DNA polymerase I
DNA polymerase I | |||||||
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UniProt P00582 | | ||||||
Other data | |||||||
EC number | 2.7.7.7 | ||||||
Chromosome | genome: 4.04 - 4.05 Mb | ||||||
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DNA polymerase I (or Pol I) is an
Discovery
In 1956,
Structure and function
General structure
Pol I mainly functions in the repair of damaged DNA. Structurally, Pol I is a member of the alpha/beta protein superfamily, which encompasses proteins in which α-helices and β-strands occur in irregular sequences.
E. coli bacteria produces 5 different DNA polymerases: DNA Pol I, DNA Pol II, DNA Pol III, DNA Pol IV, and DNA Pol V.[6]
Structural and functional similarity to other polymerases
In DNA replication, the leading DNA strand is continuously extended in the direction of replication fork movement, whereas the DNA lagging strand runs discontinuously in the opposite direction as
The X-ray crystal structures of polymerase domains of DNA polymerases are described in analogy to human right hands. All DNA polymerases contain three domains. The first domain, which is known as the "fingers domain", interacts with the dNTP and the paired template base. The "fingers domain" also interacts with the template to position it correctly at the active site.[9] Known as the "palm domain", the second domain catalyses the reaction of the transfer of the phosphoryl group. Lastly, the third domain, which is known as the "thumb domain", interacts with double stranded DNA.[10] The exonuclease domain contains its own catalytic site and removes mispaired bases. Among the seven different DNA polymerase families, the "palm domain" is conserved in five of these families. The "finger domain" and "thumb domain" are not consistent in each family due to varying secondary structure elements from different sequences.[9]
Function
Pol I possesses four enzymatic activities:
- A 5'→3' (forward) DNA-dependent DNA polymerase activity, requiring a 3' primer site and a template strand
- A 3'→5' (reverse) exonuclease activity that mediates proofreading
- A 5'→3' (forward) exonuclease activity mediating nick translation during DNA repair.
- A 5'→3' (forward) RNA-dependent DNA polymerase activity. Pol I operates on RNA templates with considerably lower efficiency (0.1–0.4%) than it does DNA templates, and this activity is probably of only limited biological significance.[11]
In order to determine whether Pol I was primarily used for DNA replication or in the repair of DNA damage, an experiment was conducted with a deficient Pol I mutant strain of E. coli. The mutant strain that lacked Pol I was isolated and treated with a mutagen. The mutant strain developed bacterial colonies that continued to grow normally and that also lacked Pol I. This confirmed that Pol I was not required for DNA replication. However, the mutant strain also displayed characteristics which involved extreme sensitivity to certain factors that damaged DNA, like
Mechanism
In the replication process,
Despite its early characterization, 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
Research applications
DNA polymerase I obtained from E. coli is used extensively for molecular biology research. However, the 5'→3' exonuclease activity makes it unsuitable for many applications. This undesirable enzymatic activity can be simply removed from the holoenzyme to leave a useful molecule called the Klenow fragment, widely used in molecular biology. In fact, the Klenow fragment was used during the first protocols of polymerase chain reaction (PCR) amplification until Thermus aquaticus, the source of a heat-tolerant Taq Polymerase I, was discovered in 1976.[15] Exposure of DNA polymerase I to the protease subtilisin cleaves the molecule into a smaller fragment, which retains only the DNA polymerase and proofreading activities.
See also
- DNA polymerase II
- DNA polymerase III
- DNA polymerase V
References
- PMID 13563462.
- ^ Voet D, Voet JG, Pratt CW (1999). Fundamentals of Biochemistry. New York: Wiley.[page needed]
- PMID 12791679.
- ^ "The Nobel Prize in Physiology or Medicine 1959". www.nobelprize.org. Retrieved 2016-11-08.
- ^ a b c Cox MM, Doudna J (2015). Molecular Biology (2nd ed.). New York: W.H. Freeman.[page needed]
- ^ a b c Cooper, Geoffrey M. Geoffrey (2000-01-01). "DNA Replication".
{{cite journal}}
: Cite journal requires|journal=
(help) - ISBN 978-981-4299-16-9.[page needed]
- ^ "DNA Polymerase I: Enzymatic Reactions".
- ^ a b "MBIO.4.14.5". bioscience.jbpub.com. Retrieved 2017-05-14.
- S2CID 3344014.
- PMID 7679988.
- S2CID 4182917.
- S2CID 39605644.
- ^ EMBL-EBI. "EMBL European Bioinformatics Institute". www.ebi.ac.uk. Retrieved 2016-11-08.
- ISBN 978-1-4020-6240-7.