RNA-Directed DNA Polymerases Reverse Transcriptases

Mechanism of Action Apurine nucleoside analog that is intracellularly converted into a triphosphate, which interferes with RNA-directed DNA polymerase (reverse transcriptase). Therapeutic Effect Inhibits replication of retroviruses, including HIV. Pharmacokinetics Variably absorbed from the GI tract. Protein binding less than 5%. Rapidly metabolized intracellularly to active form. Primarily excreted in urine. Partially (20%) removed by hemodialysis. Half-life 1.5 hr metabolite 8-24 hr. 

In 1994, six novel drimane-type sesquiterpenoids, mniopetals A-F (1-6) (Fig. (1)), were isolated by Anke and Steglich from the fermentation broth of Canadian Mniopetalum sp. 87256 [1, 2]. These natural products are known to inhibit the enzymatic activity of RNA-directed DNA-polymerases (reverse transcriptases) of the human immunodeficiency virus (HIV)-l, avian myeloblastosis virus (AMV), and moloney murine leukemia virus (MMuLV) as shown in Table 1. In addition, mniopetals exhibit moderate antimicrobial and cytotoxic activities. In the following year, a structurally and biologically similar natural product kuehnero-... 

Retroviruses are eukaryotic RNA-viruses, such as certain tumor viruses and human immunodeficiency virus (HIV) containing an RNA-directed DNA polymerase (reverse transcriptase, RT). Viral reverse transcriptase is a multifunctional enzyme possessing three enzymatic activities, which catalyze reactions essential to viral replication (Skalka and Goff, 1993) ... 

The reverse transcriptase from the avian myeloblastosis virus (AMV RTase) is a prototype of avian retroviral RTases. The enzyme is a dimer (155 kDa) composed of nonidentical subunits a and /3. AMV RTase has multiple enzymatic activities including RNA-directed DNA polymerase (reverse transcriptase), DNA-directed DNA polymerase, RNase H, and DNA endonuclease. AMV RTase does not have 3 5 - or 5 3 -exonuclease activity. AMV RTase is a key reagent... 

Many of the rifaldehyde derivatives have also been screened for inhibition of the RNA-directed DNA polymerases (reverse transcriptases) of murine sarcoma virus (MSV) and Rauscher murine leukemia virus (RLV) and the purified DNA polymerases from human normal and leukemic lymphoblasts (40,163). Surprisingly perhaps, the best compounds shown in Fig. 19 (48, 49, rifampicin) were inactive. Further testing of derivatives of rifaldehyde indicated tl at a longer, bulkier side chain is needed at C-25 for inhibition of viral polymerases. 

Because DNA polymerases extend their growing polynucleotide chains only in the 5 —> 3 direction, and because the two strands of the parental DNA duplex are antiparallel, removal of the RNA primer that is paired with the 3 end of the parental template DNA would leave an overhanging 3 DNA strand with no means of having its complement synthesized. Ordinary DNA polymerases are unable to initiate DNA chains de novo. Each round of replication would consequently shorten the DNA because a portion could not be copied. To circumvent this problem, human DNA chromosomes have a segment of repeating G-rich DNA (telomeres) at their ends. In addition, a special enzyme, telomerase, which is an RNA-dependent DNA polymerase (reverse transcriptase) that carries its own RNA template, can, in effect, extend the uncompleted end at each round of replication. The RNA template renews the repeating telomere sequence so that the DNA is not shortened (text, pp.765-766). 

DNA-directed DNA polymerases [EC 2.7.7.7], also called DNA nucleotidyltransferases (DNA-directed), are enzymes that catalyze the DNA template-directed extension of the 3 -end of a nucleic acid strand one nucleotide at a time. Thus, n deoxynucleoside triphosphates produce n pyrophosphate (or, diphosphate) ions and DNA . This enzyme cannot initiate the synthesis of a polymeric chain de novo it requires a primer which may be DNA or RNA. RNA-directed DNA polymerases [EC 2.7.7.49], also referred to as reverse transcriptases, DNA nucleotidyltransferases (RNA-directed), and revertases, are enzymes that catalyze the RNA template-directed extension of the 3 -end of a nucleic acid strand one nucleotide at a time. Thus, n deoxynucleoside triphosphates produce n pyrophosphate (or, diphosphate) ions and DNA . As was the case above, this enzyme cannot initiate the synthesis of a polymeric chain de novo it requires a primer which may be DNA or RNA. 

Complementary DNAs (cDNA) are DNA copies of mRNAs. Reverse transcriptase is the RNA-directed DNA polymerase that synthesizes DNA strand, using purified mRNA as the template. DNA polymerase is then used to copy the DNA strand forming a double-stranded cDNA, which is cloned into a suitable vector. Once a cDNA derived from a particular gene has been identified, the cDNA becomes an effective probe for screening genomic libraries (Cowell and Austin, 1997). Annotated human cDNA sequences can be accessed from HUNT at http // www.hri.co.jp/HUNT (Yudate, 2001). 

The answer is e. (Murray, pp 412—434. Scriver, pp 3-45. Sack, pp 3-29. Wilson, pp 99-121.) The AIDS treatment drug azidothymidine (AZT) exerts its effect by inhibiting viral reverse transcriptase. Thus, it prevents replication of the human immunodeficiency virus. Reverse transcriptase is an RNA-directed DNA polymerase. The RNA of retroviruses utilizes reverse transcriptase to synthesize DNA provirus, which in turn synthesizes new viral RNA. AZT inhibits DNA provirus production, but does not directly inhibit synthesis of new viral RNA. 

It is mentioned also that the presence of the enzyme revCTse transcriptase may serve to furnish a marker. (This enzyme is otherwise known as RNA-directed DNA polymerase, and is the enzyme distinguishing retroviruses.) The conversion of isolated RNA to cDNA, is involved followed by amplification, a process designated as reverse transcriptase-PCR, or simply RT-PCR. 

Some of quercetin s potentially useful vs. potentially harmful properties have been reviewed by Alan Gaby, MD., in an editorial in the May 1998 issue of the Townsend Letter for Doctors Patients. It acts as an inhibitor for the enzyme phosphodiesterase, and hence may possibly act against asthma. Also, it may act against allergies by inhibiting the release of histamine. Quercetin is also an inhibitor for the enzyme aldose reductase, which is involved in diabetic complications. Perhaps most interestingly, it inhibits the enzyme reverse transcriptase (or RNA-directed DNA polymerase), involved in the action of retroviruses such as the AIDS virus or HIV (and retroviruses may also be involved in cancer formation). 

More specifically, it may be commented in passing that an enzyme involved in the formation of cancerous cells via viruses is DNA-dependent RNA polymerase, also called DNA-directed RNA polymerase (Hoffman, 1999, p. 189). On the other hand, what are called retroviruses have notably been implicated in cancer and immunodeficiency diseases. The enzyme involved here is called reverse transcriptase, or RNA-dependent DNA polymerase (or RNA-directed DNA polymerase). 

Enzymatic reactions commonly used to synthesize double-stranded cDNA from mRNA and oligonucleotide primers such as oligo d(T). The enzyme reverse transcriptase is an RNA-directed DNA polymerase first discovered in eukaryotic retroviruses. The conversion of RNA to DNA in step 2 is accomplished by combining the endonucleolytic activity of E. coli RNase H to create nicks in the RNA-DNA hybrid with the DNA polymerizing activity of E. coli DNA pol I, which uses the residual RNA as a primer and the first-strand cDNA as a template. E. coli DNA... 

In the early 1970s, Howard Temin and David Baltimore independently discovered the retroviral enzyme reverse transcriptase, an RNA-directed DNA polymerase. This discovery not only proved once again that all rules in biology have exceptions (it was thought up to that point that genome information could only flow from DNA to RNA), but it also provided a... 

Reverse transcriptase is an RNA-directed DNA polymerase common in retroviruses. The HIV retrovirus that causes AIDS contains a reverse transcriptase. Like other RNA-dependent polymerases, reverse transcriptase is very error prone because it contains no proofreading activity. The replication scheme of retroviral RNA is as follows ... 

Relate the catalytic activity of reverse transcriptase (an RNA-directed DNA polymerase) to the replication of retroviruses. Provide an overvievc of retroviral replication. 

Often, RNA is available for analysis, rather than DNA. This is the case in cultures of some viruses e.g. the human immunodeficiency vims (HIV), which only contain RNA. PCR, however, is not capable of using RNA as a template and, therefore, amplification does not take place. In order to analyse genetic material of such species, PCR is combined with another enzymatic reaction called reverse transcription (RT). In RT, the enzyme RNA-directed DNA polymerase also known as reverse transcriptase, is used. This enzyme synthesises, or more accurately transcribes, mRNA to its complementary strand of DNA (cDNA). 

This chapter focuses on a few selected DNA polymerases that are most widely used in recombinant DNA technology. In the category of DNA-directed DNA polymerases (Section II), four DNA polymerases are described that originate from bacteria . coli and T. aquaticus) and bacteriophages (T4 and T7). In the category of RNA-directed DNA polymerases (Section III), two reverse transcriptases, one from AMV and the other from MoLV, are described. As the typical enzyme of template-independent DNA polymerases (Section IV), the terminal deoxynucleoti-dyltransferase (TDTase) from calf thymus is described. 

Retroviral RTases are multifunctional enzymes that exhibit at least three distinct enzymatic activities (i) RNA-directed DNA polymerase, (ii) DNA-directed DNA polymerase in the conversion of ssDNA to dsDNA, and (iii) RNase H that selectively removes the RNA moiety from a RNA DNA heteroduplex. Reverse transcriptases lack both 3 5 - and 5 —> 3 -exonuclease activities, and exhibit relatively high frequencies of nucleotide misincorporation. The RNA- and DNA-dependent polymerase activities are physically inseparable. Reverse transcriptases from avian retroviruses, but not from mammalian retroviruses, exhibit a fourth activity, DNA endonuclease, that is essential for the integration of the dsDNA intermediate into the host chromosome and for the productive infection of the retrovirus. 

In order to replicate the RNA viral genome, the viral RNA can act directly as the mRNA and be translated to form viral proteins. Alternatively, in the case of retrovimses, which are unique in that their genomes are transcribed into DNA from RNA by an RNA-directed DNA polymerase or reverse transcriptase, so called because information is going from RNA into DNA, which is the opposite to the conventional process of transcription (DNA into mRNA). Once transcribed into DNA, it is integrated into the cellular DNA by the viral integrase enzyme. Transcription of the viral genome by the cellular RNA polymerases yields the viral molecules that end up in virions. 

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