DNA polymerase II

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DNA Polymerase II
Pol2 structure (Based on 35KM).png
Crystal structure of DNA pol II (based on PDB entry 3K5M)
Identifiers
Organism Escherichia coli
(str. K-12 substr. MG1655)
SymbolpolB
Entrez 944779
PDB 3K5M
RefSeq (Prot) NP_414602.1
UniProt P21189
Other data
EC number 2.7.7.7
Chromosome genome: 0.06 - 0.07 Mb
Search for
Structures Swiss-model
Domains InterPro

DNA polymerase II (also known as DNA Pol II or Pol II) is a prokaryotic DNA-dependent DNA polymerase encoded by the PolB gene. [1]

Contents

DNA Polymerase II is an 89.9-kDa protein and is a member of the B family of DNA polymerases. It was originally isolated by Thomas Kornberg in 1970, and characterized over the next few years. [2] [3] [4] The in vivo functionality of Pol II is under debate, yet consensus shows that Pol II is primarily involved as a backup enzyme in prokaryotic DNA replication. The enzyme has 5′→3′ DNA synthesis capability as well as 3′→5′ exonuclease proofreading activity. DNA Pol II interacts with multiple binding partners common with DNA Pol III in order to enhance its fidelity and processivity. [1]

Discovery

DNA polymerase I was the first DNA-directed DNA polymerase to be isolated from E. coli . [5] Several studies involving this isolated enzyme indicated that DNA pol I was most likely involved in repair replication and was not the main replicative polymerase. [6] In order to better understand the in vivo role of DNA pol I, E. coli mutants deficient in this enzyme (termed Pol A1−) were generated in 1969 by De Lucia and Cairns. [7] As characterized, this new mutant strain was more sensitive to ultraviolet light, corroborating the hypothesis that DNA pol I was involved in repair replication. The mutant grew at the same rate as the wild type, indicating the presence of another enzyme responsible for DNA replication. The isolation and characterization of this new polymerase involved in semiconservative DNA replication followed, in parallel studies conducted by several labs. [2] [3] [4] The new polymerase was termed DNA polymerase II, and was believed to be the main replicative enzyme of E. coli for a time. [8] DNA pol II was first crystallized by Anderson et al. in 1994. [9]

In 2023 it was reported that ageing-related accelerated transcription causes Pol II to make more mistakes, leading to flawed copies that can cause numerous diseases. [10]

Structure

DNA Pol II is an 89.9 kD protein, composed of 783 amino acids, that is encoded by the polB (dinA) gene. A globular protein, DNA Pol II functions as a monomer, whereas many other polymerases will form complexes. There are three main sections of this monomer colloquially referred to as the palm, fingers, and thumb. This “hand” closes around a strand of DNA. The palm of the complex contains three catalytic residues that will coordinate with two divalent metal ions in order to function. DNA Pol II has a high quantity of copies in the cell, around 30-50, whereas the level of DNA Pol III in a cell is five times fewer.

General "hand" structure of a Group B Polymerase (PDB: 3NCI ) WikiHandDNAPolII.png
General "hand" structure of a Group B Polymerase ( PDB: 3NCI ​)

Similarity to other group B polymerases

Most of the polymerases have been grouped into families based on similar structure and function. DNA Pol II falls into the Group B along with human DNA Pol α, δ, ϵ, and ζ. These are all homologs of RB69, 9°N-7, and Tgo. The other members of group B do have at least one other subunit which makes the DNA Pol II unique. [11]

Function

Confirmed

Polymerases all are involved with DNA replication in some capacity, synthesizing chains of nucleic acids. DNA replication is a vital aspect of a cell's proliferation. Without replicating its DNA, a cell cannot divide and share its genetic information to progeny. In prokaryotes, like E. coli, DNA Pol III is the major polymerase involved with DNA replication. While DNA Pol II is not a major factor in chromosome replication, it has other roles to fill.

DNA Pol II does participate in DNA replication. While it might not be as fast as DNA Pol III, it has some abilities that make it an effective enzyme. This enzyme has an associated 3′→5′ exonuclease activity along with primase activity. DNA Pol II is a high fidelity enzyme with a substitution error rate of ≤ 2×10−6 and a −1 frameshift error rate of ≤ 1×10−6. DNA Pol II can proofread and process mismatches caused by the Pol III. Banach-Orlowska et al. showed that DNA Pol II is involved with replication but it is strand dependent and preferentially replicates the lagging strand. A proposed mechanism suggests that when DNA Pol III stalls or becomes non-functional, then DNA Pol II is able to be specifically recruited to the replication point and continue replication. [1]

There are many different ways that DNA can be damaged, from UV damage to oxidation, so it is logical that there are different types of polymerases to fix these damages. One important role that DNA Pol II is the major polymerase for the repairing of inter-strand cross-links. Interstrand cross-links are caused by chemicals such as nitrogen mustard and psoralen which create cytotoxic lesions. Repairing these lesions is difficult because both DNA strands have been damaged by the chemical agent and thus the genetic information on both strands is incorrect. The exact mechanism of how these interstrand cross-links are fixed is still being researched, but it is known that Pol II is highly involved. [11]

Activity

DNA Pol II is not the most studied polymerase so there are many proposed functions of this enzyme which are all likely functions but are ultimately unconfirmed: [1]

Mechanism

DNA Polymerase II Repair Active Site (PDB ID: 3K5M) DNA Pol II Active Site.png
DNA Polymerase II Repair Active Site (PDB ID: 3K5M)

During DNA replication, base pairs are subject to damage in the sequence. A damaged sequence of DNA can cause replication to be stalled. [12] In order to fix an error in the sequence, DNA Pol II catalyzes the repair of nucleotide base pairs. In vitro studies have shown that Pol II occasionally interacts with Pol III accessory proteins (β‐clamp and clamp loading complex) giving the Pol II access to the growing nascent strand. [1] [13] [14] [15] Concerning the function of DNA Pol II during DNA replication, this makes sense as any mistakes that Pol III produces will be in the growing strand and not the conservative strand. The N-terminal domain of DNA Pol II is responsible for the association and dissociation of the DNA strand to the catalytic subunit. There are most likely two sites in the N-terminal domain of DNA Pol II that recognize single-stranded DNA. One site(s) is responsible for recruiting single-stranded DNA to DNA Pol II and another site(s) is responsible for the dissociation of single-stranded DNA from DNA Pol II. [16]

DNA Pol II Active Site (PDB ID 3K5M) DNA Pol II Active Site (PDB ID 3K5M).png
DNA Pol II Active Site (PDB ID 3K5M)

Upon binding of substrate, DNA Pol II binds nucleoside triphosphates to maintain the hydrogen bonded structure of DNA. The correct dNTP is then bound and the enzyme complex undergoes conformational changes of subdomains and amino acid residues. These conformational changes allow the rate of repair synthesis to be fast. [17] The active site contains two Mg2+ ions that are stabilized by catalytic Aspartic Acids D419 and D547. [18] Magnesium ions bind to DNA along with dNTP in the open state and coordinate conformational changes of active site amino acid residues in order for catalysis to take place (closed state). After magnesium ions are released, the enzyme returns to its open state. [19]

Species distribution

Prokaryotic

DNA Polymerase II is a member of the polymerase B family and supports Polymerase III in DNA replication moving from the 3′ end to the 5′ end. [20] In the case when Polymerase III stalls during a replication error, Polymerase II can interrupt and excise the mismatched bases. Polymerase II has a much higher fidelity factor than Polymerase III, meaning that it is much less likely to create mispairings. Without Polymerase II's proofreading step, Polymerase III would extend the mispairings and thus create a mutation. [1]

In addition to protecting from mutations that could be caused by Polymerase III, Polymerase II functions to protect against mutations caused by Polymerase IV. Polymerase IV is much more error prone than Polymerase II but also functions to repair mismatched base pairings starting from the 3′ end. Polymerase II protects the 3′ end from Polymerase IV and blocks it from acting. This protection will prevent the formation of mutations while the Polymerase II is functioning normally. If the Polymerase II is knocked out by a mutation or disabled by other factors, Polymerase IV will take its place to fix the mispaired bases. [1]

Eukaryotic

While Polymerase II will not function naturally in conjunction with the eukaryotic members of Family B, it does share similar structural and functional motifs. The members of Family B include Polymerase α, ε, ζ, and δ. These polymerases all function to proofread the newly synthesized DNA in the 3′→5′ direction. These polymerases are capable of synthesizing DNA on both the leading and lagging strands. This class of polymerase tends to be very accurate which allows them to correct any mispairings that occur during DNA synthesis. [20]

Regulation

DNA Polymerase II is naturally abundant in the cell, which usually amounts to five times greater than the amount of Polymerase III. This greater abundance allows Polymerase II to overpower Polymerase III in the case of mispairings. This amount can be increased upon the inducement of the SOS response, which upregulates the polB gene so the amount of Polymerase II increases to about sevenfold greater. Although Polymerase II can work on both strands, it has been shown to prefer the lagging strand versus the leading strand. [1]

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">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

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.

<span class="mw-page-title-main">DNA polymerase I</span> Family of enzymes

DNA polymerase 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. 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. 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.

<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.

dnaQ is the gene encoding the ε subunit of DNA polymerase III in Escherichia coli. The ε subunit is one of three core proteins in the DNA polymerase complex. It functions as a 3’→5’ DNA directed proofreading exonuclease that removes incorrectly incorporated bases during replication. dnaQ may also be referred to as mutD.

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">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.

A nick is a discontinuity in a double stranded DNA molecule where there is no phosphodiester bond between adjacent nucleotides of one strand typically through damage or enzyme action. Nicks allow DNA strands to untwist during replication, and are also thought to play a role in the DNA mismatch repair mechanisms that fix errors on both the leading and lagging daughter strands.

<span class="mw-page-title-main">Slipped strand mispairing</span> Nucleotide duplications created by DNA polymerase during DNA replication

Slipped strand mispairing is a mutation process which occurs during DNA replication. It involves denaturation and displacement of the DNA strands, resulting in mispairing of the complementary bases. Slipped strand mispairing is one explanation for the origin and evolution of repetitive DNA sequences.

<span class="mw-page-title-main">Prokaryotic DNA replication</span> DNA Replication in prokaryotes

Prokaryotic DNA Replication is the process by which a prokaryote duplicates its DNA into another copy that is passed on to daughter cells. Although it is often studied in the model organism E. coli, other bacteria show many similarities. Replication is bi-directional and originates at a single origin of replication (OriC). It consists of three steps: Initiation, elongation, and termination.

<span class="mw-page-title-main">Eukaryotic transcription</span> Transcription is heterocatalytic function of DNA

Eukaryotic transcription is the elaborate process that eukaryotic cells use to copy genetic information stored in DNA into units of transportable complementary RNA replica. Gene transcription occurs in both eukaryotic and prokaryotic cells. Unlike prokaryotic RNA polymerase that initiates the transcription of all different types of RNA, RNA polymerase in eukaryotes comes in three variations, each translating a different type of gene. A eukaryotic cell has a nucleus that separates the processes of transcription and translation. Eukaryotic transcription occurs within the nucleus where DNA is packaged into nucleosomes and higher order chromatin structures. The complexity of the eukaryotic genome necessitates a great variety and complexity of gene expression control.

<span class="mw-page-title-main">POLD1</span> Protein-coding gene in the species Homo sapiens

The gene polymerase delta 1 (POLD1) encodes the large, POLD1/p125, catalytic subunit of the DNA polymerase delta (Polδ) complex. The Polδ enzyme is responsible for synthesizing the lagging strand of DNA, and has also been implicated in some activities at the leading strand. The POLD1/p125 subunit encodes both DNA polymerizing and exonuclease domains, which provide the protein an important second function in proofreading to ensure replication accuracy during DNA synthesis, and in a number of types of replication-linked DNA repair following DNA damage.

<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.

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. The proofreading mechanisms of Hopfield and Ninio are non-equilibrium active processes that consume ATP to enhance specificity of various biochemical reactions.

<span class="mw-page-title-main">Circular chromosome</span> Type of chromosome

A circular chromosome is a chromosome in bacteria, archaea, mitochondria, and chloroplasts, in the form of a molecule of circular DNA, unlike the linear chromosome of most eukaryotes.

DNA polymerase IV is a prokaryotic polymerase that is involved in mutagenesis and is encoded by the dinB gene. It exhibits no 3′→5′ exonuclease (proofreading) activity and hence is error prone. In E. coli, DNA polymerase IV is involved in non-targeted mutagenesis. Pol IV is a Family Y polymerase expressed by the dinB gene that is switched on via SOS induction caused by stalled polymerases at the replication fork. During SOS induction, Pol IV production is increased tenfold and one of the functions during this time is to interfere with Pol III holoenzyme processivity. This creates a checkpoint, stops replication, and allows time to repair DNA lesions via the appropriate repair pathway. Another function of Pol IV is to perform translesion synthesis at the stalled replication fork like, for example, bypassing N2-deoxyguanine adducts at a faster rate than transversing undamaged DNA. Cells lacking dinB gene have a higher rate of mutagenesis caused by DNA damaging agents.

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.

References

  1. 1 2 3 4 5 6 7 8 Banach-Orlowska M, Fijalkowska IJ, Schaaper RM, Jonczyk P (October 2005). "DNA polymerase II as a fidelity factor in chromosomal DNA synthesis in Escherichia coli". Molecular Microbiology. 58 (1): 61–70. doi: 10.1111/j.1365-2958.2005.04805.x . PMID   16164549.
  2. 1 2 Kornberg T, Gefter ML (September 1970). "DNA synthesis in cell-free extracts of a DNA polymerase-defective mutant". Biochemical and Biophysical Research Communications. 40 (6): 1348–55. doi:10.1016/0006-291X(70)90014-8. PMID   4933688.
  3. 1 2 Moses RE, Richardson CC (December 1970). "A new DNA polymerase activity of Escherichia coli. I. Purification and properties of the activity present in E. coli polA1". Biochemical and Biophysical Research Communications. 41 (6): 1557–64. doi:10.1016/0006-291X(70)90565-6. PMID   4922636.
  4. 1 2 Knippers R (December 1970). "DNA polymerase II". Nature. 228 (5276): 1050–3. Bibcode:1970Natur.228.1050K. doi:10.1038/2281050a0. PMID   4921664. S2CID   4211529.
  5. ↑ 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.
  6. ↑ Smith DW, Schaller HE, Bonhoeffer FJ (May 1970). "DNA synthesis in vitro". Nature. 226 (5247): 711–3. Bibcode:1970Natur.226..711S. doi:10.1038/226711a0. PMID   4910150. S2CID   1505496.
  7. ↑ 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.
  8. ↑ Kornberg T, Gefter ML (April 1971). "Purification and DNA synthesis in cell-free extracts: properties of DNA polymerase II". Proceedings of the National Academy of Sciences of the United States of America. 68 (4): 761–4. Bibcode:1971PNAS...68..761K. doi: 10.1073/pnas.68.4.761 . PMC   389037 . PMID   4927672.
  9. ↑ Anderson WF, Prince DB, Yu H, McEntee K, Goodman MF (April 1994). "Crystallization of DNA polymerase II from Escherichia coli". Journal of Molecular Biology. 238 (1): 120–2. doi:10.1006/jmbi.1994.1765. PMID   8145251.
  10. ↑ "Ageing-associated changes in transcriptional elongation influence longevity". Nature. 12 April 2023. Retrieved 22 May 2023.
  11. 1 2 Bebenek K, Kunkel TA (2004). "Functions of DNA polymerases". Advances in Protein Chemistry. 69: 137–65. doi:10.1016/S0065-3233(04)69005-X. ISBN   9780120342693. PMID   15588842.
  12. ↑ Becherel OJ, Fuchs RP (July 2001). "Mechanism of DNA polymerase II-mediated frameshift mutagenesis". Proceedings of the National Academy of Sciences of the United States of America. 98 (15): 8566–71. Bibcode:2001PNAS...98.8566B. doi: 10.1073/pnas.141113398 . PMC   37476 . PMID   11447256.
  13. ↑ Wickner S, Hurwitz J (October 1974). "Conversion of phiX174 viral DNA to double-stranded form by purified Escherichia coli proteins". Proceedings of the National Academy of Sciences of the United States of America. 71 (10): 4120–4. Bibcode:1974PNAS...71.4120W. doi: 10.1073/pnas.71.10.4120 . PMC   434340 . PMID   4610569.
  14. ↑ Hughes AJ, Bryan SK, Chen H, Moses RE, McHenry CS (March 1991). "Escherichia coli DNA polymerase II is stimulated by DNA polymerase III holoenzyme auxiliary subunits". The Journal of Biological Chemistry. 266 (7): 4568–73. doi: 10.1016/S0021-9258(20)64360-5 . PMID   1999435.
  15. ↑ Bonner CA, Stukenberg PT, Rajagopalan M, Eritja R, O'Donnell M, McEntee K, et al. (June 1992). "Processive DNA synthesis by DNA polymerase II mediated by DNA polymerase III accessory proteins". The Journal of Biological Chemistry. 267 (16): 11431–8. doi: 10.1016/S0021-9258(19)49928-6 . PMID   1534562.
  16. ↑ Maki S, Hashimoto K, Ohara T, Sugino A (August 1998). "DNA polymerase II (epsilon) of Saccharomyces cerevisiae dissociates from the DNA template by sensing single-stranded DNA". The Journal of Biological Chemistry. 273 (33): 21332–41. doi: 10.1074/jbc.273.33.21332 . PMID   9694894.
  17. ↑ Beard WA, Wilson SH (May 2014). "Structure and mechanism of DNA polymerase β". Biochemistry. 53 (17): 2768–80. doi:10.1021/bi500139h. PMC   4018062 . PMID   24717170.
  18. ↑ Wang F, Yang W (December 2009). "Structural insight into translesion synthesis by DNA Pol II". Cell. 139 (7): 1279–89. doi:10.1016/j.cell.2009.11.043. PMC   3480344 . PMID   20064374.
  19. ↑ Yang L, Arora K, Beard WA, Wilson SH, Schlick T (July 2004). "Critical role of magnesium ions in DNA polymerase beta's closing and active site assembly". Journal of the American Chemical Society. 126 (27): 8441–53. doi:10.1021/ja049412o. PMID   15238001.
  20. 1 2 Mandal A (26 February 2019). "Prokaryotic DNA Polymerases". News Medical.
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