Deoxynucleotides

In the diester method a deoxynucleoside-5 -monophosphate is condensed with the 3 -OH group of a deoxynucleotide to produce a 3, 5 -phosphodiester. This is illustrated by a general method for dinucleotide synthesis developed by H.G. Khorana (K.L. Agarwal, 1976). One N-... 

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. 

DNA polymerases normally use 3 -deoxynucleotide triphosphates as substrates for polymerization. Given an adequate concentration of substrate, DNA polymerase synthesizes a long strand of new DNA complementary to the substrate. The use of this reaction for sequencing DNA depends on the inclusion of a single 2/3 -dideoxynucleoside triphosphate (ddNTP) in each of four polymerization reactions. The dideoxynucleotides ate incorporated normally in the chain in response to a complementary residue in the template. Because no 3 -OH is available for further extension, polymerization is... 

Decau.se its longer half-life and lower energy make it more convenient to handle, is replacing "" P as the radioactive tracer of choice in. sequencing by the Sanger method. "" S-ct-labeled deoxynucleotide analogs provide die. source for incorporating radioactivity into DNA. 

Because dideoxynucleotides lack 3 -OH groups, these nucleotides cannot serve as acceptors for 5 -nucleotide addition in the polymerization reaction, and thus the chain is terminated where they become incorporated. The concentrations of the four deoxynucleotides and the single dideoxynucleotide in each reaction mixture are adjusted so that the dideoxynucleotide is incorporated infrequently. Therefore, base-specific premature chain termination is only a random, occasional event, and a population of new strands of varying length is synthesized. Four reactions are run, one for each dideoxynucleotide, so that termination, although random, can occur everywhere in the sequence. In each mixture, each newly synthesized strand has a dideoxynucleotide at its 3 -end, and its presence at that position demonstrates that a base of that particular kind was specified by the template. A radioactively labeled dNTP is included in each reaction mixture to provide a tracer for the products of the polymerization process. 

The double-stranded DNA to be amplified is heated in the presence of Taq polymerase, Mg2+ ion. the four deoxynucleotide triphosphate monomers (dNTPs), and a large excess of two short oligonucleotide primers of about 20 bases each. Each primer is complementary to the sequence at the end of one of the target DNA segments. At a temperature of 95 °C, double-stranded DNA denatures, spontaneously breaking apart into two single strands. 

All NRTIs, as exemplified for AZT (Fig. 7), act in a similar fashion following their uptake by the cells, they are phosphorylated successively to their 5 -monophosphate, 5 -diphosphate, and 5 -triphosphate form (De Clercq 2002). Unlike the first phosphorylation step in the metabolic pathway of the acyclic guanosine analogues (see above), which is carried out by a virus-encoded enzyme (thymidine kinase), the first as well as the subsequent phosphorylations of the 2, 3 -dideoxynucleosides are carried out by cellular enzymes, that is, a 2 -deoxynucleoside (e.g., dThd) kinase, a 2 -deoxynucleotide (e.g., dTMP) kinase, and a (2 -deoxy)nucleoside 5 -diphosphate (NDP) kinase. 

In mammahan cells, the polymerase is capable of polymerizing about 100 nucleotides per second, a rate at least tenfold slower than the rate of polymerization of deoxynucleotides by the bacterial DNA polymerase complex. This reduced rate may result from interference by nucleosomes. It is not known how the rephcation complex negotiates nucleosomes. 

Figure 8.24 Separation of the major deoxyribonucleosides and their 5 - monophosphate deoxynucleotides on a strong cation exchange column (column one) and a reversed-phase column. The unseparated nucleosides. A, on the ion- exchange column were switched to the reversed-ptose column. Pe2dc Identification A = nucleosides, B d-CMP, C d-AMP, D - d-GJIP, E - d-CVD, P d-UKO, G THD, and H = d-AOO. (Reproduced with permission from ref. 298. Copyright Preston Publications, Inc.)...

SCHEME 9.9 Reversible alkylation of deoxynucleotides by a metabolite of 2,6,-di-tert-butyl-4-methylphenol. 

The reversibility of QM adducts also creates numerous challenges. For example, measuring the full burden of DNA alkylation by a QM can be obscured by the loss of its labile products during or before chemical identification can be completed. Results from a deoxynucleotide model system indicated that only a small fraction of the possible adducts could be measured after the interval required for analysis of DNA. Perhaps the kinetic products of QMs also contribute to the cellular activity of these intermediates although this has yet to be explored. QM equivalents can be envisioned to migrate from one reversible nucleophile such as the N1 of adenine in such cofactors as ATP to another until quenched by a compound such as glutathione that is present in cells as a defense against undesirable electrophiles. 

The activation of the 5 -hydroxy group of synthetic deoxyoligonucleotides on a controlled porous glass support was achieved with CDI to give a 5 -imidazolide, which was subsequently converted with hexamethylenediamine to yield as the carbamate a 5 -aminoalkylated supported deoxynucleotide.t206]... 

PNP catalyzes the phosphorolysis of purine nucleosides and deoxynucleosides in mammalian cells. The absence of PNP interferes with the proper degradation of 2 -deoxyguanosine, leading to an imbalance of cellular levels of deoxynucleotides... 

Figure 9.3 A single unit of DNA polymerase III complex synthesizes both new strands of DNA, one continuously and the other in short pieces. Deoxynucleotide additon to die daughter strands is indicated by vertical lines across the strands.

F. H. Westheimer (1987) has provided a detailed survey of the multifarious ways in which phosphorus derivatives function in living systems (Table 4.7). The particular importance of phosphorus becomes clear when we remember that the daily turnover of adenosine triphosphate (ATP) in the metabolic processes of each human being amounts to several kilograms Phosphate residues bond two nucleotides or deoxynucleotides in the form of a diester, thus making possible the formation of RNA and DNA the phosphate always contains an ionic moiety, the negative charge of which stabilizes the diester towards hydrolysis and prevents transfer of these molecules across the lipid membrane. 

A sequence, in general, is the relative order of base pairs, whether in a fragment of a protein, DNA, a gene, a chromosome, or an entire genome. DNA is composed of two antiparallel strands of deoxynucleotides held together by hydrogen bonds between purine (adenine, A and guanine, G) and pyrimidine (thymidine, T uracil, U and cytosine, C) bases. 

Enzymatic techniques can employ a variety of DNA or RNA polymerases to add controlled amounts of modified nucleotides to an existing stand. However, the most common procedures utilize either DNA polymerase I or terminal deoxynucleotide transferase. The polymerase is used with a template to add modified nucleoside triphosphates to the end of a DNA molecule or to various sites within the middle of a sequence. The terminal transferase can add modified monomers to the 3 end of a chain without a template. 

The two complementary strands of the DNA double helix run in antiparallel directions (Fig. 4-1). The phosphodiester connection between individual deoxynucleotides is directional. It connects the 5 -hydroxyl group of one nucleotide with the 3 -hydroxyl group of the next nucleotide. Think of it as an arrow. If the top strand sequence is written with the 5 end on the left (this is the conventional way), the bottom strand will have a complementary sequence, and the phosphate backbone will run in the opposite direction the 3 end will be on the left. The antiparallel direc-... 

Purine Synthesis Purine Salvage Deoxynucleotides Purine Degradation... 

Deoxynucleotides for DNA synthesis are made at the nucleoside diphosphate level and then have to be phosphorylated up to the triphosphate using a kinase and ATP. The reducing equivalents for the reaction come from a small protein, thioredoxin, that contains an active site with two cysteine residues. Upon reduction of the ribose to the 2 -deoxyri-bose, the thioredoxin is oxidized to the disulfide. The thioredoxin(SS) made during the reaction is recycled by reduction with NADPH by the enzyme thioredoxin reductase. 

Ribonucleotide reductase works on ribo-A, -U, -G, -C diphosphates to give the deoxynucleotide. The deoxyuridine, which is useless for RNA synthesis, is converted to deoxythymidine by the enzyme thymidylate synthase, which uses methylene tetrahydrofolate as a one-carbon donor. The odd thing here is that ribonucleotide reductase uses the UDP as a substrate to give the dUDP. This must then be hydrolyzed to the dUMP before thymidylate synthase will use it to make dTMP. Then the dTMP has to be kinased (phosphorylated) up to dTTP before DNA can be made. 

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