And RNA-dependent DNA

Steitz . DNA- and RNA-dependent DNA polymerases. Curr Opin Struct Biol 1993 3 31-38. 

Steitz, T. A. (1993). DNA-and RNA-dependent DNA polymerases. Cure. Opin. Struct. Biol. 3, 31-38. 

Zinc is involved in many biochemical functions. Several zinc metal-loenzymes have been recognized in the past decade. Zinc is required for each step of cell cycle in microoragnisms and is essential for DNA synthesis. Thymidine kinase, DNA-dependent RNA polymerase, DNA-polymer-ase from various sources, and RNA-dependent DNA polymerase from viruses have been shown to be zinc-dependent enzymes. Zinc also regulates the activity of RNase, thus the catabolism of RNA appears to be zinc dependent. The effect of zinc on protein synthesis may be attributable to its vital role in nucleic acid metabolism. 

Amino-3 -deoxyadenosine. 3 -Amino-3 -deoxyadenosine (17) is elaborated by Cordyceps militarise Aspergillus nidulanSe and Helminthosporium (3,4). The biosynthesis proceeds direcdy from adenosine. Compound (17) inhibits RNA polymerase, but not DNA polymerase, and replaces the adenosyl residue at the 3 -terminus of tRNA. Phenylalanyl-(3 -amino-3 -deoxyadenosyl)-tRNA has acceptor but not donor activity (31,32). Compound (17) also inhibits retroviral RNA-dependent DNA polymerase (33). 

Studies have demonstrated that one such method is to examine the effects of disinfectants on endogenous RNA-dependent DNA polymerase (i.e. reverse transcriptase) activity. In essence, HIV is an RNA virus after it enters a cell the RNA is converted to DNA under the influence of reverse transcriptase. The virus induces a cytopathic effect on T lymphocytes, and in the assay reverse transcriptase activity is determined after exposure to different concentrations of various disinfectants. However, it has been suggested that monitoring residual viral reverse transcriptase activity is not a satisfactory alternative to tests whereby infectious HIV can be detected in systems employing fresh human peripheral blood mononuclear cells. 

In contrast to the effects obtained with viruses mentioned earlier, rous sarcoma virus (RSV) is inactivated by direct contact with 2 [81]. Evidence for the drug action by a chelate compound was obtained by using concentrations of 3a and copper(II) sulfate, neither of which individually affected enzyme activity or transforming abilities [82]. In a later study these workers showed that several metal complexes inhibit the RNA dependent DNA polymerases and the transforming ability of RSV, the most active compound being a 1 1 copper(II)... 

After the virus has attached to CD4 and chemokine receptors, another viral glycoprotein (gp41) assists with viral fusion to the cell and internalization of the viral contents. The viral contents include single-stranded RNA, an RNA-dependent DNA polymerase (also known as reverse transcriptase), and other enzymes. Using the single-stranded viral RNA as a template, reverse transcriptase synthesizes a complementary strand of DNA. The single-stranded viral RNA is removed from the newly formed DNA strand by ribonuclease H, and reverse transcriptase completes the synthesis of double-stranded DNA. The... 

Enzymes in viruses We have stated that virus particles do not carry out metabolic processes. Outside of a host cell, a virus particle is metabolically inert. However, some viruses do contain enzymes which play roles in the infectious process. For instance, many viruses contain their own nucleic acid polymerases which transcribe the viral nucleic acid into messenger RNA once the infection process has begun. The retroviruses are RNA viruses which replicate inside the cell as DNA intermediates. These viruses possess an enzyme, an RNA-dependent DNA popo called reverse transcriptase, which transcribes the information in the incoming RNA into a DNA intermediate. It should be noted that reverse transcriptase is unique to the retroviruses and is not found in any other viruses or in cells. 

DNA has two broad functions replication and expression. First, DNA must be able to replicate itself so that the information coded into its primary structure is transmitted faithfully to progeny cells. Second, this information must be expressed in some useful way. The method for this expression is through RNA intermediaries, which in turn act as templates for the synthesis of every protein in the body. The relationships of DNA to RNA and to protein are often expressed in a graphic syllogism called the central dogma. The concept was proposed by Crick in 1958 and was revised in 1970 to accommodate the discovery of the RNA-dependent DNA polymerase. Crick s original theory suggested that the flow of information was always from RNA to protein and could not be reversed, yet it allowed for the possibility of DNA synthesis from RNA. 

Both dogmas had to be revised or expanded, the first because of the discovery of reverse transcriptase (an RNA-dependent DNA-polymerase) by D. Baltimore (MIT) and H. M. Temin (University of Wisconsin). This is an enzyme which catalyses the synthesis of DNA from single-strand RNA ... 

Reverse transcriptase is an RNA-dependent DNA polymerase that requires an RNA template to direct the synthesis of new DNA. Retroviruses, most notably HIV, use this enzyme to repHcate their RNA genomes. DNA synthesis by reverse transcriptase in retroviruses can be inhibited by AZT. ddC, and ddl. 

The reverse transcriptase enzyme (RT) is the primary enzyme responsible for the conversion of the viral single-strand RNA to the double-strand DNA. The reverse transcriptase enzyme is a component of the virion and is encoded by the pol gene. The RT is manufactured in the HIV-infected cells as a gag-pol fusion polyprotein. The RT is not the only enzyme necessary for the translation of RNA to DNA. The other enzymes for this conversion include RNA-dependent DNA polymerase, DNA-dependent DNA polymerase, and RNase H (Gilboa and Mitra, 1978 Prasad and Gogg, 1990). The reverse transcriptase enzyme has a high error rate (1 in 2000 bases), which produces higher incidents of mutation. Some of these mutations make the virus resistant to NNRTI treatment. 

Viruses are obligate intracellular organisms as their replication is based on DNA and RNA dependent processes and protein synthesis of the host. Antiviral therapy can therefore not be as selective as antibacterial treatments and anti-viral agents tend to inhibit host cell function and can cause major toxicity. An other problem with antiviral therapy is the fact that active viral replication mostly takes place before symptoms become manifest. Our armamentarium against most viral infections is limited. 

The retroviral genomic RNA serves as the template for synthesis of a double-stranded DNA copy, the provirus (Figure 49-4). Synthesis of the provirus is mediated by a virus-encoded RNA-dependent DNA polymerase, or reverse transcriptase. The provirus is translocated to the nucleus and is integrated into host DNA. Transcription of this... 

Although the existence of this enzyme may not be surprising, the mechanism by which it acts is remarkable and unprecedented. Telomerase, like some other enzymes described in this chapter, contains both RNA and protein components. The RNA component is about 150 nucleotides long and contains about 1.5 copies of the appropriate CyKx telomere repeat. This region of the RNA acts as a template for synthesis of the T -G strand of the telomere. Telomerase thereby acts as a cellular reverse transcriptase that provides the active site for RNA-dependent DNA synthesis. Unlike retroviral reverse transcriptases, telomerase copies only a small segment of RNA that it carries within itself. Telomere synthesis requires the 3 end of a chromosome as primer and proceeds in the usual 5 —>3 direction. Having syn-... 

The process of eukaryotic DNA replication closely follows that of prokaryotic DNA synthesis. Some differences, such as the multiple origins of replication in eukaryotic cells versus single origins of replication in prokaryotes, have already been discussed. Eukaryotic single-stranded DNA-binding proteins and ATP-dependent DNA helicases have been identified, whose functions are analogous to those of the prokaryotic enzymes previously discussed. In contrast, RNA primers are removed by RNase H. 

Fig. 26-4A) synthesized DNA normally. This finding stimulated an intensive search for new polymerases. Two were found DNA polymerases II (gene pol B)264 and III. Both are present in amounts less than 25% of that of DNA polymerase I.265 266 Both have properties similar to those of polymerase I, but there are important differences. By now DNA polymerases have been isolated from many organisms, many genes have been cloned and many sequences, both of bacterial and eukaryotic polymerases are known. Comparisons of both sequences and three-dimensional structures,266a/b a few of which are shown in Fig. 27-12, suggest that the polymerases belong to at least six families (Table 27-1). These include the RNA-dependent DNA polymerases known as reverse transcriptases as well as some RNA polymerases.267 2681... 

Illimaquinone, such as avarone and avarol isolated from a Red Sea sponge, i.e., Smenospongia, has been reported to inhibit the RNase H activity associated with the HIV-1 reverse transcriptase at a concentration of 5 to 10 pg/rnl, whereas it was not active against the RNA-dependent DNA polymerase (RDDP) and DNA-dependent DNA polymerase (DDDP) activities of the enzyme at a concentration of 50 pg/rnl. 

Pretazettine (395) has been the subject of numerous biological studies, and it has been shown to exhibit a number of interesting activities (96,97,101,178-187). For example, 395 was found to inhibit HeLa cell growth as well as protein synthesis in eukaryotic cells by interfering with the peptide bond formation step (97,101). Furthermore, pretazettine inhibited the purified RNA-dependent DNA polymerase (reverse transcriptase) from avian myeloblastosis virus, a typical C-type virus (178), in an unusual fashion since it physically combined with the polymerase enzyme itself rather than interacted with the nucleic acid template. Pretazettine also exhibited antiviral activity against the Rauscher leukemia virus in mouse embryo cell cultures by suppressing viral replication (179). 

Reverse transcription (which occurs in both prokaryotes and eukaryotes) is the synthesis of DNA from an RNA template. A class of RNA viruses, called retroviruses, are characterized by the presence of an RNA-dependent DNA polymerase (reverse transcriptase). The vims that causes AIDS, Human Immunodeficiency Virus (HIV), is a retro-vims. Because nuclear cell division doesn t use reverse transcriptase, the most effective anti-HIV drugs target reverse transcriptase, either its synthesis or its activity. Telomerase, discussed in the previous section, is a specialized reverse transcriptase enzyme. See Figure 12-5. 

As has already been mentioned, some lipophilic rifamycins and some strepto-varicins and geldanamycins affect the growth of cells transformed by RNA tumour viruses or the RNA-dependent DNA polymerase (reverse transcriptase) characteristic of these viruses. Again, high drug concentrations are needed to produce an effect and only partial, but never absolute, selectivity of enzyme inhibition has been found. 

Conditions for optimal recoveries of poly(A) containing RNA, with minimal contamination from rRNA, were investigated. The poly(A) fractions isolated were effective as an RNA template for the synthesis of complementary DNA with the RNA-dependent DNA polymerase of avian myeloblastosis virus. Poly(dT)-cellulose has also been used both in the purification of a 14 S messenger RNA for the immunoglobulin light chain from microsomes of MOPC 41 mouse myeloma that appeared to code for a precursor protein [117], and in the purification of RNA-dependent DNA polymerase from RNA tumour virus [118]. An example of the use of oligo(dT)-cellulose is provided by the purification of a viral specific RNA from sarcoma virus-transformed nonproducer cells [119]. 

Bioassay-guided fractionation of an aqueous extract from a Philippine Islands collection of the soft coral Lohophytum spp. yielded cembranoid diterpenes, Fig. (3), which exhibited moderate HIV-inhibitory activity in a cell-based in vitro anti-HIV assay [44], while new isomalabaricane triterpenes. Fig. (4), have been isolated from the sponge Stelletta spp. [45]. Other anti-HIV diterpenes also included the dolabellane diterpenes isolated from the Brazilian brown algae Dictyota pfaffi [46] and Dictyota menstrualis [47]. To investigate the effect of these diterpenes in the reverse transcription of the viral genomic RNA, the recombinant HIV-1 RT was assayed in vitro in the presence of each compound. All compounds inhibited the RNA-dependent DNA-polymerase activity of HIV-1 RT and consequently virus replication. 

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