Klenow fragment of DNA

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.

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. 

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... 

Beese, L. S., Friedman, J. M., and Steitz, T. A. (1993b). Crystal structures of the Klenow fragment of DNA polymerase I complexed with deoxynucleoside triphosphate and pyrophosphate. Biochemistry 32, 14095-14101. 

All life forms require polymerases for replication of nucleic acids. DNA polymerases from various life forms [8] as well as reverse transcriptase from HIV [9] are all activated by two metal ions, which are thought to provide three types of activation, i.e. Lewis acid, metal-hydroxide and leaving group [Figure 6.26(A)], The Klenow fragment of DNA polymerase I is a 3, 5 -exonuclease that is involved in editing the growing... 

In the Klenow fragment of DNA polymerase I (the 3, 5 -exonnclease), two metal ions are nsed. One stabihzes the transientpentacovalent species and the leaving of a 3 oxyanion (Mg +), and the other facilitates the formation of an attacking water molecnle or hydroxyl gronp (Zn +). 

The three-dimensional structures of a number of DNA polymerase enzymes are known. The first such structure to be determined was that of the so-called Klenow fragment of DNA polymerase I from E. coli (Figure 27.11). This fragment comprises two main parts of the full enzyme, including the polymerase unit. This unit approximates the shape of a right hand with domains that are referred to as the fingers, the thumb, and the palm. In addition to the polymerase, the Klenow... 

A.H. Polesky, T.A. Steitz, N.D. Grindley, and C.M. Joyce. 1990. Identification of residues critical for the polymerase activity of the Klenow fragment of DNA polymerase I from Escherichia coli J. Biol. Chem. 265 14579-14591. (PubMed)... 

B2518), and 5 units/mL Klenow fragment of DNA polymerase I. The labeling solution without polymerase can be prepared in bulk and stored frozen in aliquots, but the DNA polymerase must be added immediately prior to use. The biotinylated nucleotide can be obtained from Life Technologies Ltd. (Paisley, Scotland cat. no. 19534-016). Deoxynucleotide triphosphates can be purchased as a set from Boehringer Mannheim UK, East Sussex, UK cat. no. 1277 049 (see Notes 4,5, and 7). 

A variety of enzymes can be used for incorporation of the label. Klenow fragment of DNA polymerase I is preferable to the DNA polymerase I holoenzyme because it has the identical polymerase activity, but lacks the exonuclease activity that could cause artifactual labeling (43). Alternatively one can use TdT (33), which adds on long tails of nucleotides to the 3 hydroxyl ends of DNA without the need for a template strand (43). It is extremely important to use the correct concentration of enzyme, as increased amounts will lead to nonspecific staining of morphologically normal nuclei (34,35). Obviously, insufficient enzyme will lead to a reduction in the staining of apoptotic nuclei. 

Enzymatic Synthesis.—It has been demonstrated that, where the synthesis of longer double-stranded DNA segments is required, the amount of chemical synthesis involved may be reduced by more than 40% by synthesizing chemically two 40-mers (say), each of which is part of the sequence of the two different strands but has an overlap sequence nine or ten bases long at the 3 -terminus complementary to that on the other strand, annealing them, and using the resultant part-duplex as a template-primer for Klenow fragment of DNA polymerase I to complete the duplex by copy synthesis. ... 

Klenow fragment of DNA polymerase 1 from E.colr E.coli alkaline phosphatase is also... 

We should note that, given the difference in quantum yield between the free and bound probe, the fractional intensities utilized in Fig. 7 actually represent small percentages of bound probe on a molar basis. In fact, considering the accuracy of the differential phase measurement (better than O.r) one can detect, in this system, on the order of 0.1% bound probe. This phenomenon also occurs in time-domain measurements. Specifically, if one monitors the anisotropy decay of a system which displays multiple lifetimes associated with multiple rotational diffusion rates then one may observe a decline at short times of the anisotropy followed by a rise at latter times and subsequent decrease. This dip and rise effect has been observed by Millar and co-workers in studies on protein-DNA interactions, specifically in the case of the interaction of a fluorescent DNA duplex with the Klenow fragment of DNA polymerase. 

Why is the Klenow fragment of DNA polymerase, rather than the whole enzyme, used in DNA sequencing ... 

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