DNA polymerases are enzymes that catalyze the formation of DNA by adding deoxyribonucleotides to the 3′-OH end of a pre-existing primer. There are different types of DNA polymerases. Most DNA polymerases used in molecular biology are prokaryotic DNA polymerases. Klenow fragment and T4 DNA polymerase are two types of DNA polymerases that have a prokaryotic origin. Klenow fragment is the large fragment that is generated from the cleavage of E.coli DNA polymerase 1 into two fragments by bacterial protease subtilisin. T4 DNA polymerase is the DNA polymerase 1 of bacteriophage T4. Both Klenow and T4 DNA polymerase have 5′→3′ polymerase and 3′→5′ exonuclease activity. They both lack 5′→3′ exonuclease activity, making them more suitable for many applications in molecular biology.
1. Overview and Key Difference
2. What is Klenow
3. What is T4 DNA Polymerase
4. Similarities Between Klenow and T4 DNA Polymerase
5. Side by Side Comparison – Klenow vs T4 DNA Polymerase in Tabular Form
What is Klenow?
Klenow fragment is a DNA polymerase that is useful in molecular biology. When E. coli DNA polymerase 1 undergoes proteolytic digestion by bacterial protease, subtilisin, it results in two fragments: one is a large fragment, and the other one is a small fragment. Klenow fragment is the large fragment which is 68 kDa in size. Klenow fragment has 5′→3′ polymerase and 3′→5′ exonuclease (proofreading) activities of DNA Pol I. The 3′→5′ exonuclease activity of the Klenow fragment facilitates the removal of incorrectly added bases as polymerization progresses. Klenow fragment does not contain 5′→3′ exonuclease activity, which is shown by full-length or intact E. coli DNA polymerase 1. It is advantageous when only the polymerization activity is needed alone. Klenow fragments are used to fill 5’ overhangs, synthesis of probes, sequencing DNA, synthesis of double-stranded DNA, synthesis of cDNA second strand and site-directed mutagenesis.
In certain applications, the 3′→5′ exonuclease activity of the Klenow fragment also becomes undesirable. It can be removed by introducing a mutation to the gene that codes for Klenow fragment. The resulting Klenow fragment is known as exo-Klenow fragment. Therefore, exo-Klenow fragment has only the 5′→3′ polymerase activity of E. coli polymerase 1.
What is T4 DNA Polymerase?
T4 DNA polymerase is a DNA polymerase that catalyzes the synthesis of DNA. It is a protein coded by Bacteriophage T4. Structurally, T4 DNA polymerase is an 898 amino acid residue protein (molecular weight of 103.6 kDa). It requires a template and a primer to catalyze the synthesis.
Similar to the Klenow fragment, T4 DNA polymerase has both 5′→3′ polymerase and 3′→5′ exonuclease activities. Moreover, it lacks 5′→3′ exonuclease activity as well. T4 DNA polymerase is useful in the filling of 5′-protruding ends of DNA fragments. It is also often used to 5′-end or 3′-end-labelling of double-stranded DNA. T4 DNA polymerase is frequently used in blunt cloning. The 3′→5′ exonuclease (proofreading) activity of T4 DNA polymerase is stronger (more than 200 times) than the Klenow fragment. Most importantly, T4 DNA polymerase has high processivity (400 nucleotides per second).
What are the Similarities Between Klenow and T4 DNA Polymerase?
- Klenow and T4 DNA polymerase are two prokaryotic polymerases.
- Both polymerases have 5′→3′ polymerase and 3′→5′ exonuclease activity.
- They do not exhibit 5′→3′ exonuclease activity.
- Both enzymes require a template and a primer to catalyze the synthesis of DNA.
- These enzymes can be heat inactivated at 75 0
What is the Difference Between Klenow and T4 DNA Polymerase?
The key difference between Klenow and T4 DNA polymerase is the origin of each enzyme. Klenow fragment is originated from a bacterium while T4 DNA polymerase is originated from a bacteriophage which is a virus. T4 DNA polymerase has a high processivity than Klenow fragment. Moreover, T4 DNA polymerase has a strong proofreading activity than the Klenow fragment. Thus, this is another difference between Klenow and T4 DNA polymerase.
The below info-graphic lists more differences between Klenow and T4 DNA polymerase in tabular form.
Summary – Klenow vs T4 DNA Polymerase
Klenow fragment is a large fragment of E. coli DNA polymerase 1. It has only 5′→3′ polymerase and 3′→5′ exonuclease activities. It lacks 5′→3′ exonuclease activity of intact E. coli DNA pol 1. Therefore, Klenow fragment is a bacterial polymerase. On the other hand, T4 DNA polymerase is a polymerase coded by the bacteriophage T4. Similar to the Klenow fragment, it contains both 5′→3′ polymerase and 3′→5′ exonuclease activities, and it lacks 5′→3′ exonuclease activity. However, T4 DNA polymerase has high processivity and high proofreading activity than the Klenow fragment. So, this is the key difference between Klenow and T4 DNA polymerase.
1. “DNA Polymerase, T4.” DNA Polymerase, T4 – Worthington Enzyme Manual, Available here.
2. Rittié, Laure, and Bernard Perbal. “Enzymes Used in Molecular Biology: a Useful Guide.” Journal of Cell Communication and Signaling, Springer Netherlands, June 2008, Available here.
1. “PolymeraseDomains” – Protein Data Bank (Public Domain) via Commons Wikimedia
2. “T4-DNA-Polymerase” By ChemPro – J. Wang, P. Yu, T. C. Lin, W. H. Konigsberg, T. A. Steitz (1996). “Crystal structures of an NH2-terminal fragment of T4 DNA polymerase and its complexes with single-stranded DNA and with divalent metal ions” Biochemistry 35: 8110–8119. DOI:10.1021/bi960178r. (CC0) via Commons Wikimedia