Deoxyribonucleoside triphosphate replication

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. 

DNA Replication Proceeds by the Polymerization of Deoxyribonucleoside Triphosphates Along a Template... 

The base sequences of newly synthesized DNA must faithfully match the sequences of parent DNA. To achieve faithful replication, each strand within ihe parent double helix acts as a template for the synthesis of a new DNA strand with a complementary sequence. The building blocks for the synthesis of the new strands are deoxyribonucleoside triphosphates, d hey are added, one at a time, to the 3 end of an existing strand of DNA. 

The major enzyme involved in replication is a DNA polymerase that copies each parental template strand in the 3 to 5 direction, producing new strands in a 5 to 3 direction. Deoxyribonucleoside triphosphates serve as the precursors. 

E. coli has three DNA polymerases, Pol I, Pol II, and Pol III. Pol III is the major replicative enzyme (Table 13.1). All DNA polymerases that have been studied copy a DNA template strand in its 3 to 5 direction, producing a new strand in the 5 to 3 direction (Fig. 13.4). Deoxyribonucleoside triphosphates (dATP, dGTP, dCTP, and dTTP) serve as substrates for the addition of nucleotides to the growing chain. 

You have a double-strand linear DNA molecule, the appropriate primers, all the enzymes required for DNA replication, four P-labeled deoxyribonucleoside triphosphates, Mg " ion, and the means to detect newly synthesized radioactive DNA. Why is this system not sufficient to distinguish between conservative and semiconservative replication of the DNA molecule ... 

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... 

An in-vitro nuclear system prepared from HeLa cells, described by Friedman and Mueller (1968), appears to continue the DNA replication process observed in vivo. The system requires intact nuclei, the four deoxyribonucleoside triphosphates, magnesium ion, ATP, and, in addition, a heat-labile cytoplasmic factor. The activity of the system was similar to the DNA synthetic activity observed in intact cells in synchronized culture (Friedman and Mueller, 1968). Cytoplasmic factors also appear to stimulate in-vitro nuclear systems prepared from normal and regenerating rat livers (De Beilis, 1969). The cytoplasmic factors are present in both normal and regenerating liver cytoplasm and stimulate nuclear DNA synthesis in both systems. The stimulation was most marked using regenerating liver factors and normal liver nuclei (De Beilis, 1969). When mouse liver nuclei are recombined with cell free cytoplasmic extracts from mouse ascitic or L-cells active in DNA synthesis there is a marked stimulation of DNA synthesis in the isolated nuclei (Thompson and McCarthy, 1968). Cytoplasmic preparations from HeLa cells also stimulated DNA synthesis in mouse liver nuclei. 

In the replication of DNA, the two strands of a double helix unwind and separate as new chains are synthesized. Each parent strand acts as a template for the formation of a new complementary strand. Thus, the replication of DNA is semiconservative—each daughter molecule receives one strand from the parent DNA molecule. The replication of DNA is a complex process carried out by many proteins, including several DNA polymerases. The activated precursors in the synthesis of DNA are the four deoxyribonucleoside 5 -triphosphates, line new strand is synthesized in the 5 3 direction by a nucleophilic attack by the 3 -hydrox-... 

DNA replication uses deoxyribonucleoside-5 -triphosphates (dNTPs) to build the DNA chains. 

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