2 -deoxyribonucleoside-5 -triphosphate incorporation

Deoxyribonucleoside triphosphate dependence All four required All four work, but single nucleotide will incorporate All four... 

Gemcitabine is phosphorylated initially by the enzyme deoxycytidine kinase and then by other nucleoside kinases to the di- and triphosphate nucleotide forms, which then inhibit DNA synthesis. Inhibition is considered to result from two actions inhibition of ribonucleotide reductase by gemcitabine diphosphate, which reduces the level of deoxyribonucleoside triphosphates required for the synthesis of DNA and incorporation of gemcitabine triphosphate into DNA. Following incorporation of gemcitabine nucleotide, only one additional nucleotide can be added to the growing DNA strand, resulting in chain termination. 

DNA repair is best studied in a system where the background levels of replication are low. Suitable systems are cultures which have come to rest at high density, or unstimulated lymphocyte preparations where less than 1% of the cells are in S-phase. The low levels of replicative incorporation can be further repressed by 1-2 mM hydroxyurea which selectively inhibits replication (Cleaver, 1969b). This selective effect may simply be a result of the very small pools of deoxyribonucleoside triphosphates required for repair. 

Biotinylated dUTP can also be used to label DNA probes by a different method, namely random-primed labeling (4). The principle of this method is based on the reannealing of hexadeoxyribonucleotide primers, which have random specificity, to the denatured DNA strands. The DNA to be labeled has to be linearized and denatured before the strands are used as templates in the labeling reaction. The complementary strands are synthesized from the 3 OH termini of the reannealed hexanucleotides by the Klenow fragment of E. coli DNA polymerase I. The primers reanneal at random sites of the template strands, so that the synthesis of the complementary strands is primed at random sites. If one of the deoxyribonucleoside triphosphates present in the reaction mixture is labeled, the newly synthesized strands will become labeled by the incorporation of the labeled nucleotides. The end product of this reaction is a mixture of unlabeled (template) and labeled... 

A FIGURE 9-22 Structures of deoxyribonucleoside triphosphate (dNTP) and dideoxyribonucleoside triphosphate (ddNTP). Incorporation of a ddNTP residue into a growing DNA strand terminates elongation at that point. 

A single (template) strand of the DNA to be sequenced (blue letters) is hybridized to a synthetic deoxyribonucleotide primer (black letters). The primer is elongated in a reaction mixture containing the four normal deoxyribonucleoside triphosphates plus a relatively small amount of one of the four dideoxyribonucleoside triphosphates. In this example, ddGTP (yellow) is present. Because of the relatively low concentration of ddGTR incorporation of a ddGTP and thus chain termination, occurs at a given position in the sequence only about 1 percent of the time. Eventually the reaction mixture will contain a mixture of prematurely terminated... 

The deoxyribonucleoside triphosphate dUTP can be used as a substrate for DNA polymerase during DNA replication because the structure and hydrogen-bonding properties of uracil are very similar to those of thymine. When incorporated into a doubfe-strand DNA pofymer, uracif pairs with adenine, as does thymine. For a discussion of the reasons uracif is not normaffy incorporated into DNA, see page 771 of the text. 

What is known concerning the activities of DNA polymerases isolated from various eukaryotes DNA polymerase (replicative deoxynucleotidyl transferase) has been isolated from calf thymus (Bollum, 1960) and catalyzes the addition of deoxyribonucleoside triphosphates to the 3 4iydroxyl terminus of a primer DNA in a reaction which has an absolute requirement for template DNA. In addition, terminal deoxynucleotidyl transferases (end addition enzymes) have been recognized as separate enzymes from calf thymus (Krakow et al., 1962) and physically separated from the enzyme DNA polymerase (Yoneda and Bollum, 1965). The terminal deojQ nucleotidyl transferase enzymes catalyze the incorporation of mononucleotide units from the nucleoside-5 -triphosphates into 3 -terminal positions of DNA in a reaction not template directed. The template requirements of the calf thymus polymerases have been discussed by Bollum... 

Giller, G. Tasara, T. Angerer, B. Muhlegger, K. Amacker, M. Winter, H. Incorporation of reported molecule-labeled nucleotides by DNA polymerases. I. Chemical synthesis of various reporter group-labeled 2 -deoxyribonucleoside-5 -triphosphates. Nucleic Acids Res. 2003, 31, 2630-2635. 

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