferase. Another difference between prokaryotes and eukaryotes is that eukaryotic pre-tRNA molecules often contain a short intron in the loop of the anticodon arm (Fig. 4). 

Polynucleotide polymerases, or nucleotidyl transferases, are enzymes that catalyze the template-instructed polymerization of deoxyribo- or ribonu-cleoside triphosphates into polymeric nucleic acid - DNA or RNA. Depending on their substrate specificity, polymerases are classed as RNA- or DNA-dependent polymerases which copy their templates into RNA or DNA (all combinations of substrates are possible). Polymerization, or nucleotidyl transfer, involves formation of a phosphodiester bond that results from nucleophilic attack of the 3 -OH of primer-template on the a-phosphate group of the incoming nucleoside triphosphate. Although substantial diversity of sequence and function is observed for natural polymerases, there is evidence that many employ the same mechanism for DNA or RNA synthesis. On the basis of the crystal structures of polymerase replication complexes, a two-metal-ion mechanism of nucleotide addition was proposed [1] during this two divalent metal ions stabilize the structure and charge of the expected pentacovalent transition state (Figure B.16.1). 

Enzymes which catalyze the reaction type (a) include phosphodiesterases, phospholipases (C and D), nucleotidyl transferases, nucleases, and pyrophos-phokinases. The type (b) reaction involves mainly phosphokinases and phos-phomutases. The hydrolysis of phosphomonoesters (reaction type c) is catalyzed by phosphatases, nucleotidases, ATPases, and so on. Most phosphatases also catalyze the phosphoryl transfer reaction, type (b), if an alcohol is used as an acceptor. 

Kanamycin nucleotidyl transferase Thermostability Increase 20 C DNA shuffling + screen-ing/selection T. thermophilus [171]... 

D Nucleotidyl transferases Transfer of nucleotidyl moieties 2.7.7. Nucleotidyltransferases... 

Posttranscriptional processing of tRNA requires several distinct steps, as summarized in Figure 25.8. First, the 50 and 30 ends must be cleaved to release the tRNA sequence from the larger precursor transcript and introns must be removed if they are present. Second, the required CCA charging sequence at the 30 end of tRNA must sometimes be added by a nucleotidyl transferase. Third, all tRNAs contain a large number of modified bases which result from reductions, methylations, and deaminations. These modifications can affect codon recognition by the tRNAs during protein synthesis (Chapter 26). 

The bifiinctional nature of the AlgA protein can be clearly discerned in the structure of the gene that encodes it (Figure 5). The region that encodes amino acids 2—287 is a member of the Pfam family of nucleotidyl transferases, and the region from 298 to 464 is a member of the Pfam family of PMIs. Amino acids 396-404 contain the sequence that was identified as a conserved motif in type M I and II PMIs. ... 

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