DNA polymerase reaction

FIGURE 12.3 The chain termination or dideoxy method of DNA sequencing, (a) DNA polymerase reaction, (b) Structure of dideoxynucleotide. (c) Four reaction mixtures with nucleoside triphosphates plus one dideoxynucleoside triphosphate, (d) Electro-phoretogram. Note that the nucleotide sequence as read from the bottom to the top of the gel is the order of nucleotide addition carried out by DNA polymerase. 

Double-stranded products were purified using centrifugation dialysis following the protocol described by Allard et al.31 Purified double-stranded products were subsequently dried under vacuum and resuspended in 15 /A of IX Tris/EDTA buffer (TE). To obtain single-stranded DNA, we used 2 n 1 of the purified double-stranded PCR product as the template. The concentrations of Taq DNA polymerase, reaction buffer, dNTPs, and... 

The interaction of tilorone hydrochloride to DNA encouraged us to study the template activity of the complexes in DNA- and RNA- polymerase systems from E. coli. Both the activities were found to be inhibited by tilorone the DNA polymerase reaction being more sensitive towards tilorone. The inhibition of the RNA-polymerase reaction by tilorone was dependent on the A-T content of DNA-template. The template activity of poly (dA-dT) in the RNA polymerase reaction is distinctly more sensitive towards tilorone than that of DNA particularyly, at low drug concentrations the poly (dA-dT)-catalyzed reaction was three times more sensitive than the DNA-catalyzed activity of RNA polymerase reaction60. ... 

We have conducted some model studies to analyze the products of the FLV-DNA-polymerase reaction under the influence of tilorone. The procedure we adopted was based on a recent report by Kotler and Becker64 on distamycin A, which has been shown by us to react with ss-DNA and ds-DNA65,66. ... 

The product analysis of the DNA-polymerase reaction (FLV) in the absence and in the presence of tilorone (1 x 10-4 M) is depicted in Fig. 10. The products of the viral DNA-polymerase reaction were, under these conditions, eluted in three species. The first species to be eluted from the column contained ss-DNA, the second contained the RNA-DNA hybrid-molecules (hy-DNA) and finally, the ds-DNA, eluted in the last species. Analysis of products synthesized in the presence of tilorone showed that the ss-DNA and the hybrid species, but not the ds-DNA species were synthesized. This indicates that tilorone has a low affinity to viral RNA, but can block the synthesis of ds-DNA by interacting with ss-DNA or hy-DNA. 

Biotin can be chemically or biochemically incorporated into DNA. Phos-phoramidite derivatives of biotin are used in the chemical synthesis of DNA to add biotin to the 5 end, or internally (Fig. 3e). Biotin-dUTP (Fig. 3f) is used in DNA polymerase reactions to add biotin to DNA enzymatically. 

Figure 1 Chemical mechanism of DNA polymerase and 3 -5 exonuclease, (a) DNA polymerase reaction. The enzyme chelates two metal Ions using three aspartic acid residues (only two are shown). Metal ion A abstracts the 3 hydroxyl proton of the primer terminus to generate a nucleophile that attacks the a-phosphate of an incoming dNTP substrate. The phosphoryl transfer results In production of a pyrophosphate leaving group, which is stabilized by metal Ion B. (b) The 3 -5 exonuclease proofreading activity is located in a site that is distinct from the polymerase site yet it uses two-metal-ion chemistry similar to DNA synthesis. The reaction type is hydrolysis in which metal ion A activates water to form the hydroxy anion nucleophile. Nucleophile attack on the phosphate of the mismatched nucleotide releases it as dNMP (dGMP in the case shown).

Figure 27.12. DNA Polymerase Mechanism. Two metal ions (typically, Mg2+) participate in the DNA polymerase reaction. One metal ion coordinates the 3 -hydroxyl group of the primer, whereas the phosphate group of the nucleoside triphosphate bridges between the two metal ions. The hydroxyl group of the primer attacks the phosphate group to form a new 0-P bond.

The endogenic reaction is catalyzed by the viral RNA (70s), template for the reverse transcriptase. It is not clear whether the higher activity of distamycin/Gly in this system, compared to the template activity of DNA in bacterial system (Table 10), is due to its higher affinity for viral RNA. This was investigated by using various exogenous templates in the DNA-polymerase reaction of FL-virions. 

Synthetic polymers containing either deoxyribonucleotide or ribonucleotide strands can be used as templates by the DNA polymerases of RNA tumor viruses. Table 12 shows the activity of distamycin/A and its amino acid derivatives on the DNA polymerase reaction of FL-virions, catalyzed by poly rA ... 

Table 1 3. Inhibition of DNA-dependent DNA polymerase reaction (E. coli B) by distamycin A and its structural analogues...

The activity of these derivatives on the DNA-polymerase reaction of bacterial cells, catalyzed by denatured DNA is shown in Table 13. These studies are important to compare the sensitivities of viral and bacterial DNA polymerases towards these antibiotics. As follows from results the DNA polymerase reaction of bacterial cells is highly sensitive to distamycin/A and distamycin/Gly. The molar concentration of the antibiotics used in this reaction (1 x 10 4M) is slightly higher, compared to the DNA-dependent RNA-polymerase reaction (8 x 10-5M) however, the inhibitory effect in the former reaction is much more pronounced. 

Table 17 shows how template-dependent DNA polymerase activity of FLV is inhibited by various daunomycin derivatives. The reactions catalyzed by poly-(dA-dT) and poly (rA) (dT)i2 are highly sensitive to the action of daunomycin and its derivatives. Here again, daunomycin and adriamycin are most effective, and the N-acetyl derivative is completely inactive. It is interesting that the poly (dA-dT)-and poly (rA) (dT)i2-dependent reactions are more sensitive to these antibiotics than the endogenous reaction (see Table 17), while the DNA polymerase reaction catalyzed by poly (dl-dC) is completely insensitive. The most active derivatives (daunomycin, adriamycin and dihydro daunomycin) slightly stimulate 3H-dGMP incorporation catalyzed by poly (dl-dC). This stimulation is particularly noticeable in the case of dihydro daunomycin. Surprisingly, the N-acetyl derivative was found to inhibit this reaction. The mechanism of this inhibition is not understood. 

Table 19. Inhibition of DNA-dependent DNA polymerase reaction (E. coli B)by tilorone hydrochloride. The calf thymus DNA primed assay system contained (total vol 0.3 ml) 0.07 M glycine buffer, pH 9.2,7 mM MgClj, 1 mM (3-mercaptoethanol, 10 m/Umoles each of dTTP, dCTP and dGTP, 2 flCi of 3H-dATP, 20 jig of denatured calf thymus DNA. The reaction was started by adding 0.02 ml (approx. 50 ftg protein) of the enzyme preparation...

The inhibiting activity of tilorone hydrochloride on the DNA-dependent RNA-polymerase reaction is shown in Table 20. Compared to the DNA-poly-merase reaction, the RNA-polymerase reaction requires large amounts of tilorone hydrochloride for its inhibition no significant inhibition was observed below 15 pg/re act ion mixture of tilorone hydrochloride. Whereas this amount of tilorone was able to completely inhibit the DNA polymerase reaction (see Table 19). One explanation is that in the RNA-polymerase reaction the DNA concentration is approx. 2.5 times more than that used in the DNA-polymerase reaction. However, this may not be the only reason for such differences. Our spectrophotometric data show that Mg2+ions influence the tilorone binding to DNA. Since the Mg2+ion concentrations in both systems are different, this may account for the variable sensitivity of both systems towards tilorone hydrochloride. 

Synthetic polymers containing either desoxyribonucleotide or ribonucleotide strands are known to stimulate the in vitro DNA synthesis by RNA tumor viruses. Some inhibitors of the DNA-polymerase reaction in RNA tumor viruses are known to exhibit a template-primer specificity6, 7 34 Table 22 shows the inhibition of template-dependent DNA polymerase activity of FLV by DEAE-F at various concentrations. The reaction catalyzed by poly (dl - dC) is most strongly stimulated by DEAE-F. Thus at 20 pg/reaction mixt. of DEAE-F the incorporation of 3H—dGMP is almost 4 times that of control. 

Thus, the binding constant of KF to the template was 100 times larger than that to the produced flat end. This is the first example of investigating both kinetically and quantitatively the binding, catalysis, and release processes of DNA polymerase reactions in situ on the same device. 

Mature mRNA transcripts (sense strand) from eukaryotic cells can be purified and then reverse transcribed, with the assistance of a reverse transcriptase enzyme (from Moloney murine leukemia virus, MMLV), into complementary DNAs (cDNAs) that will anneal with the mRNA transcripts by Watson-Crick base pairing to give anti-parallel DNA/RNA duplexes or double helices. The poly(A) tail in each mature mRNA transcript is actually a usefiil handle for each reverse transcriptase reaction. Thereafter, DNA/RNA duplexes must be broken down with the assistance ofRNAse enzymes (specific for the hydrolysis of RNA phospho diester links) and a sense strand of DNA constructed instead on each cDNA single strand so that equivalent, more stable antiparallel DNA/DNA duplexes are generated instead, with the assistance of a DNA polymerase enzyme. In this instance, the poly(T) tail in each cDNA molecule turns out to be important for the DNA polymerase reaction ... 

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