DNA polymerase Klenow fragment

Ribonucleotide reductase activity was assayed based on CDP reduction, using a modified method of Jong et al. (1998), with the [ CICDP reduction product determined as radioactivity incorporated into DNA in a series of two coupled reactions, catalyzed by nucleoside diphosphate kinase and DNA polymerase (Klenow fragment). A 40 pi reaction mixture contained 50 mM Hepes pH 7.2, 10 mM dithiothreitol. 

Restriction enzymes, T4-DNA ligase, DNA polymerase (Klenow fragment), T4 DNA kinase and dideoxynucleotides were from Boehringer Mannheim and used as specified by the supplier. Goat anti-rabbit IgG conjugated to alkaline phosphatase was obtained from Sigma. 

Another enzyme, which shares many of the properties of the DNA polymerase (Klenow sub-fragment) is the phage T4 encoded polymerase. The enzyme is a single polypeptide chain similar in size to Pol I of E. coli but which totally lacks the 5 ->3 ... 

Two photoreactive dATP analogues (115, 116) have been incorporated into DNA using Klenow fragment. Subsequent UV irradiation of the primer extension reaction allowed specific cross-linking of the analogues to the polymerase. ... 

DNA polymerase I has been purified to homogeneity. When the pure enzyme is treated with subtilisin, a proteolytic enzyme from Bacillus subtilis, the polymerase is cleaved into two pieces. The small fragment retains the 5 to 3 nuclease activity, whereas the larger piece, called a Klenow fragment, has both polymerase activity and the 3 to 5 exonuclease activity. The Klenow fragment is sold commercially for use in labeling DNA for use in detecting recombinant DNA. 

The Klenow fragment. The DNA polymerase 1 molecule contains its polymerase and nuclease activities on different parts of the enzyme molecule. These two parts can be separated by treatment with the enzyme sub-tilisin. The part which retains the polymerase function is known as the Klenow fragment. This enzyme sythesizes a new DNA strand complementary to the single strand of DNA (the template) only. It is used to create blunt ends in dsDNA and in the dideoxy method of DNA sequencing. 

Biolabs (Pickering, ON), and Promega (Madison, WI). It is important to note that restriction enzymes producing 3 -overhangs should be avoided if possible (e.g., Psfl, Sfil, Kpnl). The use of such enzymes has been reported to result in the production of additional, nonspecific transcripts (Schenborn and Mierendorf, 1985). If these enzymes must be used, an exonuclease such as DNA Polymerase 1 Large (Klenow) Fragment can be utilized to convert the overhang to a blunt end before the template is transcribed. 

Results from testing against a variety of polymerase enzymes show that efavirenz is inactive up to 300 p,M for a 50% inhibition (Young et al., 1995). The polymerase enzymes studied were Moloney murine leukemia virus RT, human DNA polymerases a, 3, and 7, Escherichia coli RNA polymerase, and the Klenow fragment. Cytotoxicity studies in their primary cells and in a T-cell line reveal that efavirenz has a selectivity index of 80,000. 

Fig. 28. Synthesis of labeled DNA probes. A Labeled DNA can be generated using different enzymes (Klenow fragment of DNA polymerase or a terminal transferase) to incorporate labeled nucleotides into specific DNA sequences. Probes can be labeled using radioactive nucleotides or nucleotides labeled with an immunogenic molecule such as biotin. B The labeled probe is then hybridized to the target nucleic acid, which is either bound to a membrane or in a tissue section or cell. An antibody is then used to detect the non-radioactively-labeled probe. C The antibody may be conjugated to a fluorescent or chemiluminescent dye, or an enzyme that produces a color reaction. The target nucleic acid is thus visualized.

Further reaction of 53 with diphenyl phosphate afforded a phospho-diester-bridged dinuclear magnesium(II) complex 54. The Mg-Mg distance of 4.11 A is comparable to similar distances in the Klenow fragment of E. coli DNA polymerase I (3.9 A) (39), rat DNA polymerase j8 (4 A) (46), and inositol monophosphatase (3.8 A) (35). The flexibility of the bridging carboxylates in 52 is manifested by the ca. 0.75-A range of Mg-Mg distances in these complexes, which can readily adjust the metal coordination environment. 

DNA polymerase I, then, is not the primary enzyme of replication instead it performs a host of clean-up functions during replication, recombination, and repair. The polymerase s special functions are enhanced by its 5 —>3 exonuclease activity. This activity, distinct from the 3 —>5 proofreading exonuclease (Fig. 25-7), is located in a structural domain that can be separated from the enzyme by mild protease treatment. When the 5 —>3 exonuclease domain is removed, the remaining fragment (Afr 68,000), the large fragment or Klenow fragment (Fig. 25-8), retains the polymerization and... 

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