Question

The Klenow fragment is produced when DNA polymerase 1 from E.coli is enzymatically cleaved by the protease subtilisin. Using diagrams, explain what the Klenow fragment is and why the fragment is better suited for site-directed mutagenesis than intact DNA polymerase 1?

Answer 1

Ans) The Klenow Fragment is a cleavage product of the E. coli DNA Polymerase I with the protease, subtilisin. It lacks the 5’-3’ exonuclease activity of DNA Pol I, a feature that makes the enzyme unsuitable for experimental purposes. However, it retains the original polymerase activity as well as the 3’-5’ proofreading activity. This enzyme is thus suitable for DNA synthesis applications such in probe generation, dideoxy sequencing, DNA blunting of overhangs, and DNA labeling, among others.

Klenow Fragment is the large fragment of DNA Polymerase I that retains its 5'→3' polymerase, 3'→5' exonuclease and strand displacement activities. The enzyme lacks the 5'→3' exonuclease activity of intact DNA polymerase I. Klenow retains the polymerization fidelity of the holoenzyme without degrading 5' termini. As in site-directed mutagenesis, we change, insert or delete single amino acid residues, multiple residues or even entire structural elements so that's why fragment is better suited than DNA pol 1 and also DNA pol 1 posses an additional exonuclease activity at N terminal domain which makes it less suitable