DNA polymerase I

Last updated
DNA polymerase I
PolymeraseDomains.jpg
Functional domains in the Klenow Fragment (left) and DNA Polymerase I (right).
Identifiers
Organism Escherichia coli
(str. K-12 substr. MG1655)
SymbolpolA
Entrez 948356
PDB 1DPI
RefSeq (Prot) NP_418300.1
UniProt P00582
Other data
EC number 2.7.7.7
Chromosome genome: 4.04 - 4.05 Mb
Search for
Structures Swiss-model
Domains InterPro

DNA polymerase I (or Pol I) is an enzyme that participates in the process of prokaryotic DNA replication. Discovered by Arthur Kornberg in 1956, [1] it was the first known DNA polymerase (and the first known of any kind of polymerase). It was initially characterized in E. coli and is ubiquitous in prokaryotes. In E. coli and many other bacteria, the gene that encodes Pol I is known as polA. The E. coli Pol I enzyme is composed of 928 amino acids, and is an example of a processive enzyme — it can sequentially catalyze multiple polymerisation steps without releasing the single-stranded template. [2] The physiological function of Pol I is mainly to support repair of damaged DNA, but it also contributes to connecting Okazaki fragments by deleting RNA primers and replacing the ribonucleotides with DNA.

Contents

Discovery

In 1956, Arthur Kornberg and colleagues discovered Pol I by using Escherichia coli (E. coli) extracts to develop a DNA synthesis assay. The scientists added 14C-labeled thymidine so that a radioactive polymer of DNA, not RNA, could be retrieved. To initiate the purification of DNA polymerase, the researchers added streptomycin sulfate to the E. coli extract. This separated the extract into a nucleic acid-free supernatant (S-fraction) and nucleic acid-containing precipitate (P-fraction). The P-fraction also contained Pol I and heat-stable factors essential for the DNA synthesis reactions. These factors were identified as nucleoside triphosphates, the building blocks of nucleic acids. The S-fraction contained multiple deoxynucleoside kinases. [3] In 1959, the Nobel Prize in Physiology or Medicine was awarded to Arthur Kornberg and Severo Ochoa "for their discovery of the mechanisms involved in the biological synthesis of Ribonucleic acid and Deoxyribonucleic Acid." [4]

Arthur Kornberg 1969 Arthur Kornberg 1969.jpg
Arthur Kornberg 1969

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 DNA Pol I consists of multiple domains with three distinct enzymatic activities. Three domains, often referred to as thumb, finger and palm domain work together to sustain DNA polymerase activity. [5] A fourth domain next to the palm domain contains an exonuclease active site that removes incorrectly incorporated nucleotides in a 3' to 5' direction in a process known as proofreading. A fifth domain contains another exonuclease active site that removes DNA or RNA in a 5' to 3' direction and is essential for RNA primer removal during DNA replication or DNA during DNA repair processes.

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 Okazaki fragments. [7] DNA polymerases also cannot initiate DNA chains so they must be initiated by short RNA or DNA segments known as primers. [5] In order for DNA polymerization to take place, two requirements must be met. First of all, all DNA polymerases must have both a template strand and a primer strand. Unlike RNA, DNA polymerases cannot synthesize DNA from a template strand. Synthesis must be initiated by a short RNA segment, known as RNA primer, synthesized by Primase in the 5' to 3' direction. DNA synthesis then occurs by the addition of a dNTP to the 3' hydroxyl group at the end of the preexisting DNA strand or RNA primer. Secondly, DNA polymerases can only add new nucleotides to the preexisting strand through hydrogen bonding. [6] Since all DNA polymerases have a similar structure, they all share a two-metal ion-catalyzed polymerase mechanism. One of the metal ions activates the primer 3' hydroxyl group, which then attacks the primary 5' phosphate of the dNTP. The second metal ion will stabilize the leaving oxygen's negative charge, and subsequently chelates the two exiting phosphate groups. [8]

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:

  1. A 5'→3' (forward) DNA-dependent DNA polymerase activity, requiring a 3' primer site and a template strand
  2. A 3'→5' (reverse) exonuclease activity that mediates proofreading
  3. A 5'→3' (forward) exonuclease activity mediating nick translation during DNA repair.
  4. 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 UV light. Thus, this reaffirmed that Pol I was more likely to be involved in repairing DNA damage rather than DNA replication. [6]

Mechanism

In the replication process, RNase H removes the RNA primer (created by primase) from the lagging strand and then polymerase I fills in the necessary nucleotides between the Okazaki fragments (see DNA replication ) in a 5'→3' direction, proofreading for mistakes as it goes. It is a template-dependent enzyme—it only adds nucleotides that correctly base pair with an existing DNA strand acting as a template. It is crucial that these nucleotides are in the proper orientation and geometry to base pair with the DNA template strand so that DNA ligase can join the various fragments together into a continuous strand of DNA. Studies of polymerase I have confirmed that different dNTPs can bind to the same active site on polymerase I. Polymerase I is able to actively discriminate between the different dNTPs only after it undergoes a conformational change. Once this change has occurred, Pol I checks for proper geometry and proper alignment of the base pair, formed between bound dNTP and a matching base on the template strand. The correct geometry of A=T and G≡C base pairs are the only ones that can fit in the active site. However, it is important to know that one in every 104 to 105 nucleotides is added incorrectly. Nevertheless, Pol I can fix this error in DNA replication using its selective method of active discrimination. [5]

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 replication forks in E. coli. Additionally, it is insufficiently processive to copy an entire genome, as it falls off after incorporating only 25–50 nucleotides. Its role in replication was proven when, in 1969, John Cairns isolated a viable polymerase I mutant that lacked the polymerase activity. [12] 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 mutant, which was named by Cairns to credit "Paula" [De Lucia]. [13] It was not until the discovery of DNA polymerase III that the main replicative DNA polymerase was finally identified.

Research applications

DNA Polymerase I: Klenow Fragment (PDB 1KLN EBI) PDB 1kln EBI.jpg
DNA Polymerase I: Klenow Fragment (PDB 1KLN EBI)

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

Related Research Articles

<span class="mw-page-title-main">DNA replication</span> Biological process

In molecular biology, DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule. DNA replication occurs in all living organisms acting as the most essential part of biological inheritance. This is essential for cell division during growth and repair of damaged tissues, while it also ensures that each of the new cells receives its own copy of the DNA. The cell possesses the distinctive property of division, which makes replication of DNA essential.

<span class="mw-page-title-main">Primer (molecular biology)</span> Short strand of RNA or DNA that serves as a starting point for DNA synthesis

A primer is a short single-stranded nucleic acid used by all living organisms in the initiation of DNA synthesis. A synthetic primer may also be referred to as an oligo, short for oligonucleotide. DNA polymerase enzymes are only capable of adding nucleotides to the 3’-end of an existing nucleic acid, requiring a primer be bound to the template before DNA polymerase can begin a complementary strand. DNA polymerase adds nucleotides after binding to the RNA primer and synthesizes the whole strand. Later, the RNA strands must be removed accurately and replace them with DNA nucleotides forming a gap region known as a nick that is filled in using an enzyme called ligase. The removal process of the RNA primer requires several enzymes, such as Fen1, Lig1, and others that work in coordination with DNA polymerase, to ensure the removal of the RNA nucleotides and the addition of DNA nucleotides. Living organisms use solely RNA primers, while laboratory techniques in biochemistry and molecular biology that require in vitro DNA synthesis usually use DNA primers, since they are more temperature stable. Primers can be designed in laboratory for specific reactions such as polymerase chain reaction (PCR). When designing PCR primers, there are specific measures that must be taken into consideration, like the melting temperature of the primers and the annealing temperature of the reaction itself. Moreover, the DNA binding sequence of the primer in vitro has to be specifically chosen, which is done using a method called basic local alignment search tool (BLAST) that scans the DNA and finds specific and unique regions for the primer to bind.

<span class="mw-page-title-main">RNA polymerase</span> Enzyme that synthesizes RNA from DNA

In molecular biology, RNA polymerase, or more specifically DNA-directed/dependent RNA polymerase (DdRP), is an enzyme that catalyzes the chemical reactions that synthesize RNA from a DNA template.

Protein engineering is the process of developing useful or valuable proteins through the design and production of unnatural polypeptides, often by altering amino acid sequences found in nature. It is a young discipline, with much research taking place into the understanding of protein folding and recognition for protein design principles. It has been used to improve the function of many enzymes for industrial catalysis. It is also a product and services market, with an estimated value of $168 billion by 2017.

<span class="mw-page-title-main">DNA polymerase</span> Form of DNA replication

A DNA polymerase is a member of a family of enzymes that catalyze the synthesis of DNA molecules from nucleoside triphosphates, the molecular precursors of DNA. These enzymes are essential for DNA replication and usually work in groups to create two identical DNA duplexes from a single original DNA duplex. During this process, DNA polymerase "reads" the existing DNA strands to create two new strands that match the existing ones. These enzymes catalyze the chemical reaction

DNA primase is an enzyme involved in the replication of DNA and is a type of RNA polymerase. Primase catalyzes the synthesis of a short RNA segment called a primer complementary to a ssDNA template. After this elongation, the RNA piece is removed by a 5' to 3' exonuclease and refilled with DNA.

<span class="mw-page-title-main">Nuclease</span> Class of enzymes which cleave nucleic acids

In biochemistry, a nuclease is an enzyme capable of cleaving the phosphodiester bonds between nucleotides of nucleic acids. Nucleases variously effect single and double stranded breaks in their target molecules. In living organisms, they are essential machinery for many aspects of DNA repair. Defects in certain nucleases can cause genetic instability or immunodeficiency. Nucleases are also extensively used in molecular cloning.

DnaG is a bacterial DNA primase and is encoded by the dnaG gene. The enzyme DnaG, and any other DNA primase, synthesizes short strands of RNA known as oligonucleotides during DNA replication. These oligonucleotides are known as primers because they act as a starting point for DNA synthesis. DnaG catalyzes the synthesis of oligonucleotides that are 10 to 60 nucleotides long, however most of the oligonucleotides synthesized are 11 nucleotides. These RNA oligonucleotides serve as primers, or starting points, for DNA synthesis by bacterial DNA polymerase III. DnaG is important in bacterial DNA replication because DNA polymerase cannot initiate the synthesis of a DNA strand, but can only add nucleotides to a preexisting strand. DnaG synthesizes a single RNA primer at the origin of replication. This primer serves to prime leading strand DNA synthesis. For the other parental strand, the lagging strand, DnaG synthesizes an RNA primer every few kilobases (kb). These primers serve as substrates for the synthesis of Okazaki fragments.

<span class="mw-page-title-main">DNA polymerase III holoenzyme</span> Primary enzyme complex involved in prokaryotic DNA replication

DNA polymerase III holoenzyme is the primary enzyme complex involved in prokaryotic DNA replication. It was discovered by Thomas Kornberg and Malcolm Gefter in 1970. The complex has high processivity and, specifically referring to the replication of the E.coli genome, works in conjunction with four other DNA polymerases. Being the primary holoenzyme involved in replication activity, the DNA Pol III holoenzyme also has proofreading capabilities that corrects replication mistakes by means of exonuclease activity reading 3'→5' and synthesizing 5'→3'. DNA Pol III is a component of the replisome, which is located at the replication fork.

In molecular biology and biochemistry, processivity is an enzyme's ability to catalyze "consecutive reactions without releasing its substrate".

<span class="mw-page-title-main">Okazaki fragments</span> Transient components of lagging strand of DNA

Okazaki fragments are short sequences of DNA nucleotides which are synthesized discontinuously and later linked together by the enzyme DNA ligase to create the lagging strand during DNA replication. They were discovered in the 1960s by the Japanese molecular biologists Reiji and Tsuneko Okazaki, along with the help of some of their colleagues.

<span class="mw-page-title-main">Klenow fragment</span>

The Klenow fragment is a large protein fragment produced when DNA polymerase I from E. coli is enzymatically cleaved by the protease subtilisin. First reported in 1970, it retains the 5' → 3' polymerase activity and the 3’ → 5’ exonuclease activity for removal of precoding nucleotides and proofreading, but loses its 5' → 3' exonuclease activity.

<span class="mw-page-title-main">Exonuclease</span> Class of enzymes; type of nuclease

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 the endonuclease, which cleaves phosphodiester bonds in the middle (endo) of a polynucleotide chain. Eukaryotes and prokaryotes have three types of exonucleases involved in the normal turnover of mRNA: 5′ to 3′ exonuclease (Xrn1), which is a dependent decapping protein; 3′ to 5′ exonuclease, an independent protein; and poly(A)-specific 3′ to 5′ exonuclease.

<i>Taq</i> polymerase Thermostable form of DNA polymerase I used in polymerase chain reaction

Taq polymerase is a thermostable DNA polymerase I named after the thermophilic eubacterial microorganism Thermus aquaticus, from which it was originally isolated by Chien et al. in 1976. Its name is often abbreviated to Taq or Taq pol. It is frequently used in the polymerase chain reaction (PCR), a method for greatly amplifying the quantity of short segments of DNA.

<span class="mw-page-title-main">DNA polymerase II</span>

DNA polymerase II is a prokaryotic DNA-dependent DNA polymerase encoded by the PolB gene.

<span class="mw-page-title-main">Replisome</span> Molecular complex

The replisome is a complex molecular machine that carries out replication of DNA. The replisome first unwinds double stranded DNA into two single strands. For each of the resulting single strands, a new complementary sequence of DNA is synthesized. The total result is formation of two new double stranded DNA sequences that are exact copies of the original double stranded DNA sequence.

<span class="mw-page-title-main">T7 DNA polymerase</span>

T7 DNA polymerase is an enzyme used during the DNA replication of the T7 bacteriophage. During this process, the DNA polymerase “reads” existing DNA strands and creates two new strands that match the existing ones. The T7 DNA polymerase requires a host factor, E. coli thioredoxin, in order to carry out its function. This helps stabilize the binding of the necessary protein to the primer-template to improve processivity by more than 100-fold, which is a feature unique to this enzyme. It is a member of the Family A DNA polymerases, which include E. coli DNA polymerase I and Taq DNA polymerase.

Φ29 DNA polymerase is an enzyme from the bacteriophage Φ29. It is being increasingly used in molecular biology for multiple displacement DNA amplification procedures, and has a number of features that make it particularly suitable for this application. It was discovered and characterized by Spanish scientists Luis Blanco and Margarita Salas.

DNA Polymerase V is a polymerase enzyme involved in DNA repair mechanisms in bacteria, such as Escherichia coli. It is composed of a UmuD' homodimer and a UmuC monomer, forming the UmuD'2C protein complex. It is part of the Y-family of DNA Polymerases, which are capable of performing DNA translesion synthesis (TLS). Translesion polymerases bypass DNA damage lesions during DNA replication - if a lesion is not repaired or bypassed the replication fork can stall and lead to cell death. However, Y polymerases have low sequence fidelity during replication. When the UmuC and UmuD' proteins were initially discovered in E. coli, they were thought to be agents that inhibit faithful DNA replication and caused DNA synthesis to have high mutation rates after exposure to UV-light. The polymerase function of Pol V was not discovered until the late 1990s when UmuC was successfully extracted, consequent experiments unequivocally proved UmuD'2C is a polymerase. This finding lead to the detection of many Pol V orthologs and the discovery of the Y-family of polymerases.

<span class="mw-page-title-main">DNA polymerase alpha</span> Family of protein complexes

DNA polymerase alpha also known as Pol α is an enzyme complex found in eukaryotes that is involved in initiation of DNA replication. The DNA polymerase alpha complex consists of 4 subunits: POLA1, POLA2, PRIM1, and PRIM2.

References

  1. ↑ Lehman IR, Bessman MJ, Simms ES, Kornberg A (July 1958). "Enzymatic synthesis of deoxyribonucleic acid. I. Preparation of substrates and partial purification of an enzyme from Escherichia coli". The Journal of Biological Chemistry. 233 (1): 163–70. doi: 10.1016/S0021-9258(19)68048-8 . PMID   13563462.
  2. ↑ Voet D, Voet JG, Pratt CW (1999). Fundamentals of Biochemistry . New York: Wiley.[ page needed ]
  3. ↑ Lehman IR (September 2003). "Discovery of DNA polymerase". The Journal of Biological Chemistry. 278 (37): 34733–8. doi: 10.1074/jbc.X300002200 . PMID   12791679.
  4. ↑ "The Nobel Prize in Physiology or Medicine 1959". www.nobelprize.org. Retrieved 2016-11-08.
  5. 1 2 3 Cox MM, Doudna J (2015). Molecular Biology (2nd ed.). New York: W.H. Freeman.[ page needed ]
  6. 1 2 3 Cooper, Geoffrey M. Geoffrey (2000-01-01). "DNA Replication".{{cite journal}}: Cite journal requires |journal= (help)
  7. ↑ Hübscher U, Spadari S, Villani G, Maga G (2010). DNA Polymerases. doi:10.1142/7667. ISBN   978-981-4299-16-9.[ page needed ]
  8. ↑ "DNA Polymerase I: Enzymatic Reactions".
  9. 1 2 "MBIO.4.14.5". bioscience.jbpub.com. Retrieved 2017-05-14.
  10. ↑ Loeb LA, Monnat RJ (August 2008). "DNA polymerases and human disease". Nature Reviews Genetics. 9 (8): 594–604. doi:10.1038/nrg2345. PMID   18626473. S2CID   3344014.
  11. ↑ Ricchetti M, Buc H (February 1993). "E. coli DNA polymerase I as a reverse transcriptase". The EMBO Journal. 12 (2): 387–96. doi:10.1002/j.1460-2075.1993.tb05670.x. PMC   413221 . PMID   7679988.
  12. ↑ De Lucia P, Cairns J (December 1969). "Isolation of an E. coli strain with a mutation affecting DNA polymerase". Nature. 224 (5225): 1164–6. Bibcode:1969Natur.224.1164D. doi:10.1038/2241164a0. PMID   4902142. S2CID   4182917.
  13. ↑ Friedberg EC (February 2006). "The eureka enzyme: the discovery of DNA polymerase". Nature Reviews Molecular Cell Biology. 7 (2): 143–7. doi:10.1038/nrm1787. PMID   16493419. S2CID   39605644.
  14. ↑ EMBL-EBI. "EMBL European Bioinformatics Institute". www.ebi.ac.uk. Retrieved 2016-11-08.
  15. ↑ van Pelt-Verkuil E, van Belkum A, Hays JP (2008). "Taq and Other Thermostable DNA Polymerases". Principles and Technical Aspects of PCR Amplification. pp. 103–18. doi:10.1007/978-1-4020-6241-4_7. ISBN   978-1-4020-6240-7.
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