Klenow fragment

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. 

End filling is a more gentle method but can only be used on DNA molecules that have sticky ends. It uses the Klenow fragment, which fills in the sticky ends by synthesizing the complementary strand. Again if the reaction is carried out in the presence of labelled deoxynucleotides the result is a strand of DNA which is radioactively labelled. 

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. 

Extensive kinetic experiments on the Klenow fragment (and other replicases) have resulted in the general mechanism illustrated in Figure Except for variations... 

A detailed analysis of solid-state structures of several unrelated systems that catalyze the hydrolysis of diphosphate esters (alkaline phosphatase and the Klenow fragment , among others) revealed that their active sites invariably contain conserved carboxylate... 

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... 

FIGURE 25-8 Large (Klenow) fragment of DNA polymerase I. This polymerase is widely distributed in bacteria. The Klenow fragment, produced by proteolytic treatment of the polymerase, retains the polymerization and proofreading activities of the enzyme. The Klenow fragment shown here is from the thermophilic bacterium Bacillus stearothermophilus (PDB ID 3BDP). The active site for addition of nucleotides is deep in the crevice at the far end of the bound DNA. The dark blue strand is the template. 

A sample of double-stranded DNA is denatured. One of the resulting single strands is used as a template to direct the synthesis of a complementary strand of radioactive DNA using a suitable DNA polymerase. The "Klenow fragment" of E. coli, DNA polymerase I, reverse transcriptase from a retrovirus, bacteriophage T7 DNA polymerase, Taq polymerase, and specially engineered enzymes produced from cloned genes have all been used. 

E. coli DNA polymerase I and Klenow fragment E. coli 5 —> 3 chain growth 3 —> 5 exonuclease 5 —> 3 exonuclease (lacking in Klenow fragment)... 

Some bacteriophage encode their own DNA polymerases. However, they usually rely on the host cell to provide accessory proteins. The sequence of the DNA polymerase from phage T7 is closely homologous to that of the Klenow fragment and the 3D structures are similar. The 80-kDa T7 polymerase requires the 12-kDa thioredoxin from the host cell as an additional subunit. It has been genetically engineered to improve its usefulness in DNA sequencing 278... 

About 45% of the sequence of the RNA polymerase encoded by phage T7, which transcribes RNA from the phage DNA, is also similar to that of the Klenow fragment. Sequences of these DNA polymerases are distantly related to those of reverse transcriptases.279280 The 136-kDa polymerase y functions in mitochondria but is encoded in a nuclear gene. It is the only DNA polymerase that is inhibited by antiviral nucleotide analogs such as AZT (Box 28-C).280a b... 

The so-called Klenow fragment of DNA polymerase 1 of E. coli (Chapter 14, section Al) contains the 5 -3 -polymerization and the 3 -5 -exonuclease domains. Detailed pre-steady state kinetics have been made of the polymerization and exonuclease activities.39-43 The editing site is 35 A away from the polymerization site.32 The mechanism of the polymerization activity (Figure 13.7) is very similar to that for hydrolysis (Figure 13.8). The key to both is the presence of two metal ions, 3.9 A apart, that stabilize the developing charges on the transition state and metal-bound HO- or RO ions (see Chapter 2, section B7).44,45... 

There is an equilibrium between DNA binding to the two sites. Time-resolved fluorescence spectroscopy of labeled duplex DNA bound to the Klenow fragment shows that 12% occupies the editing, site and 88% the polymerization.42 The presence of mismatches both slows down elongation and increases the occupancy of the editing site 43 Comparison of all the steps for correct and incorrect nucleotide incorporation confirms that the fidelity stems from discrimination in... 

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