Klenow fragment

The Klenow fragment is a large protein fragment produced when DNA polymerase I from E. coli is enzymatically cleaved by the protease subtilisin. First reported in 1970, it retains the 5’ → 3’ polymerase activity and the 3’ → 5’ exonuclease activity for removal of precoding nucleotides and proofreading, but loses its 5' → 3' exonuclease activity.

The other smaller fragment formed when DNA polymerase I from E. coli is cleaved by subtilisin retains the 5'→ 3' exonuclease activity but does not have the other two activities exhibited by the Klenow fragment (i.e. 5'-> 3' polymerase activity, and 3'->5' nuclease activity).

Because the 5' → 3' exonuclease activity of DNA polymerase I from E. coli makes it unsuitable for many applications, the Klenow fragment, which lacks this activity, can be very useful in research. The Klenow fragment is extremely useful for research-based tasks such as:

Synthesis of double-stranded DNA from single-stranded templates

Filling in recessed 3' ends of DNA fragments

Digesting away protruding 3' overhangs

Preparation of radioactive DNA probes

The Klenow fragment was also the original enzyme used for greatly amplifying segments of DNA in the polymerase chain reaction (PCR) process, before being replaced by thermostable enzymes such as Taq polymerase

Just as the 5' → 3' exonuclease activity of DNA polymerase I from E.coli can be undesirable, the 3' → 5' exonuclease activity of Klenow fragment can also be undesirable for certain applications. This problem can be overcome by introducing mutations in the gene that encodes Klenow. This results in forms of the enzyme being expressed that retain 5' → 3' polymerase activity, but lack any exonuclease activity (5' → 3' or 3' → 5'). This form of the enzyme is called the exo- Klenow fragment.

Text Source: Wikipedia Liscence NGU