Enzyme exonuclease

This assay used the enzyme exonuclease III (32) which digests... 

In particular, an exonuclease protection assay has been presented for the detection of thrombin (Wang et al., 2004). Also, in this case ligation has been used in connection with an aptamer but with the addition of a further technique, based on the enzyme exonuclease I. This enzyme can degrade DNA by releasing deoxyri-bonucleoside 5 -monophosphates from the 3 termini of single-stranded DNA chains. The assay is based on the interaction between the DNA aptamer and its specific target, thrombin (Figure 8.12). 

NUCLEOSIDASES AND RELATED ENZYMES - EXONUCLEASES ACTIVE WITH EITHER RIBO- OR DEOXYRIBONUCLEIC ACIDS AND PRODUCING 5 -PHOSPHOMONOESTERS... 

Electroactive labels introduced into DNA also possess electrochemical signals at less extreme potentials than intrinsic DNA responses. An example is electroactive osmium tetraoxide with 2,2 -bipyridine bound to free 3 -ends of the ss regions created by a DNA repair enzyme exonuclease III, which responds to the extent of DNA damage [25]. The technique is capable of detection of one lesion per 10 nucleotides in supercoiled plasmid DNA. DNA-hybridization biosensors were proposed for studies of DNA damage by common toxicants and pollutants where voltammetric transduction was achieved by coupling ferrocene moiety to streptavidin linked to biotinylated target DNA [26]. 

Exonucleases. Like the endonucleases they are restriction enzymes which act at the 3 or 5 ends of linear DNA by hydrolysing off the nucleotides. Although they are highly specific for hydrolysing nucleotides at the 3 or 5 ends of linear DNA, the number of nucleotides cleaved are time dependent and usually have to be estimated from the time allocated for cleavage. Commercially available exonucleases are used without further purification. 

Poly(A) tails are added to the S end of mRNA molecules in a posttranscriptional processing step. The mRNA is first cleaved about 20 nucleotides downstream from an AAUAA recognition sequence. Another enzyme, poly(A) polymerase, adds a poly(A) tail which is subsequently extended to as many as 200 A residues. The poly(A) tail appears to protect the S end of mRNA from S —> S exonuclease attack. The presence or absence of the poly(A) tail does not determine whether a precursor molecule in the nucleus appears in the cytoplasm, because all poly(A)-tailed hnRNA molecules do not contribute to cytoplasmic mRNA, nor do all cytoplasmic mRNA molecules contain poly(A) tails... 

Exonuclease An enzyme that cleaves nucleotides from either the 3 or 5 ends of DNA or RNA. Fingerprinting The use of RFLPs or repeat sequence DNA to establish a unique pattern of DNA fragments for an individual. 

DNA polymerase I is a nonessential enzyme, since viable E. coli mutants lack it (pol A). This conclusion is complicated, however, since the enzyme catalyzes three separate chemical reactions. It polymerizes deoxyribonucleoside triphosphates, and it has two exonucleolytic activities, a 3 to 5 activity and a 5 to 3 activity. The pol A - mutants lack only the polymerization activity. Other mutants lacking both the polymerase and the 5 to 3 exonuclease activity are lethal. Thus the exonuclease function is the more important one. This fits with the role of this enzyme in removing damaged DNA segments (DNA repair) and in removing covalently attached RNA from DNA chains. We will later see that small RNAs serve as primers of DNA synthesis. 

DNA polymerase I has been purified to homogeneity. When the pure enzyme is treated with subtilisin, a proteolytic enzyme from Bacillus subtilis, the polymerase is cleaved into two pieces. The small fragment retains the 5 to 3 nuclease activity, whereas the larger piece, called a Klenow fragment, has both polymerase activity and the 3 to 5 exonuclease activity. The Klenow fragment is sold commercially for use in labeling DNA for use in detecting recombinant DNA. 

Under certain circumstances DNA has both primer and template activities. For example, the addition of mononucleotides is to the 3 end of the growing DNA primer. This presents a problem with regard to how the other strand is synthesized. Biochemists have looked hard but unsuccessfully for an enzyme that can add deoxyribonucleotides onto the 5 end of DNA primers. Such a primer should contain a triphosphate on the hydroxyl group of the 5 end. Although a very active 5 -exonuclease, actually part of DNA polymerase I, has made the search for such an activated 5 end extremely difficult, investigators conclude that a polymerase able to use such a primer probably does not exist. On the contrary, good evidence suggests that the synthesis of both strands is by the known DNA poly-merases. 

Using phosphotriester methods, dinucleoside (3 - 50-monophosphates containing 6-methyl-2,-deoxyuridine at the 3 - or 5 -end have been prepared.44 N.m.r. spectroscopy indicates that this nucleoside possesses the syn conformation in these compounds, and, on treatment with snake venom phosphodiesterase, d(m6UpT) is degraded, while d(Apm6U) is not, indicating that this enzyme, a 3 -exonuclease, requires the anti conformation to be present in the substrate. Two modified nucleo-side-5 -monophosphates, (20) and (21), which are resistant to 5 -nucleotidase, have been isolated from tRNA snake venom hydrolysates.45 A synthesis of (20) has been reported.46... 

Biolabs (Pickering, ON), and Promega (Madison, WI). It is important to note that restriction enzymes producing 3 -overhangs should be avoided if possible (e.g., Psfl, Sfil, Kpnl). The use of such enzymes has been reported to result in the production of additional, nonspecific transcripts (Schenborn and Mierendorf, 1985). If these enzymes must be used, an exonuclease such as DNA Polymerase 1 Large (Klenow) Fragment can be utilized to convert the overhang to a blunt end before the template is transcribed. 

Answer E. The 3 to 5 exonuclease activity of DNA pol 8 represents the proofreading activity of an enzyme required for the replication of human chromosomal DNA. DNA pol y (mitochondrial) and DNA pol III (prokaryotic) do not participate in this process, short RNA primers are replaced with DNA during replication, and new DNA strands are always... 

Comparison between DNA repair and phospholipid repair The processes that can lead to DNA damage and the type of damage are described in Chapter 9 and Appendix 9.6. The repair processes involve removal of the specific nucleotide(s) by an exonuclease and replacement of the nucleotide by a DNA polymerase. Since the strand must be broken to remove the damage (by an endonuclease) these parts of the strand must be repaired by a ligase. The process is known as excision-repair. Of interest, there is a degree of similarity between the removal of damaged polyunsaturated fatty acids from phospholipids in membranes and replacement with a new fatty acid by two enzymes, a deacylase and an acyltransferase (see above and Chapter 11), and excision-repair of DNA. 

Utilising a reversion assay in Salmonella enterica, Prieto et al reported an increased frequency of point mutations following bile-salt exposure. Mutations were predominantly nucleotide substitutions (GC to AT transitions) and -1 frameshift mutations.The frameshifts were dependent on SOS induction and linked to the activity of DinB polymerase (Pol IV). The authors proposed that the GC to AT transitions stimulated by bile, could have arisen from oxidative processes giving rise to oxidised cytosine residues. Consistent with this hypothesis, the authors demonstrated that strains of S. enterica-lacking enzymes required for base-excision repair (endonuclease III and exonuclease IV) and the removal of oxidised bases, demonstrated increased bile-acid sensitivity compared with competent strains. In another study using E. coli, resistance to the DNA-damaging effects of bile was associated with Dam-directed mismatch repair, a pathway also involved with the repair of oxidative DNA lesions. ... 

Any enzyme that catalyzes breakage of a phosphodiester linkage at one or the other end of a polynucleotide chain, resulting in the release of single nucleotides or small oligonucleotides. See specific exonuclease... 

Venom exonuclease [EC 3.1.15.1], also known as venom phosphodiesterase, catalyzes the exonucleolytic cleavage of RNA or DNA (preferring single-stranded substrates) in the 3 to 5 direction to yield 5 -phosphomononucleo-tides. Similar enzymes include hog kidney phosphodiesterase and the Lactobacillus exonuclease. See also specific phosphodiesterase J. A. Gerit (1992) The Enzymes, 3rd ed., 20, 95. 

Fig. 4. The use of arsonomethyl phosphonate as an analogue of diphosphate by RNA polymerase. The enzyme accepts the analogue in the reverse of polymerase action. Since the product hydrolyzes, the overall effect is that of an exonuclease, producing nucleoside 5 -phosphates (59).

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