DNA polymerase
| Dictionary: DNA polymerase |
n.
Any of various enzymes that function in the replication and repair of DNA by catalyzing the linking of dATP, dCTP, dGTP, and dTTP in a specific order, using single-stranded DNA as a template.
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| Genetics Encyclopedia: DNA Polymerases |
DNA polymerases are proteins that synthesize new DNA strands using preexisting DNA strands as templates. Before one cell divides to produce two cells, the DNA containing the genetic information in it must be duplicated for the new cell, in a process known as polymerization. In human cells, duplicating the DNA genome requires the polymerization of 2.91 billion nucleotides, the building blocks of DNA. In the bacterium Escherichia coli, the polymerization of 4.64 million nucleotides is necessary to duplicate the genome for the new cell. In all cells, the DNA polymerases are the protein catalysts that link together the nucleotide building blocks of the new DNA polymer in an accurate and timely process that occurs during replication.
The DNA polymerases are also required to repair the DNA of the genome. The genome's DNA can be damaged by highly reactive molecules that are either produced in the cell during normal metabolic processes or brought into the cell from external sources. The damage, if not repaired, could result in the production of mutations in the genome or possibly cell death. Several DNA repair processes occurring in the cell have been identified that preserve the integrity of the genome by removing the damaged nucleotides and resynthesizing DNA by the DNA polymerases.
The Dna Polymerase Mechanism
All DNA polymerases share a common mechanism for DNA chain synthesis. The polymerization of DNA occurs by the linkage of one nucleotide at a time to the end of a preexisting DNA chain. The sequence fluctuations of the nucleotides on the DNA template upon which the DNA polymerase is moving determines which nucleotide is added onto the end of the growing DNA chain. If a thymine (T) nucleotide is positioned in the DNA template, for example, then an adenine (A) is polymerized onto the DNA chain opposite the thymine in the DNA template. If a guanine (G) nucleotide is positioned in the template, a cytosine (C) is linked to the growing DNA chain opposite the guanine. This polymerization process results in the synthesis of a DNA chain that is complementary, rather than identical, to the template strand of DNA, and is sequenced according to the proper Watson-Crick nucleotide base pairing rules. During replication, both strands of the duplex DNA molecule serve as templates. The DNA strands are separated, and each of the DNA strands is copied by the DNA polymerases. This process results in two identical copies of the original duplex DNA molecule being produced for the two cells.
The DNA polymerase uses the nucleoside triphosphate form of the deoxynucleotides to build the DNA polymer. The monophosphate form of the deoxynucleotide is incorporated into the growing DNA chain, and a pyrophosphate molecule, a kind of salt, is released. The DNA polymerase can add nucleotides only to the 3′-OH end of the growing DNA chain (see above diagram). Therefore, DNA polymerization occurs in only one direction. Some DNA polymerases are highly processive, polymerizing many nucleotides to the 3′ end of the DNA chain before falling off the DNA template. Other DNA polymerases are distributive in nature, incorporating just one nucleotide and then falling off the DNA template.
Occasionally, the DNA polymerase will incorrectly polymerize a nucleotide onto the growing DNA chain. Removal of this misinserted nucleotide must be performed by a "proofreading" exonuclease, which is a substance that removes nucleotides from the 3′ end of the DNA molecule. The combined actions of DNA polymerases and proofreading exonucleases improve the accuracy of DNA synthesis and thus minimize introduction of errors into the genome.
The Variety of Dna Polymerases
Like all proteins, the DNA polymerases are encoded in genes. The genes that encode the human DNA polymerases are contained in the genomic DNA at various positions on several different chromosomes. In February 2001 the first "working draft" sequence of the human genome was published. Analysis of this sequence shows us that there are perhaps as many as fifteen different DNA polymerase genes in the human genome. Each of these genes encodes a different DNA polymerase protein. However, biochemists have not yet isolated all of these enzymes. A similar analysis of the Escherichia coli genome shows us that there are five different DNA polymerase genes present in this bacterium. The multitude of DNA polymerases in human and bacterial cells indicates a specialized role for the different enzymes in various aspects of DNA replication and repair, many of which have yet to be identified.
The human DNA polymerase α is encoded in the POLA (polymerase alpha) gene located on the human X chromosome. The DNA polymerases δ and ε are encoded in the POLD1 (polymerase delta 1) and POLE1 (polymerase epsilon 1) genes, which are located on chromosomes 19 and 12, respectively. These three DNA polymerases are most frequently associated with replication of the human genome. The DNA polymerase β is encoded by the POLB (polymerase beta) gene on chromosome 8 and is involved in DNA repair.
The DNA polymerase γ is encoded by the POLG (polymerase gamma) gene on chromosome 15 and replicates the DNA of the mitochondria. In Escherichia coli the DNA polymerase I is the most active. This enzyme functions in the bacterial cell to repair DNA, while the DNA polymerase III is responsible for replicating the genome. There are several additional DNA polymerases in human and in bacterial cells of which the precise function is not known. Some of these enzymes might be necessary to replicate genomic DNA that has been damaged. The ability to replicate damaged DNA could lead to mutations introduced into the genome but would preserve the life of the cell.
The amino acid sequences of the DNA polymerase proteins can be deduced from the genetic code contained in the DNA polymerase genes. Based on the amino acid sequences of the DNA polymerases, these proteins have been classified into several families. Analysis of these sequences reveals a relatively diverse collection of proteins with some very important similarities in specific amino acid regions along the length of the protein.
The similarities in amino acid sequences in certain parts of the DNA polymerase proteins tell us that these regions of the protein have been conserved throughout evolution. These specific amino acids are those that are important in the catalytic function of DNA polymerization by these proteins. Some of the similar amino acids are necessary for binding metal atoms that are needed by the DNA polymerase to carry out the polymerization reaction. Other amino acid sequences allow the DNA polymerase to hold on to the DNA and the four different deoxynucleoside triphosphates as the enzyme polymerizes the new DNA chain. Some DNA polymerases have the necessary amino acid sequences to generate a 3′ proofreading exonuclease domain (region), allowing the DNA polymerase to remove mistakes, or proofread, as it builds the DNA polymer.
The DNA polymerases range in size from just over three hundred amino acids in length to more than two thousand amino acids in length. Three-dimensional studies of these enzymes have shown that the DNA polymerases have a common protein fold that resembles the shape of a "right hand" (see diagram). The "thumb," "fingers," and "palm" form a pocket along which the DNA can move. The DNA molecule interacts with specific amino acids located in the "palm" region of the DNA polymerase, and the "thumb" clamps down on the DNA to hold it as the DNA chain-elongation reaction proceeds. The amino acids that are common in many of the DNA polymerases are found in the regions where the enzyme contacts the DNA molecule.
Bibliography
Alberts, Bruce, et al. Molecular Biology of the Cell, 4th ed. New York: Garland Science, 2002.
Baltimore, D. "Our Genome Unveiled." Nature 409 (Feb. 15, 2001): 814-816.
Blattner, F. R., et al. "The Complete Genome Sequence of Escherichia coli K-12." Science 277 (1997): 1453-1462.
Venter, J. C., et al. "The Sequence of the Human Genome." Science 291 (2001): 1304-1351.
Wood, R. D., M. Mitchell, J. Sgouros, and T. Lindahl. "Human DNA Repair Genes." Science 291 (2001): 1284-1289.
—Fred W. Perrino
| Science Dictionary: DNA polymerase |
(pol-uh-muh-rays)
An enzyme that assembles new DNA by copying an existing strand.
| Wikipedia: DNA polymerase |
A DNA polymerase is an enzyme that catalyzes the polymerization of deoxyribonucleotides into a DNA strand. DNA polymerases are best-known for their role in DNA replication, in which the polymerase "reads" an intact DNA strand as a template and uses it to synthesize the new strand. The newly-polymerized molecule is complementary to the template strand and identical to the template's original partner strand. DNA polymerases use a magnesium ion for catalytic activity.
Contents |
Function
DNA polymerase can add free nucleotides to only the 3’ end of the newly-forming strand. This results in elongation of the new strand in a 5'-3' direction. No known DNA polymerase is able to begin a new chain (de novo). DNA polymerase can add a nucleotide onto only a preexisting 3'-OH group, and, therefore, needs a primer at which it can add the first nucleotide. Primers consist of RNA and DNA bases with the first two bases always being RNA, and are synthesized by another enzyme called primase. An enzyme known as a helicase is required to unwind DNA from a double-strand structure to a single-strand structure to facilitate replication of each strand consistent with the semiconservative model of DNA replication.
Error correction is a property of some, but not all, DNA polymerases. This process corrects mistakes in newly-synthesized DNA. When an incorrect base pair is recognized, DNA polymerase reverses its direction by one base pair of DNA. The 3'->5' exonuclease activity of the enzyme allows the incorrect base pair to be excised (this activity is known as proofreading). Following base excision, the polymerase can re-insert the correct base and replication can continue.
Variation across species
DNA polymerases have highly-conserved structure, which means that their overall catalytic subunits vary, on a whole, very little from species to species. Conserved structures usually indicate important, irreplicable functions of the cell, the maintenance of which provides evolutionary advantages.
Some viruses also encode special DNA polymerases, such as Hepatitis B virus DNA polymerase. These may selectively replicate viral DNA through a variety of mechanisms. Retroviruses encode an unusual DNA polymerase called reverse transcriptase, which is an RNA-dependent DNA polymerase (RdDp). It polymerizes DNA from a template of RNA.
DNA polymerase families
Based on sequence homology, DNA polymerases can be further subdivided into seven different families: A, B, C, D, X, Y, and RT.
Family A
Polymerases contain both replicative and repair polymerases. Replicative members from this family include the extensively-studied T7 DNA polymerase, as well as the eukaryotic mitochondrial DNA Polymerase γ. Among the repair polymerases are E. coli DNA pol I, Thermus aquaticus pol I, and Bacillus stearothermophilus pol I. These repair polymerases are involved in excision repair and processing of Okazaki fragments generated during lagging strand synthesis.
Family B
Polymerases mostly contain replicative polymerases and include the major eukaryotic DNA polymerases α, δ, ε, (see Greek letters) and also DNA polymerase ζ. Family B also includes DNA polymerases encoded by some bacteria and bacteriophages, of which the best-characterized are from T4, Phi29, and RB69 bacteriophages. These enzymes are involved in both leading and lagging strand synthesis. A hallmark of the B family of polymerases is remarkable accuracy during replication; and many have strong 3'-5' exonuclease activity (except DNA polymerase α and ζ, which have no proofreading activity).
Family C
Polymerases are the primary bacterial chromosomal replicative enzymes. DNA Polymerase III alpha subunit from E. coli is the catalytic subunit [1] and possesses no known nuclease activity. A separate subunit, the epsilon subunit, possesses the 3'-5' exonuclease activity used for editing during chromosomal replication. Recent research has classified Family C polymerases as a subcategory of Family X.
Family D
Polymerases are still not very well characterized. All known examples are found in the Euryarchaeota subdomain of Archaea and are thought to be replicative polymerases.
Family X
Contains the well-known eukaryotic polymerase pol β, as well as other eukaryotic polymerases such as pol σ, pol λ, pol μ, and terminal deoxynucleotidyl transferase (TdT). Pol β is required for short-patch base excision repair, a DNA repair pathway that is essential for repairing abasic sites. Pol λ and Pol μ are involved in non-homologous end-joining, a mechanism for rejoining DNA double-strand breaks. TdT is expressed only in lymphoid tissue, and adds "n nucleotides" to double-strand breaks formed during V(D)J recombination to promote immunological diversity. The yeast Saccharomyces cerevisiae has only one Pol X polymerase, Pol4, which is involved in non-homologous end-joining.
Family Y
Polymerases differ from others in having a low fidelity on undamaged templates and in their ability to replicate through damaged DNA. Members of this family are hence called translesion synthesis (TLS) polymerases. Depending on the lesion, TLS polymerases can bypass the damage in an error-free or error-prone fashion, the latter resulting in elevated mutagenesis. Xeroderma pigmentosum variant (XPV) patients for instance have mutations in the gene encoding Pol η (eta), which is error-free for UV-lesions. In XPV patients, alternative error-prone polymerases, e.g., Polζ (zeta) (polymerase ζ is a B Family polymerase a complex of the catalytic subunit REV3L with Rev7, which associates with Rev1[2]), are thought to be involved in mistakes that result in the cancer predisposition of these patients. Other members in humans are Pol ι (iota), Pol κ (kappa), and Rev1 (terminal deoxycytidyl transferase). In E.coli, two TLS polymerases, Pol IV (DINB) and PolV (UmuD'2C), are known.
Family RT
The reverse transcriptase family contains examples from both retroviruses and eukaryotic polymerases. The eukaryotic polymerases are usually restricted to telomerases. These polymerases use an RNA template to synthesize the DNA strand.
Prokaryotic DNA polymerases
Bacteria have 5 known DNA polymerases:
- Pol I: implicated in DNA repair; has 5'->3' (Polymerase) activity and both 3'->5' exonuclease (Proofreading) and 5'->3' exonuclease activity (RNA Primer removal).
- Pol II: involved in reparation of damaged DNA; has 3'->5' exonuclease activity.
- Pol III: the main polymerase in bacteria (elongates in DNA replication); has 3'->5' exonuclease proofreading ability.
- Pol IV: a Y-family DNA polymerase.
- Pol V: a Y-family DNA polymerase; participates in bypassing DNA damage.
Eukaryotic DNA polymerases
Eukaryotes have at least 15 DNA Polymerases:[3]
- Pol α (synonyms are RNA primase, DNA polymerase): forms a complex with a small catalytic (PriS) and a large noncatalytic (PriL) subunit[4], with the Pri subunits acting as a primase (synthesizing an RNA primer), and then with DNA Pol α elongating that primer with DNA nucleotides. After around 20 nucleotides[5] elongation is taken over by Pol ε (on the leading strand) and δ (on the lagging strand).
- Pol β: Implicated in repairing DNA, in base excision repair and gap-filling synthesis.
- Pol γ: Replicates and repairs mitochondrial DNA and has proofreading 3'->5' exonuclease activity.
- Pol δ: Highly processive and has proofreading 3'->5' exonuclease activity. Thought to be the main polymerase involved in lagging strand synthesis, though there is still debate about its role[6].
- Pol ε: Also highly processive and has proofreading 3'->5' exonuclease activity. Highly related to pol δ, and thought to be the main polymerase involved in leading strand synthesis[7], though there is again still debate about its role[6].
- η, ι, κ, and Rev1 are Y-family DNA polymerases and Pol ζ is a B-family DNA polymerase. These polymerases are involved in the bypass of DNA damage.[8]
- There are also other eukaryotic polymerases known, which are not as well characterized: θ, λ, φ, σ, and μ. There are also others, but the nomenclature has become quite jumbled.
None of the eukaryotic polymerases can remove primers (5'->3' exonuclease activity); that function is carried out by other enzymes. Only the polymerases that deal with the elongation (γ, δ and ε) have proofreading ability (3'->5' exonuclease). Nuclease is a great advocate for DNA replication.
See also
References
- ^ doi:10.1016/j.cell.2006.07.028
- ^ Gan GN; Wittschieben JP; Wittschieben BØ; Wood RD (2008). "DNA polymerase zeta (pol zeta) in higher eukaryotes". Cell Research 18: 174-83. PMID 18157155.
- ^ I. Hubscher, U.; Maga, G.; Spadari, S. (2002). "Eukaryotic DNA polymerases". Annual Review of Biochemistry 71: 133. doi:. PMID 12045093.
- ^ Elizabeth R. Barry; Stephen D. Bell (12/2006). "DNA Replication in the Archaea". Microbiology and Molecular Biology Reviews 70: 876-887. doi:. PMID 17158702.
- ^ J. M. Berg; J. L. Tymoczko; L. Stryer "Biochemie", Springer, Heidelberg/Berlin 2003
- ^ a b Scott D McCulloch; Thomas A Kunkel (01/2008). "The fidelity of DNA synthesis by eukaryotic replicative and translesion synthesis polymerases". Cell Research 18: 148. doi:. PMID 18166979.
- ^ Pursell, Z.F. et al. (2007). "Yeast DNA Polymerase ε Participates in Leading-Strand DNA Replication". Science 317: 127. doi:. PMID 17615360.
- ^ I. Prakash, S.; Johnson, R. E.; Prakash, L. (2005). "Eukaryotic translesion synthesis DNA polymerases: specificity of structure and function". Annual Review of Biochemistry 74: 317. doi:. PMID 15952890.
External links
- Burgers P, Koonin E, Bruford E et al. (2001). "Eukaryotic DNA polymerases: proposal for a revised nomenclature". J. Biol. Chem. 276 (47): 43487–90. doi:. PMID 11579108.
- PDB Molecule of the Month pdb3_1
- Unusual repair mechanism in DNA polymerase lambda, Ohio State University, July 25, 2006.
- MeSH DNA+polymerases
- EC 2.7.7.7
- A great animation of DNA Polymerase from WEHI
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