And RNA-dependent DNA polymerases

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... 

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. 

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... 

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. 

The discovery of reverse transcriptase in oncogenic RNA viruses1, 2 and human leukemic cells3 opens a new horizon for the study of the role of viruses in cancer. It may eventually enable us to design useful drugs for the selective chemotherapy of cancer. The RNA-dependent DNA polymerase, or reverse transcriptase, of virions is responsible for the synthesis of DNA chains on the RNA template, which give rise to a hybrid molecule (RNA—DNA). These chains are released from the RNA template as single-stranded DNA molecules and serve as the template for the synthesis of double-stranded DNA (Fig. 1). 

The inhibitory activity of daunomycin and its structural analogues on viral oncogenesis by FLV and RSV, and on in vitro transformation by MSV (M) suggests that it is the activity of the virus-associated enzymes which is sensitive to these antibiotics. The RNA-dependent DNA polymerase of the virions is responsible for the synthesis of viral DNA. Table 16 shows how the reverse-transcriptase activity of MSV (M), FLV and RSV is inhibited by various daunomycin derivatives. 

An unusual feature of the retrovirus family, of which HIV is a member, is the RT enzyme. This enzyme is essential for replication and has the capacity to generate DNA from RNA. The RNA-dependent DNA polymerase function of the RT provides at least one target for drug discovery. Nucleoside analogs once again proved to be effective inhibitors. Wellcome s experience in the nucleo-... 

The DHBV pol gene product is produced by in vitro transcription of a cDNA copy of the DHBV genome and subsequent translation of the RNA using an in vitro rabbit reticulocyte translation system. Aliquots of the translated polymerase are monitored, without further purification, for RNA-dependent DNA polymerase activity. The activity is monitored by measuring the incorporation of [35S]-labeled deoxynucleotide triphosphate (dNTP) into nascent DNA. 

Hepadnaviral Pol proteins are multifunctional and contain four domains, namely (in order from amino to carboxy terminus) terminal protein, spacer, polymerase, and RNaseH, respectively. Known functions include (1) acting as a primer for first (-) strand DNA synthesis (2) synthesizing first-strand DNA from RNA pregenome (reverse transcriptase or RNA-dependent DNA polymerase [RDDP] activity) (3) degrading viral RNA in resulting RNA-DNA hybrids (RNase H activity) and (4) copying second- (+) strand DNA from the (-) strand DNA template (DNA-dependent DNA polymerase [DDDP] activity). In addition, multiple Pol domains appear to be required for proper assembly of the pregenomic RNA molecule and associated Pol into cytoplasmic core particles (I). 

All replication-competent retroviruses possess a characteristic enzyme, reverse transcriptase (RT), which is present at 20-70 mol/virus particle (1-3). The enzyme is cleaved, and thereby activated, from an inactive precursor by the action of another retroviral enzyme, the viral protease. All RTs possess three distinct enzymatic activities (1) an RNA-dependent DNA polymerase, which is the RT in the strict sense of the word, (2) an RNase H, and (3) a DNA-dependent DNA polymerase. After infection of a new host cell, these different activities serve in turn to synthesize a cDNA of the viral RNA, to degrade RNA from the cDNA-RNA heteroduplex, and to duplicate the cDNA strand (reviewed in ref. 4). 

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