Biotechnology Index Glossary

Large (Klenow) Fragment of E. coli DNA Polymerase I

The 5' -> 3' exonuclease activity of E. coli's DNA polymerase I makes it unsuitable for many applications. However, this pesky enzymatic activity can readily be removed from the holoenzyme. Exposure of DNA polymerase I to the protease subtilisin cleaves the molecule into a small fragment, which retains the 5' -> 3' exonuclease activity, and a large piece called Klenow fragment. The large or Klenow fragment of DNA polymerase I has DNA polymerase and 3' -> 5' exonuclease activities, and is widely used in molecular biology.

In addition to generating Klenow fragment by proteolysis, it can be expressed in bacteria from a truncated form of the DNA polymerase I gene. The enzyme you purchase is almost certainly produced in this manner.

Klenow fragment is useful for several tasks:

Synthesis of double-stranded DNA from single-stranded templates: The function of DNA polymerases is to synthesize complementary strands during DNA replication. Performing that task in the lab is integral to such processes as synthesizing the second strand DNA in cDNA cloning and generating radioactive probes for hybridization reactions.

DNA polymerases require a primer to provide a free 3' hydroxyl group for initiation of synthesis. The primers used for most in vitro polymerization reactions are single-stranded DNAs, typically 6 to 20 bases in length, called oligonucleotides. The oligonucleotides must be complemenary to some section of template DNA. To use Klenow to synthesize a complementary strand of DNA, one simply mixes single-stranded template (usually denatured double-standed DNA), primers and the enzyme in the presence of an appropriate buffer (most restriction enzyme buffers work well). The reaction proceeds are depicted below:

One item of some significance in the above reaction is that as Klenow proceeds, it can displace primers downstream and continue synthesizing new DNA.

Filling in recessed 3' ends of DNA fragments: A "fill-in" reaction is used to create blunt ends on fragments created by cleavage with restriction enzymes that leave 5' overhangs. This reaction is conceptually identical to the one described above, but with a huge primer and a very short segment of single-stranded template.

Digesting away protruding 3' overhangs: This is another method for producing blunt ends on DNA, in this with ends generated from restriction enzymes that cleave to produce 3' overhangs. The 3' -> 5' exonuclease activity of Klenow will digest away the protruding overhang. Removal of nucleotides from the 3' ends will continue, but, in the presence of nucleotides, the polymerase activity will balance the exonuclease activity, yielding blunt ends. This reaction is more efficienty conducted with T4 DNA polymerase, which has much more potent exonuclease activity.

Preparation of radioactive DNA probes: Examine each of the reactions depicted above. What if the nucleotides used were in the reaction were radioactive? That's correct - the radioactive nucletides would be incorporated into the DNA fragment. Klenow fragment is used frequently to prepare DNA that is labeled with radionuclides or other markers.

In some situations, the 3' -> 5' exonuclease activity of Klenow fragment is either undesirable or not necessary. By introducing mutations in the gene that encodes Klenow, forms of the enzyme can be expressed that retain polymerase activity, but lack any exonuclease activity. These forms are the enzyme are usually called exo- Klenow fragment.

Back to the index of Restriction Endonucleases and DNA Modifying Enzymes

Last updated on October 20, 1999
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