The Klenow fragment is a large

protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residue (biochemistry), residues. Proteins perform a vast array of functions within organisms, including Enzyme catalysis, catalysing metab ...

fragment produced when

DNA polymerase I DNA polymerase I (or Pol I) is an enzyme that participates in the process of prokaryotic DNA replication. Discovered by Arthur Kornberg in 1956, it was the first known DNA polymerase (and the first known of any kind of polymerase). It was init ...

from ''

E. coli ''Escherichia coli'' ( )Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. is a gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus ''Escherichia'' that is commonly foun ...

'' is

enzymatically An enzyme () is a protein that acts as a biological catalyst by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as produc ...

cleaved by the

protease A protease (also called a peptidase, proteinase, or proteolytic enzyme) is an enzyme that catalysis, catalyzes proteolysis, breaking down proteins into smaller polypeptides or single amino acids, and spurring the formation of new protein products ...

subtilisin Subtilisin is a protease (a protein-digesting enzyme) initially obtained from ''Bacillus subtilis''. Subtilisins belong to subtilases, a group of serine proteases that – like all serine proteases – initiate the nucleophilic attack on the ...

. First reported in 1970, it retains the 5' → 3'

polymerase In biochemistry, a polymerase is an enzyme (Enzyme Commission number, EC 2.7.7.6/7/19/48/49) that synthesizes long chains of polymers or nucleic acids. DNA polymerase and RNA polymerase are used to assemble DNA and RNA molecules, respectively, by ...

activity and the 3’ → 5’

exonuclease Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end (exo) of a polynucleotide chain. A hydrolyzing reaction that breaks phosphodiester bonds at either the 3′ or the 5′ end occurs. Its close relative is th ...

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 ''

'' 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' exonuclease activity).

Research

Because the 5' → 3' exonuclease activity of DNA polymerase I from ''

'' 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 receded 3' ends of DNA fragments to make 5' overhang blunt * Digesting away protruding 3' overhangs * Preparation of

radioactive Radioactive decay (also known as nuclear decay, radioactivity, radioactive disintegration, or nuclear disintegration) is the process by which an unstable atomic nucleus loses energy by radiation. A material containing unstable nuclei is conside ...

DNA probe In molecular biology, a hybridization probe (HP) is a fragment of DNA or RNA, usually 15–10000 nucleotides long, which can be radioactively or fluorescently labeled. HPs can be used to detect the presence of nucleotide sequences in analyzed ...

s The Klenow fragment was also the original enzyme used for greatly amplifying segments of DNA in the

polymerase chain reaction The polymerase chain reaction (PCR) is a method widely used to make millions to billions of copies of a specific DNA sample rapidly, allowing scientists to amplify a very small sample of DNA (or a part of it) sufficiently to enable detailed st ...

(PCR) process, before being replaced by

thermostable DNA polymerase Thermostable DNA polymerases are DNA polymerases that originate from thermophiles, usually bacterial or archaeal species, and are therefore thermostable. They are used for the polymerase chain reaction and related methods for the amplification a ...

s such as

Taq polymerase ''Taq'' polymerase is a thermostable DNA polymerase I named after the thermophilic eubacterial microorganism ''Thermus aquaticus,'' from which it was originally isolated by master's student Alice Chien et al. in 1976. Its name is often abbr ...

The exo-Klenow fragment

Just as the 5' → 3'

activity of

from ''

E.coli ''Escherichia coli'' ( )Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. is a gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus ''Escherichia'' that is commonly foun ...

'' can be undesirable, the 3' → 5'

activity of Klenow fragment can also be undesirable for certain applications. This problem can be overcome by introducing

mutation In biology, a mutation is an alteration in the nucleic acid sequence of the genome of an organism, virus, or extrachromosomal DNA. Viral genomes contain either DNA or RNA. Mutations result from errors during DNA or viral replication, ...

s in the gene that encodes Klenow. This results in forms of the enzyme being expressed that retain 5' → 3' polymerase activity, but lack any

activity (5' → 3' or 3' → 5'). This form of the enzyme is called the exo-Klenow fragment. The exo-Klenow fragment is used in some fluorescent labeling reactions for microarray, and also in dA and dT tailing, an important step in the process of ligating DNA adapters to DNA fragments, frequently used in preparing DNA libraries for Next-Gen sequencing. (for instance se

References

External links

*
Diagram at vivo.colostate.edu
{{DNA replication DNA replication