Nucleic acids polymerase enzymes

The nonnucleoside reverse transcriptase inhibitors (NNRTIs), used in the treatment of AIDS, provide interesting examples of clinically relevant noncompetitive inhibitors. The causative agent of AIDS, HIV, belongs to a virus family that relies on an RNA-based genetic system. Replication of the vims requires reverse transcription of the viral genomic RNA into DNA, which is then incorporated into the genome of the infected host cell. Reverse transcription is catalyzed by a virally encoded nucleic acid polymerase, known as reverse transcriptase (RT). This enzyme is critical for viral replication inhibition of HIV RT is therefore an effective mechanism for abrogating infection in patients. 

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. 

The enzymes that synthesise RNA and DNA are known as nucleic acid polymerases. They are classified as nucleotidyl transferases (Chapter 3). The basic reaction can be represented as follows ... 

Although some depolymerases act processively in cleaving their polymeric substrates, others act by what can be described as multiple attack which results in nonselective scission or random scission. The analysis of cleavage products during the course of enzyme-catalyzed depolymerization can provide important clues about the nature of the reaction. With random scission, the rate of bond scission must be proportional to the total number of unbroken bonds present in the solution. Thomas measured the rate of base addition in a pH-Stat (a device with an automated feedback servomotor that expels ti-trant from a syringe to maintain pH) to follow the kinetics of DNA bond scission by DNase. The number of bonds cleaved was linear with time, and this was indicative of random scission. In other cases, one may apply the template challenge method to assess the processivity of nucleic acid polymerases. See Processivity... 

Several other major classes of enzymes, among them the nucleic acid polymerases, activate ATP (and other NTPs) in a completely different manner. Similar to transphos-phoiylation enzymes, they utilize two metal ions for catalysis. However, steric interactions are purposely employed in order to reverse the preferred binding situation. A MaMp y motif is generated which weakens the P —O—P,5 linkage This allows a nucleoside monophosphate group to be transferred (under liberation of PPi), a process which is essential in the biosynthesis of DNA and RNA sequences. 

Polynucleotide phosphorylase was the first nucleic acid-synthesizing enzyme discovered (Arthur Kornberg s discovery of DNA polymerase followed soon thereafter). 

In many cases nucleic acid polymerases are zinc-dependent enzymes. Hutchinson et al.45 have drawn upon the use of phosphonoacetic acid and phosphonoformic acid and introduced mono- and bis-thiopyrophosphate. They propose that the inhibition of influenza virus occurs by inhibition of RNA transcriptase45. By using 31P-NMR they... 

An alternative to the mirror image-approach is the direct selection of an aptamer from libraries of chemically modified RNAs. Many modifications in the ribose moiety of nucleic acids have been shown to dramatically increase their nuclease resistance. Modifications have to be chosen so as to be compatible with nucleic acid replicating enzymes such as reverse transcriptase, or DNA- and RNA-polymerases. The modifications most commonly used are those in which the 2 -OH group of pyrimidines is substituted by a 2 -fluoro-, or a 2 -amino group (1) [64,65],... 

Some typical biological interactions, frequently used in affinity chromatography are enzyme to substrate analogue, inhibitor, or cofactor antibody to antigen, virus, or cell lectin to polysaccharide, glycoprotein, cell surface receptor, or cell nucleic acid to complementary base sequence, histones, or nucleic acid polymerase hormone or vitamin to receptor, or carrier protein glutathione to glutathione-S-transferase (GST) or GST fusion proteins and metal ions to poly (His) fusion proteins, or native proteins with histidine, cysteine and/or tryptophan residues on their surfaces. 

In contrast to nucleic acid polymerases, polynucleotide processing enzymes often act by mechanisms that involve covalent polynucleotide enzymes as compulsory intermediates (53, 54). The covalent linkages are through phosphodies-ters comprising an enzymic nucleophile, usually the phenolic group of tyrosine, and a nucleotidyl moiety of the nucleic acid. These enzymes are not classified as nucleotidyltransferases, but they catalyze nucleotidyl group transfer as the basic reaction in isomerization processes. Examples are topoisomerases and site-specific recombinases. These enzymes utilize the enzymic nucleophile to cleave the polynucleotide in such a way as to preserve the energy of the covalent bond... 

Another group of RNA viruses called retroviruses also contain a unique nucleic acid polymerizing enzyme called reverse transcriptase or RNA-dependent DNA polymerase, since the genetic code here is taken from RNA, which is reverse of the host cell s normal mechanism. The uniqueness in this case should also theoretically permit selective control, thereby preventing retrovirus replication.12... 

When a virus infects a cell, several events occur that are specific for the invader and hence offer opportunities for selective attack. First of all, there is the contact with the cell, then the penetration of the cell s plasma membrane and the (often simultaneous) rejection of the viral coating-protein. If the virus is of the RN A type, reverse transcriptase is soon in manufacture, but in any case the synthesis of nucleic acid polymerases dominates this early stage of invasion. Next follows the synthesis of viral nucleic acids, structural proteins, and yet more enzymes, followed by the assembly of these components to form the complete virus. Finally, some thousands of these virions are liberated from each cell. Apart from the possibilities for finding selective inhibitors for each of these stages, the patient could also be helped by other drugs to control the secondary (non-viral) symptoms, which are often of an inflammatory or anaphylactic character. 

Many crystal structures have been reported involve nucleic acid interactions with DNA and RNA polymerases as well as other nucleic acid replicative anzymes. " Other structures involve tRNA and tRNA synthetases, and many nucleic acid-modifying enzymes including methyl transferases, " " repair enzymes, " endonucleases, the RNase R enzyme Rrp44 and topoisomerases and helicases. Other protein-DNA structures reported include Soxl7 bound to DNA, " the Mosl paired-end complex, TAR DNA-binding protein (TDP-43) and the tumor suppressor protein p53. A number of protein-RNA structures are also reported including pseudouridine synthase, the E.coli SeqA... 

DNA polymerase enzymes all synthesize DNA by adding deoxynucleotides to the free 3 -OH group of an RNA or DNA primer sequence. The identity of the inserted nucleotide is deterrnined by its abiHty to base-pair with the template nucleic acid. The dependence of synthesis on a primer oligonucleotide means that synthesis of DNA proceeds only in a 5%o V direction if only one primer is available, all newly synthesized DNA sequences begin at the same point. 

The helicases are enzymes central to life itself. The nature of double-stranded DNA means that before a polymerase can begin to copy the appropriate region of the nucleic acid, the two strands have to be unwound the separation of the two strands is the function of the helicase (Fig. 2). An indication of the significance of this family of enzymes is seen in the so-called Werner syndrome, where the helicase function required in the suppression of inappropriate recombination events is defective and causes genomic instability and cancer (for a review see Cobb and Bjergbaek 2006). 

Epirubicin inhibits both DNA and RNA polymerases and thus inhibits nucleic acid synthesis and topoisomerase II enzymes. Epirubicin pharmacokinetics are best described by a three-compartment model, with an a half-life of 4 to 5 minutes, a... 

Both target and signal amplification systems have been successfully employed to detect and quantitate specific nucleic acid sequences in clinical specimens. Polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), strand displacement amplification (SDA), and ligase chain reaction (LCR) are all examples of enzyme-mediated, target amplification strategies that are capable of producing billions of... 

The Li+-induced inhibition of the production of the HSV virus may be related to its actions upon viral DNA polymerase production and activity. Li+ reduces both the synthesis of DNA polymerase in tissue culture and the activity of DNA polymerase in vitro, each by about 50%. It has been proposed that Li+ reduces the biosynthesis of viral polypeptides and nucleic acids, and hence inhibits viral DNA replication by competition with Mg2+, a cofactor of many enzymes [243]. However, the inhibitory effect of Li+ on HSV replication in tissue culture is not affected by Mg2+ levels. A more likely hypothesis is the alteration of the intracellular K+ levels, possibly modifying levels of the high-energy phosphate compounds by replacement of either Na+ or K+ in Na+/K+-ATPase [244]. In tissue culture, HSV replication has been shown to be affected by the... 

The relationship between the DNA in the nucleus and proteins in the cytoplasm is not direct. The information in the DNA molecule is transmitted to the protein-synthesizing machinery of the cell via another informational nucleic acid, called messenger RNA (mRNA), which is synthesized by an enzyme called RNA polymerase. Although similar to DNA, mRNAs are single-stranded, and possess the base uracil instead of thymine and the sugar ribose rather than deoxyribose. These molecules act as short-lived copies of the genes being expressed. 

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