DNA polymerase I
Encyclopedia
DNA Polymerase I is an enzyme
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...

 that participates in the process of DNA replication
DNA replication
DNA replication is a biological process that occurs in all living organisms and copies their DNA; it is the basis for biological inheritance. The process starts with one double-stranded DNA molecule and produces two identical copies of the molecule...

 in prokaryote
Prokaryote
The prokaryotes are a group of organisms that lack a cell nucleus , or any other membrane-bound organelles. The organisms that have a cell nucleus are called eukaryotes. Most prokaryotes are unicellular, but a few such as myxobacteria have multicellular stages in their life cycles...

s. It is composed of 928 amino acids, and is an example of a processive
Processivity
In molecular biology, processivity is a measure of the average number of nucleotides added by a DNA polymerase enzyme per association/disassociation with the template. DNA polymerases associated with DNA replication tend to be highly processive, while those associated with DNA repair tend to have...

 enzyme - it can sequentially catalyze multiple polymerisations. Discovered by Arthur Kornberg
Arthur Kornberg
Arthur Kornberg was an American biochemist who won the Nobel Prize in Physiology or Medicine 1959 for his discovery of "the mechanisms in the biological synthesis of deoxyribonucleic acid " together with Dr. Severo Ochoa of New York University...

 in 1956, it was the first known DNA polymerase
DNA polymerase
A DNA polymerase is an enzyme that helps catalyze in the polymerization of deoxyribonucleotides into a DNA strand. DNA polymerases are best known for their feedback role in DNA replication, in which the polymerase "reads" an intact DNA strand as a template and uses it to synthesize the new strand....

 (and, indeed, the first known of any kind of polymerase
Polymerase
A polymerase is an enzyme whose central function is associated with polymers of nucleic acids such as RNA and DNA.The primary function of a polymerase is the polymerization of new DNA or RNA against an existing DNA or RNA template in the processes of replication and transcription...

). It was initially characterized in E. coli, although it is ubiquitous in prokaryote
Prokaryote
The prokaryotes are a group of organisms that lack a cell nucleus , or any other membrane-bound organelles. The organisms that have a cell nucleus are called eukaryotes. Most prokaryotes are unicellular, but a few such as myxobacteria have multicellular stages in their life cycles...

s. In E. coli and many other bacteria, the gene
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...

 which encodes Pol I is known as polA.

Pol I possesses three enzymatic activities:
  1. A 5' -> 3'
    Directionality (molecular biology)
    Directionality, in molecular biology and biochemistry, is the end-to-end chemical orientation of a single strand of nucleic acid. The chemical convention of naming carbon atoms in the nucleotide sugar-ring numerically gives rise to a 5′-end and a 3′-end...

     (forward) DNA polymerase activity, requiring a 3' primer
    Primer (molecular biology)
    A primer is a strand of nucleic acid that serves as a starting point for DNA synthesis. They are required for DNA replication because the enzymes that catalyze this process, DNA polymerases, can only add new nucleotides to an existing strand of DNA...

     site and a template strand
  2. A 3' -> 5' (reverse) exonuclease
    Exonuclease
    Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end of a polynucleotide chain. A hydrolyzing reaction that breaks phosphodiester bonds at either the 3’ or the 5’ end occurs. Its close relative is the endonuclease, which cleaves phosphodiester bonds in the middle ...

     activity that mediates proofreading
    Proofreading (biology)
    The term proofreading is used in genetics to refer to the error-correcting processes, first proposed by John Hopfield and Jacques Ninio, involved in DNA replication, immune system specificity, enzyme-substrate recognition among many other processes that require enhanced specificity...

  3. A 5' -> 3' (forward) exonuclease activity mediating nick translation
    Nick translation
    Nick translation was developed in 1977 by Rigby and Paul Berg. It is a tagging technique in molecular biology in which DNA Polymerase I is used to replace some of the nucleotides of a DNA sequence with their labeled analogues, creating a tagged DNA sequence which can be used as a probe in...

     during DNA repair
    DNA repair
    DNA repair refers to a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. In human cells, both normal metabolic activities and environmental factors such as UV light and radiation can cause DNA damage, resulting in as many as 1...

    .


In the replication process, DNA Polymerase I removes the RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....

 primer (created by Primase
Primase
DNA primase is an enzyme involved in the replication of DNA.Primase catalyzes the synthesis of a short RNA segment called a primer complementary to a ssDNA template...

) from the lagging strand and fills in the necessary nucleotides of the Okazaki fragments (see DNA replication
DNA replication
DNA replication is a biological process that occurs in all living organisms and copies their DNA; it is the basis for biological inheritance. The process starts with one double-stranded DNA molecule and produces two identical copies of the molecule...

) in 5' -> 3' direction, proofreading for mistakes as it goes. It is a template-dependent enzyme - it only adds nucleotides that correctly base pair
Base pair
In molecular biology and genetics, the linking between two nitrogenous bases on opposite complementary DNA or certain types of RNA strands that are connected via hydrogen bonds is called a base pair...

 with an existing DNA strand acting as a template. Ligase
Ligase
In biochemistry, ligase is an enzyme that can catalyse the joining of two large molecules by forming a new chemical bond, usually with accompanying hydrolysis of a small chemical group dependent to one of the larger molecules...

 then joins the various fragments together into a continuous strand of DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...

.

Despite its early characterisation, it quickly became apparent that Polymerase I was not the enzyme responsible for most DNA synthesis — DNA replication in E. coli proceeds at approximately 1,000 nucleotides/second, while the rate of base pair synthesis by Polymerase I averages only between 10 and 20 nucleotides/second. Moreover, its cellular abundance of approximately 400 molecules per cell did not correlate with the fact that there are typically only two replication fork
Replication fork
The replication fork is a structure that forms within the nucleus during DNA replication. It is created by helicases, which break the hydrogen bonds holding the two DNA strands together. The resulting structure has two branching "prongs", each one made up of a single strand of DNA...

s in E. coli. Moreover, it is insufficiently processive
Processivity
In molecular biology, processivity is a measure of the average number of nucleotides added by a DNA polymerase enzyme per association/disassociation with the template. DNA polymerases associated with DNA replication tend to be highly processive, while those associated with DNA repair tend to have...

 to copy an entire genome
Genome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....

, as it falls off after incorporating only 25-50 nucleotides. Its role in replication was proven when, in 1969, John Cairns
John Cairns (biochemist)
John Forster Cairns FRS is a British physician and molecular biologist who made significant contributions to molecular genetics, cancer research, and public health....

 isolated a viable Polymerase I mutant
Mutant
In biology and especially genetics, a mutant is an individual, organism, or new genetic character, arising or resulting from an instance of mutation, which is a base-pair sequence change within the DNA of a gene or chromosome of an organism resulting in the creation of a new character or trait not...

 that lacked the polymerase activity. Cairns' lab assistant Paula De Lucia created thousands of cell free extracts from E.coli colonies and assayed them for DNA-polymerase activity. The 3,478th clone contained the polA
Pola
Pola may refer to:In places:*Pola, Lesser Poland Voivodeship, in south Poland*Pola, Oriental Mindoro, a municipality in the Philippines*Pola de Allande, a town and a parish in Allande, a municipality within the province of Asturias, in northern Spain...

 mutant, which was named by Cairns to credit "Paula" [De Lucia]. It was not until the discovery of DNA polymerase III that the main replicative DNA polymerase was finally identified.

Research applications

DNA polymerase I obtained from E. coli is used extensively for molecular biology
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...

 research. However, the 5' -> 3' exonuclease activity makes it unsuitable for many applications. Fortunately this undesirable enzymatic activity can be simply removed from the holoenzyme to leave a useful molecule called the Klenow fragment
Klenow fragment
right|thumb|450px|Functional domains in the Klenow Fragment and DNA Polymerase I .The Klenow fragment is a large protein fragment produced when DNA polymerase I from E. coli is enzymatically cleaved by the protease subtilisin...

, widely used in molecular biology
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...

. Exposure of DNA polymerase I to the protease subtilisin
Subtilisin
Subtilisin is a non-specific protease initially obtained from Bacillus subtilis.Subtilisins belong to subtilases, a group of serine proteases that initiate the nucleophilic attack on the peptide bond through a serine residue at the active site. They are physically and chemically...

cleaves the molecule into a smaller fragment, which retains only the DNA polymerase and proofreading activities.
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