DNA polymerase definition
DNA polymerases are a group of enzymes that are used to make copies of DNA templates, essentially used in DNA replication mechanisms. These enzymes make new copies of DNA from existing templates and also function by repairing the synthesized DNA to prevent mutations. DNA polymerase catalyzes the formation of the phosphodiester bond which makes up the backbone of DNA molecules. It uses a magnesium ion in catalytic activity to balance the charge from the phosphate group.
What are DNA polymerases?
- DNA polymerase was first identified by Arthur Kornberg in lysates of Escherichia coli, in 1956.
- The enzyme is found and used in the DNA replication of both prokaryotic and eukaryotic cells.
- Several types of DNA polymerase enzymes have been discovered with the first one to be discovered named DNA polymerase I.
- Each of these types plays a major role in replication and DNA repair mechanisms.
- However, DNA polymerases are not used for initiating the synthesis of new strands, but in the extension of already existing DNA or RNA strands which are paired with a template strand.
- DNA polymerase starts its mechanism after a short RNA fragment is known as a primer is created and paired with a template DNA strand.
- DNA polymerase acts by synthesizing the new DNA strand by adding new nucleotides that match those of the template, extending the 3′ end of the template chain. Each nucleotide is linked with a phosphodiester bond.
- The DNA polymerase uses energy from the hydrolysis of the phosphoanhydride bond that is between the three phosphates (nucleoside triphosphates) attached to each free base (nucleotides).
- The addition of a nucleotide to a growing DNA strand forms a phosphodiester bond between the phosphate of the nucleotide to the growing chain using the high-energy phosphate bond of hydrolysis, releasing two distal phosphates known as pyrophosphate.
- DNA polymerases are very accurate in their mechanism with minimal errors of less than one error for every 107 nucleotides.
- Some types of DNA polymerase have the ability to proofread and remove unmatched bases of nucleotides and correct them.
- They also correct post-replication mismatches by monitoring and repairing the errors, by distinguishing mismatches of the new strand from the template strand sequences.
- The eukaryotic cell contains five DNA polymerase α, β, γ, δ, and ε. Polymerase γ is found in the cell mitochondria and it actively replicates the mitochondrial DNA, while polymerase α, β, δ are found in the cell nucleus hence are involved in the nuclear DNA replication.
- Polymerase α and δ are majorly applied and active in diving cells hence involved in replication while polymerase β is active in both diving and nondividing cells hence it is involved in the repair of DNA damage.
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Structure of DNA polymerase
- The DNA polymerases generally have a conserved structure, and therefore, defining its vital role in the cell function which can not be replaced.
- DNA polymerases are made up of subdomains resembling an open right hand as palm, fingers, and thumb.
- The palm contains the catalytic essential amino acids in its active sites.
- The fingers play a major role in nucleotide recognition and binding.
- The thumb is for binding of the DNA substrate
- There is a domain found between the finger and the thumb known as a pocket, which is made up of two regions i.e the insertion site and the post-insertion site.
- The incoming nucleotides bind to the insertion site while the new base pair bind in the post-insertion site.
- Other subdomains along with these domains are specific for each family, and each has essential functions in DNA replication.
- However, these subdomains are different for each polymerase.
Structure of Family A
- Besides the already discussed subdomains, Family A polymerase additionally has a 5′ to 3′ exonuclease which is used to remove the RNA primers from the Okazaki fragments.
- Some Family A groups also have a 3′ to 5′ exonuclease activity which functions in proofread the DNA.
Structure of Family B
- They also possess the basic subdomains with extremely active 3′ to 5; exonuclease to correct the errors of DNA replication.
Structure of Family X
- These family groups have the thumb, palm, and finger subdomains which are structurally part of the N-terminal or on the 31-kDA polymerase fragment.
- The palm in this family contains three aspartic acid motifs, the fingers have helices M and N containing amino acid residues.
- The N-terminal is connected to an 8kDa amino-terminal domain which contains a 5′ deoxyribose phosphate lyase, which is essential in base excision repair.
Structure of Family Y
- The N-terminal of this group contains the catalytic core of the palm, fingers, and thumb.
- They also have a C-terminal which has a conserved tertiary structure of a four-stranded beta-sheet supported on one side by two-alpha helices, which are also known as little finger domain. They play a role in DNA binding and it is essential for complete polymerase activity.
- However, this family lacks flexibility, unlike the other families.
DNA polymerase types
Basically, the types of DNA polymerase are also divided depending on the organism that posses them i.e. eukaryotic and prokaryotic DNA polymerases. These types of DNA polymerase are classified based on their characteristics including structural sequences, and functions.
Eukaryotic DNA polymerase types
- Polymerase γ is a Type A polymerase, whose main function is to replicate and repair mitochondrial DNA.
- It also functions by proofreading 3′ to 5′ exonuclease activity.
- Mutations on Poly γ significantly affect the mitochondrial DNA causing autosomal mitochondrial disorders.
Polymerase α, Polymerase δ, and Polymerase ε
- These are the type B Polymerase enzymes and they are the main polymerases applied in DNA replication.
- Pol α works by binding to the primase enzyme, forming a complex, where they both play a role in initiating replication. Primase enzyme creates and places a short RNA primer which allows Pol α to start the replication process.
- Pol δ starts the synthesis of the lagging strand from Pol α, while Pol ε is believed to synthesize the leading strand during replication.
- Studies indicate that Pol δ replicates both the lagging and leading strand.
- Pol δ and ε also have a 3′ to 5′ exonuclease activity.
Polymerase β, Polymerase μ, and Polymerase λ
- These are type 3 or Family X of polymerase enzymes.
- Pol β has a short-patch base excision repair mechanism where it repairs alkylated or oxidized bases.
- Pol λ and Pol μ are important for rejoining DNA double-strand breaks due to hydrogen peroxide and ionizing radiation, respectively.
Polymerases η, Polymerase ι, and Polymerase κ
- They are type 4 or family Y polymerases majorly used in repairing of DNA by a mechanism known as translesion synthesis.
- They are prone to errors during DNA synthesis.
- Pol η functions by accurately ensuring the translesion synthesis of DNA damages that is caused by ultraviolet radiation.
- Pol κ is still understudies but one of its known functions is to extend or insert specific bases at certain DNA lesions.
- Translesion synthesis polymerases are activated by stalled replicative DNA polymerase.
Terminal deoxynucleotidyl transferase (TdT)
- TdT functions by catalyzing the polymerization of deoxynucleoside triphosphate to the 3′-hydroxyl group of a preformed polynucleotide chain.
- TdT is a non-template directed DNA polymerase.
- It was first detected in the thymus gland.
Prokaryotic DNA polymerase types
DNA Polymerase I
- This is a type A or Family A polymerase enzyme that was initially isolated from E. coli and most abundantly found in E. coli.
- Its main function is excision repair of DNA strands from the 3′-5′ direction to the 5′-3 direction, as an exonuclease.
- It also helps with the maturation of Okazaki fragments, which are short DNA strands that make up the lagging strand during DNA replication.
- Its role during replication is the addition of nucleotide at the RNA primer and it moves along the 5′-3′ direction.
- The binding site for DNA polymerase I is known as octylglucoside.
DNA Polymerase II
- It belongs to Type B or Family B of the polymerases.
- Its major function is the 3′ – 5′ exonuclease activity and to also restart replication after replication stops due to DNA strand damages.
- The DNA polymerase II is found in the replication fork, to help in directing the activities of other polymerases.
DNA Polymerase III
- This is the primary enzyme that is used in DNA replication, belonging to the Family C or Type C.
- It is responsible for the synthesis of new strands by adding nucleotides to the 3′- OH group of the primer.
- It has a 3′-5′ exonuclease activity hence it can also proofread the errors that may arise during DNA strand replication.
DNA Polymerase IV
- It belongs to the Family Y and it is involved in the non-targeted mutagenesis.
- Its activation is based on the stalling activity of the replication fork.
- When it is activated, it creates a checkpoint, stops replication, and gives time to properly repair lesions in the new DNA strand.
- It is also involved in the repair mechanism of translesion synthesis.
- It does not have nuclease activity, therefore it is prone to errors in DNA replication.
DNA Polymerase V
- It belongs to Family Y, with high regulatory activity.
- It is produced only when DNA is damaged and it requires translesion synthesis.
- It also lacks exonuclease functions and hence it can not proofread the synthesis of DNA replicas making it less efficient.
Taq DNA polymerase
- Taq polymerase is a thermostable type of DNA polymerase 1 that was initially isolated from a thermophilic eubacterial known as Thermus aquaticus.
- It is abbreviated as Taq or Taq pol.
- It is commonly used in Polymerase Chain Reaction to amplify short strands of DNA.
- Due to its thermophilic nature, it is able to withstand denaturation that is required during PCR, hence it replaced DNA polymerase from E. coli.
Mechanism of DNA polymerase
The mechanism of DNA polymerase is referred to as a two metal ion mechanism i.e two metal ions act as active sites for stabilizing the transmission of replication. The first metal ion acts by activating the hydroxyl group which attacks the phosphate group of the dNTP and the second metal ion stabilizes the negative charges and builds on the left oxygen and chelating phosphate groups.
DNA polymerase vs RNA polymerase
|1.||Definition||It is an enzyme that synthesizes the DNA||It is an enzyme synthesizes the RNA|
|2.||Mechanism||DNA polymerase’s mechanism is during replication whereby it synthesizes new DNA strands||RNA polymerase functions during transcription, which is the synthesis of RNA|
|3.||Strands||It synthesizes a double-stranded DNA molecule||It synthesizes a single-stranded RNA molecule|
|4.||Presence or absence of Primer||Its replication mechanism is initiated by a short-RNA primer||It does not need a primer to initiate transcription|
|5.||Nucleotide insertion||It inserts nucleotides after finding the free 3’ OH end by the assistance of the primer-synthesizer, primase enzyme||It adds nucleotides directly.|
|6.||Amino acid bases||It adds dATP (Adenine-Thymine), dGTP, dCTP and dTTP to the growing new DNA strand||It inserts dATP, dGTP, dCTP and dUTP to the growing RNA strand.|
|7.||Functionality||It has polymerization and proofreading activity||RNA polymerase only has a polymerization activity.|
|8.||Polymerization rate||The rate of polymerization by DNA polymerase is about 1000 nucleotides per second in prokaryotes||The rate of RNA polymerase is 40 to 80 nucleotides per second.|
|9.||Efficiency||DNA polymerase enzyme is faster, efficient, and more accurate considering its proofreading activity.||RNA polymerase is slower, inefficient, and inaccurate.|
|10.||Subtypes||DNA polymerase has three different subtypes: Type 1, 2, and 3.||RNA polymerase has five different subtypes in eukaryotes|
|11.||Termination||The DNA synthesis continues until the end when the strand ends, that is when polymerization stops, thus the entire chromosomal DNA is synthesized.||The polymerization is terminated when RNA polymerase finds the stop codon or termination codon on the nucleic acid strand.|
References and Sources
- Microbiology by Prescott, 5th Edition
3 thoughts on “DNA Polymerase- definition, structure, types (vs RNA polymerase)”
Very informative thank you so much
Really enjoyed the notes thank you
Good way of describing, and nice post to take information on the topic
of my presentation topic, which i am going to present in college.