DNA-dependent RNA synthesis

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-... 

Newly synthesized rDNA in the oocytes of Xenopus is associated with a complex which is at least partly resistant to RNase activity and which contain an RNA molecule close to the molecular weight of the transcript of one repetitive unit (Mahdavi and Crippa, 1972 Brown and Tocchini-Valentini, 1972). Brown and Tocchini-Valentini (1972) have reported a DNA polymerase from Xenopus ovaries which, in the presence of all four deoxynucleotide triphosphates, may act on RNA from the dissociated complex. It is, therefore, very tempting to suggest that amplification through RNA-dependent DNA synthesis allows the cell to use transcription controls for DNA base sequence amplification processes. 

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 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... 

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. 

DNA synthesis in eukaryotes is more complex than in prokaryotes. Eukaryotes require thousands of origins of replication to complete replication in a timely fashion. A special RNA-dependent DNA polymerase called telomerase is responsible for the replication of the ends of linear chromosomes. 

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]. 

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. 

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. 

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). 

The answer is b. (Murray, pp 412—434. Scriver, pp 3-45. Sack, pp 3—29. Wilson, pp 99-121.) A special DNA polymerase called telomerase is responsible for replication of the telomeric DNA. Telomerase contains an RNA molecule that guides the synthesis of complementary DNA. Telomerase is therefore an RNA-dependent DNA polymerase in a category with reverse transcriptase. Telomerase does not require an RNA primer, initiating synthesis of the leading strands at 3 ends within the telomeric DNA. Synthesis of the lagging strands uses primase, DNA polymerase III, and DNA polymerase I, as with the replication of other chromosomal regions. 

After internalization, the virus is uncoated in preparation for replication. The genetic material of HIV is positive-sense singlestrand RNA (ssRNA) the virus must transcribe this RNA into DNA to optimally replicate in human cells (transcription normally occurs from DNA to RNA—HIV works backward, hence the name retrovirus). To do so, HIV is equipped with a unique enzyme, RNA-dependent DNA polymerase (reverse transcriptase). Reverse transcriptase first synthesizes a complementary strand of DNA using the viral RNA as a template. The RNA portion of this DNA-RNA hybrid is then partially removed by ribonuclease H (RNase H), allowing reverse transcriptase to complete the synthesis of a double-stranded DNA (dsDNA) molecule. Unfortunately, the fidelity of reverse transcriptase is poor. 

Reverse transcription PCR (RT-PCR) is a modification of the standard PCR technique that can be used to amplify mRNA. The first step is to convert isolated mRNA to a complementary DNA (cDNA) molecule using an RNA-dependent DNA polymerase (also known as reverse transcriptase) during a process called reverse transcription (RT). The complementary DNA can be used as any other DNA molecule for PCR amplification. The primers used for cDNA synthesis can be either nonsequence-specific primers (a mixture of random hexa-mers or oligo-dT primers) or sequence-specific primers (Fig. 2.4). Random hexamers are a mixture of all possible combinations of six nucleotide sequences that can attach randomly to mRNA and initiate reverse transcription of the entire RNA pool. Oligo-dT primers are complementary to the poly-A tail of mRNA molecules and allow synthesis of cDNA only from mRNA molecules. Sequence-specific primers are the most restricted because they are designed to bind selectively to mRNA molecules of interest, which makes reverse transcription a target-specific process. 

Retroviruses are RNA-DNA viruses that require host-cell genome integration to complete their replication cycle. The retroviral enzyme reverse transcriptase is an RNA-dependent DNA polymerase that has important uses in recombinant DNA methodologies such as the synthesis of complementary DNA (cDNA). The human immunodeficiency virus (HIV) is the etiologic agent that causes acquired immunodeficiency syndrome (AIDS) in humans. HIV infects and destroys human T cells, which are required for immune system functions. 

AMV Reverse transcriptase isolated from avian myeloblastosis virus. This enzyme catalyzes the polymerization of nucleotides and characterized by RNA-dependent DNA polymerase, DNA-dependent DNA polymerase activity besides the RNase H activity but lacks the 3 -5 exonuclease activity. The RNase H activity can cause the degradation of the RNA strand of an RNA DNA duplex, which is a disadvantage that can limit the complete synthesis of the total cDNA. The AMV Reverse Transcriptase is suitable for reverse transcription of fragments containing secondary structure due to its high optimum temperature (42 °C) for its activity. 

M-MulV Reverse transcriptase isolated from moloney murine leukemia virus. This enzyme is also characterized by RNA-dependent DNA polymerase, DNA-dependent DNA polymerase and weak RN ase H activities but lacks the 3 -5 exonuclease activity. M -MulV reverse transcriptase is able to synthesize full-length cDNA fragments from longmRNA.Thetemperaturefortheoptimalenzymeactivity is 37 °C. Becauseofthe weak RNase H activities of the M-MulV reverse transcriptase, it is more suitable for the synthesis of long cDNA fragments. 

Narciclasine (22) halts protein synthesis by blockage of peptide-bond formation on the 60-S ribosomal subunit. The effect is specific for eucaryotic cells. Narciclasine inhibits Rauscher virus NIH/3T3 (Wink, 1993). Lycorine (8) blocks mitosis in the broad bean (Viciafaba). The mechanism appears to be related to inhibition of protein synthesis (Suffness and Cordell, 1985). Pretazettine (15) has been used in combination with DNA-binding and alkylating agents in the treatment of the Rauscher leukemia virus (Cordell, 1981 Martin, 1987). This alkaloid inhibits purified RNA-dependent DNA polymerase (reverse transcriptase) from avian myelo-... 

---