Klenow fragment

The Klenow fragment is a large protein fragment which is produced when DNA polymerase I from E. coli is cleaved by the protease enzyme subtilisin. It exhibits the 5’ → 3’ polymerase activity and the 3’ → 5’ exonuclease activity for removal of precoding nucleotides, but does not retain the 5' → 3' exonuclease activity.

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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).

Research

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 for 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 exo- Klenow fragment

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.