DNA Polymerase – Definition, Mechanism, Structure, Types

What is DNA Polymerase?

• DNA polymerases are crucial enzymes involved in the replication of DNA molecules. These enzymes catalyze the synthesis of DNA by adding nucleotides to the growing DNA strand. The process of DNA replication ensures that genetic information is accurately passed down from one generation to the next.
• During DNA replication, DNA polymerases work in groups to create two identical DNA duplexes from a single original DNA duplex. These enzymes “read” the existing DNA strands and use them as templates to create two new strands that match the original ones. By catalyzing the chemical reaction between deoxynucleoside triphosphates and the DNA molecule, DNA polymerases add nucleotides to the 3′-end of the DNA strand, one nucleotide at a time.
• The replication process starts with an enzyme called helicase, which unwinds the DNA molecule and separates the two strands by breaking the hydrogen bonds between nucleotide bases. This creates two single strands of DNA that can serve as templates for replication. DNA polymerases then come into action, synthesizing new DNA strands complementary to the templates.
• The discovery of DNA polymerases played a significant role in understanding DNA replication. In 1956, Arthur Kornberg and his colleagues discovered the first DNA polymerase, DNA polymerase I (Pol I), in Escherichia coli. They unraveled the process by which this enzyme copies the base sequence of a template DNA strand, leading to Arthur Kornberg being awarded the Nobel Prize in Physiology or Medicine in 1959.
• Since then, additional DNA polymerases have been discovered. DNA polymerase II was identified in 1970 by Thomas Kornberg and Malcolm E. Gefter while studying E. coli DNA replication. Further research unveiled the existence of DNA polymerase III in the 1970s, and DNA polymerases IV and V were found in 1999, all in E. coli.
• In summary, DNA polymerases are enzymes responsible for the replication of DNA. They add nucleotides to the growing DNA strand and ensure accurate transmission of genetic information from one generation to the next. The discovery of DNA polymerases, starting with DNA polymerase I, has significantly contributed to our understanding of DNA replication and its role in inheritance.

DNA polymerases Definition

DNA polymerases are a vital group of enzymes involved in DNA replication processes. Their primary function is to create exact copies of DNA templates. Additionally, they play a crucial role in repairing synthesized DNA to prevent errors or mutations. These enzymes facilitate the formation of phosphodiester bonds, which are responsible for forming the backbone of DNA molecules. In their catalytic activity, DNA polymerases utilize magnesium ions to maintain charge balance from the phosphate group. The coordinated action of DNA polymerases ensures the accurate replication and integrity of DNA molecules.

Properties of DNA Polymerase

DNA polymerases possess several important properties that contribute to their role in DNA replication. These properties ensure the accuracy and efficiency of DNA synthesis. Let’s explore these key properties of DNA polymerases.

1. Proofreading: DNA polymerases have a remarkable ability to proofread their work. They possess an exonuclease activity that allows them to detect and remove incorrect nucleotides that have been added to the growing DNA chain. This proofreading function helps maintain the fidelity of DNA replication and minimizes errors.
2. Primer Requirement: DNA polymerases can only initiate the addition of nucleotides if there is a preexisting 3′ end. Thus, they rely on a short stretch of nucleotides called a primer, which provides the necessary 3′ end for DNA synthesis to commence. This primer requirement ensures that DNA polymerases accurately extend the existing DNA strands.
3. Structural and Functional Diversity: DNA polymerases vary in structure, function, rate of polymerization, and processivity based on the type of cell. In prokaryotic cells, there are three main DNA polymerases, namely DNA polymerase I to III. Among them, DNA polymerase III plays a central role in DNA replication in Escherichia coli. In contrast, eukaryotic cells possess five major DNA polymerases: DNA Polymerase α, ε, δ, γ, and β. Each DNA polymerase has specific functions and is responsible for DNA replication in different cellular compartments.
4. Compartment-Specific Replication: Eukaryotic cells have distinct compartments for DNA replication, including the nucleus and mitochondria. This necessitates precise DNA polymerases with specific functions in each compartment. The presence of separate DNA polymerases ensures the accurate replication of nuclear DNA and mitochondrial DNA, reflecting the complex nature of eukaryotic DNA replication.

Understanding these properties of DNA polymerases sheds light on their crucial role in DNA replication. Their ability to proofread, dependence on primers, and specialization based on cellular compartments all contribute to the faithful transmission of genetic information. DNA polymerases are indispensable enzymes that facilitate the accurate synthesis and maintenance of DNA molecules in living organisms.

Structure of DNA Polymerase

The structure of DNA polymerases is crucial for their function in DNA replication. Understanding their structural components provides insights into their mechanisms. Let’s delve into the structure of DNA polymerases.

DNA polymerases exhibit a conserved structure that is essential for their role in cellular functions, making them irreplaceable enzymes. They consist of subdomains that resemble an open right hand, including the palm, fingers, and thumb.

The palm domain contains the catalytic amino acids in its active sites, where the polymerization of DNA occurs. It plays a vital role in the enzymatic activity of DNA polymerases.

The fingers subdomain is responsible for nucleotide recognition and binding. It interacts with the incoming nucleotides and ensures the correct base pairing during DNA synthesis.

The thumb subdomain is involved in binding the DNA substrate. It stabilizes the interaction between the DNA template and the DNA polymerase.

Between the fingers and the thumb, there is a domain called the pocket. This pocket consists of two regions, namely the insertion site and the post-insertion site. The incoming nucleotides bind to the insertion site, while the newly formed base pairs bind to the post-insertion site.

In addition to these main subdomains, DNA polymerases have other specific subdomains that vary for each family. These subdomains contribute to the overall structure and have essential functions in DNA replication.

Let’s explore the structures of different families of DNA polymerases:

1. Family A: In addition to the previously mentioned subdomains, Family A polymerases possess a 5′ to 3′ exonuclease activity, which is involved in removing RNA primers from Okazaki fragments. Some Family A groups also exhibit a 3′ to 5′ exonuclease activity, which aids in proofreading DNA during replication.
2. Family B: Family B polymerases possess the basic subdomains found in DNA polymerases, along with an exceptionally active 3′ to 5′ exonuclease activity. This exonuclease activity helps correct errors that may occur during DNA replication.
3. Family X: Family X polymerases have the thumb, palm, and finger subdomains, which are structurally part of the N-terminal or the 31-kDa polymerase fragment. The palm in this family contains three aspartic acid motifs, while the fingers consist of helices M and N with specific amino acid residues. The N-terminal region is connected to an 8 kDa amino-terminal domain that contains a 5′ deoxyribose phosphate lyase, an essential component for base excision repair.
4. Family Y: Family Y polymerases have a catalytic core consisting of the palm, fingers, and thumb in the N-terminal region. They also possess a C-terminal region with a conserved tertiary structure, featuring a four-stranded beta-sheet supported by two alpha helices, known as the little finger domain. This domain plays a critical role in DNA binding and is essential for complete polymerase activity. Unlike other families, Family Y polymerases lack flexibility.

DNA polymerase types

Essentially, DNA polymerase varieties are divided based on the organism that possesses them, i.e., eukaryotic and prokaryotic DNA polymerases. Based on their characteristics, including structural sequences and functions, these varieties of DNA polymerase are categorised.

Eukaryotic DNA polymerase types

Eukaryotic cells employ various types of DNA polymerases that play essential roles in DNA replication and repair processes. Let’s explore the different types of eukaryotic DNA polymerases:

1. Polymerase γ (Pol γ): Pol γ is a Type A polymerase primarily responsible for replicating and repairing mitochondrial DNA. It also possesses proofreading abilities through its 3′ to 5′ exonuclease activity. Mutations in Pol γ can have significant impacts on mitochondrial DNA, leading to autosomal mitochondrial disorders.
2. Polymerase α (Pol α), Polymerase δ (Pol δ), and Polymerase ε (Pol ε): These are Type B polymerases and are the main enzymes involved in DNA replication. Pol α forms a complex with the primase enzyme to initiate replication. The primase enzyme creates a short RNA primer that allows Pol α to begin the replication process. Pol δ is responsible for synthesizing the lagging strand, while Pol ε is believed to synthesize the leading strand during replication. Studies suggest that Pol δ can also replicate both the lagging and leading strands. Pol δ and Pol ε also possess 3′ to 5′ exonuclease activity.
3. Polymerase β (Pol β), Polymerase μ (Pol μ), and Polymerase λ (Pol λ): These enzymes belong to Type 3 or Family X of polymerases. Pol β is involved in short-patch base excision repair, specifically repairing alkylated or oxidized bases. Pol λ and Pol μ play crucial roles in rejoining DNA double-strand breaks caused by hydrogen peroxide and ionizing radiation, respectively.
4. Polymerase η (Pol η), Polymerase ι (Pol ι), and Polymerase κ (Pol κ): These are Type 4 or Family Y polymerases primarily utilized in a process called translesion synthesis, which repairs DNA damage. Translesion synthesis polymerases have an increased propensity for errors during DNA synthesis. Pol η accurately carries out translesion synthesis of DNA damages caused by ultraviolet radiation. The specific function of Pol κ is still being studied, but it is known to extend or insert specific bases at certain DNA lesions. Translesion synthesis polymerases are activated when the replicative DNA polymerase encounters obstacles during replication.
5. Terminal deoxynucleotidyl transferase (TdT): TdT catalyzes the polymerization of deoxynucleoside triphosphates to the 3′-hydroxyl group of a preformed polynucleotide chain. It is a non-template directed DNA polymerase and was initially discovered in the thymus gland.

Prokaryotic DNA polymerase types

1. DNA polymerase I

• DNA polymerase I (Pol I) is a vital enzyme involved in prokaryotic DNA replication and repair processes. Discovered by Arthur Kornberg in 1956, it was the first-known DNA polymerase. Pol I is widely present in prokaryotes and was initially characterized in Escherichia coli (E. coli). It belongs to the alpha/beta protein superfamily and exhibits processive enzymatic activity, allowing for sequential catalysis without releasing the single-stranded template.
• The primary physiological function of Pol I is to support DNA repair. It contributes to the repair of damaged DNA by deleting RNA primers and replacing them with DNA, thus connecting Okazaki fragments. Pol I consists of multiple domains, including the thumb, finger, and palm domains, which work collaboratively to sustain DNA polymerase activity. Additionally, it possesses exonuclease active sites that remove incorrectly incorporated nucleotides in a process called proofreading.
• In E. coli, there are several DNA polymerases, including Pol I, Pol II, Pol III, Pol IV, and Pol V. While eukaryotic cells also have multiple DNA polymerases, DNA polymerase β shares similarities with Pol I in terms of its involvement in DNA repair rather than replication. Eukaryotes have fifteen identified DNA polymerases.
• DNA polymerases require both a template strand and a primer strand for DNA replication. They initiate synthesis using short RNA segments called primers. DNA synthesis occurs by adding deoxynucleotide triphosphates (dNTPs) to the 3′ hydroxyl group at the end of the preexisting DNA strand or RNA primer. The polymerization mechanism involves two-metal ion catalysis, where one metal ion activates the primer hydroxyl group, initiating the reaction, and the other stabilizes the leaving oxygen’s negative charge.
• The structure of DNA polymerases resembles a human right hand, consisting of three domains: fingers, palm, and thumb. The fingers domain interacts with dNTPs and the template base, while the palm domain catalyzes the phosphoryl group transfer. The thumb domain interacts with double-stranded DNA. DNA polymerases also possess an exonuclease domain responsible for removing mispaired bases.
• Pol I exhibits four enzymatic activities. It has a 5’→3′ DNA-dependent DNA polymerase activity, requiring a primer and template strand, and a 3’→5′ exonuclease activity for proofreading. The enzyme also performs a 5’→3′ exonuclease activity during DNA repair and a 5’→3′ RNA-dependent DNA polymerase activity, although with lower efficiency compared to DNA templates.
• Experiments with Pol I-deficient E. coli mutants have demonstrated its crucial role in DNA repair rather than replication. The mutants displayed sensitivity to DNA-damaging factors, further supporting its involvement in repairing DNA damage.
• Overall, DNA polymerase I plays a critical role in prokaryotic DNA replication and repair processes, contributing to the maintenance and integrity of the genome.

Mechanism of DNA polymerase I

• The mechanism of DNA polymerase I (Pol I) involves its role in DNA replication and repair processes. During replication, the RNA primer on the lagging strand is removed by RNase H, and then Pol I fills in the necessary nucleotides between the Okazaki fragments in a 5’→3′ direction. As it adds nucleotides, Pol I proofreads for mistakes, ensuring accurate base pairing with the template strand.
• Pol I is a template-dependent enzyme, meaning it adds nucleotides that correctly base pair with the existing DNA template. The nucleotides must be in the correct orientation and geometry to properly align with the template strand. This enables DNA ligase to join the fragments and form a continuous DNA strand. Research on Pol I has shown that different deoxynucleotide triphosphates (dNTPs) can bind to the same active site on the enzyme. However, Pol I actively discriminates between dNTPs only after undergoing a conformational change. Once the change occurs, Pol I checks for the proper geometry and alignment of the base pair formed between the bound dNTP and the template strand. Only the correct geometry of A=T and G≡C base pairs can fit in the active site. Despite these mechanisms, there is still a low error rate of approximately one incorrect nucleotide added per every 10^4 to 10^5 nucleotides. However, Pol I has the ability to correct these errors through its selective active discrimination process.
• Although Pol I was one of the first DNA polymerases discovered, it was found to have limited involvement in most DNA synthesis. The rate of base pair synthesis by Pol I is relatively slow, averaging between 10 and 20 nucleotides per second, while DNA replication in Escherichia coli proceeds at approximately 1,000 nucleotides per second. Additionally, the cellular abundance of Pol I is not proportional to the number of replication forks in E. coli, as there are typically only two. Pol I is also not highly processive, falling off after incorporating only 25–50 nucleotides. These observations led to the discovery of DNA polymerase III as the main replicative DNA polymerase.
• In summary, DNA polymerase I plays a role in DNA replication and repair by adding nucleotides to fill in gaps between Okazaki fragments. It proofreads for errors and discriminates between different nucleotides to maintain accuracy. However, it is not the primary polymerase responsible for DNA replication, as it has lower processivity and synthesis rates compared to other polymerases like DNA polymerase III.

2. DNA Polymerase II

• DNA polymerase II, also known as DNA Pol II or Pol II, is an essential enzyme involved in DNA replication in prokaryotes. Encoded by the PolB gene, DNA Pol II is a member of the B family of DNA polymerases, with a molecular weight of 89.9-kDa. Initially isolated by Thomas Kornberg in 1970, this polymerase has since been extensively characterized.
• While the exact in vivo role of DNA Pol II is still a subject of debate, it is generally accepted that this enzyme acts as a backup polymerase in prokaryotic DNA replication. It possesses both 5’→3′ DNA synthesis capability and 3’→5′ exonuclease proofreading activity, ensuring accuracy during replication. To enhance its fidelity and processivity, DNA Pol II interacts with multiple binding partners shared with DNA Pol III, another important DNA polymerase in prokaryotes.
• DNA polymerase I, the first DNA-directed DNA polymerase isolated from E. coli, was found to be involved in repair replication rather than serving as the primary replicative polymerase. Mutant strains of E. coli lacking DNA Pol I were generated in 1969, confirming its role in repair replication. However, this raised the question of the existence of another enzyme responsible for DNA replication. Parallel studies conducted by different laboratories led to the discovery of DNA polymerase II, initially believed to be the main replicative enzyme in E. coli. In 1994, Anderson et al. successfully crystallized DNA Pol II, further advancing its structural understanding.
• In recent developments, it has been reported in 2023 that accelerated transcription associated with aging can lead to an increased error rate of DNA Pol II. These errors can result in flawed DNA copies, potentially leading to various age-related diseases.
• The structure of DNA Pol II consists of an 89.9 kDa protein composed of 783 amino acids, encoded by the polB (dinA) gene. Unlike many other polymerases that form complexes, DNA Pol II functions as a monomer. It comprises three main sections known as the palm, fingers, and thumb, which collectively form a “hand” that encloses around a DNA strand. The palm region contains three catalytic residues that coordinate with divalent metal ions to facilitate enzymatic activity. In terms of cellular abundance, DNA Pol II is present in higher quantities, with approximately 30-50 copies per cell, compared to the significantly lower levels of DNA Pol III.[9]
• DNA Pol II belongs to the Group B family of polymerases, alongside human DNA Pol α, δ, ϵ, and ζ. These polymerases share structural and functional similarities with homologs such as RB69, 9°N-7, and Tgo. Notably, DNA Pol II is unique among the Group B polymerases due to its functioning as a monomer, whereas others have at least one additional subunit.
• In conclusion, DNA polymerase II plays a crucial role in DNA replication in prokaryotes, serving as a backup enzyme. It possesses both synthesis and proofreading capabilities, ensuring accurate DNA replication. While its exact in vivo function is still under investigation, DNA Pol II contributes to the fidelity and processivity of DNA replication, working in conjunction with other DNA polymerases in prokaryotic cells.

Mechanism of DNA polymerase II

• The mechanism of DNA polymerase II involves its role in repairing damaged nucleotide base pairs during DNA replication. When errors or damage occur in the DNA sequence, replication can be stalled. DNA Pol II catalyzes the repair process by interacting with Pol III accessory proteins, such as the β-clamp and clamp loading complex, which provide access to the growing nascent strand.
• During DNA replication, DNA Pol III may produce mistakes in the growing strand. DNA Pol II functions to fix these errors in the sequence, ensuring the fidelity of DNA replication. The N-terminal domain of DNA Pol II plays a crucial role in the association and dissociation of the DNA strand to the catalytic subunit. Within the N-terminal domain, there are likely two sites that recognize single-stranded DNA—one responsible for recruiting single-stranded DNA to DNA Pol II and another for dissociating it from the enzyme.
• Upon binding of the substrate, DNA Pol II binds nucleoside triphosphates to maintain the hydrogen-bonded structure of DNA. The correct deoxyribonucleotide triphosphate (dNTP) is then selected and bound by the enzyme. This triggers conformational changes in subdomains and amino acid residues of the DNA Pol II complex, facilitating fast repair synthesis. The active site of DNA Pol II contains two magnesium ions, which are stabilized by catalytic aspartic acids D419 and D547. These magnesium ions, along with the dNTP, bind to the DNA in the open state of the enzyme. They coordinate the conformational changes of active site amino acid residues, enabling catalysis to occur in the closed state. Once the magnesium ions are released, the enzyme returns to its open state, ready for subsequent repair synthesis.
• Overall, DNA polymerase II plays a critical role in repairing damaged DNA during replication. It interacts with other proteins, recognizes single-stranded DNA, and undergoes conformational changes to ensure accurate repair synthesis. The coordination of magnesium ions and nucleotide binding is essential for the catalytic activity of DNA Pol II, allowing for efficient DNA repair.

3. DNA Polymerase III

• DNA Polymerase III is a crucial enzyme complex involved in prokaryotic DNA replication. It was first discovered by Thomas Kornberg and Malcolm Gefter in 1970 and plays a central role in replicating the E. coli genome. The DNA Polymerase III holoenzyme, as the primary replication enzyme, exhibits high processivity, meaning it adds numerous nucleotides per binding event. It works in conjunction with other DNA polymerases, including Pol I, Pol II, Pol IV, and Pol V, to ensure efficient and accurate DNA replication.
• One of the notable features of DNA Polymerase III holoenzyme is its proofreading capability. It possesses exonuclease activity, which allows it to detect and correct replication errors. This proofreading activity reads the DNA strand in the 3’→5′ direction and synthesizes the new strand in the 5’→3′ direction, ensuring fidelity in DNA replication. The DNA Polymerase III holoenzyme is an integral component of the replisome, a complex located at the replication fork.
• The DNA Polymerase III holoenzyme is comprised of various subunits that work together harmoniously. Each DNA Pol III holoenzyme contains two DNA Pol III enzymes, consisting of α, ε, and θ subunits. The α subunit possesses polymerase activity, while the ε subunit exhibits 3’→5′ exonuclease activity. The θ subunit stimulates the proofreading function of the ε subunit. Additionally, two β units, known as sliding DNA clamps (dnaN), keep the polymerase stably bound to the DNA during replication. Two τ units (dnaX) dimerize the core enzymes (α, ε, and θ subunits), enhancing their functionality.
• The DNA Polymerase III holoenzyme also includes a γ unit (dnaX) that serves as a clamp loader for the lagging strand Okazaki fragments. It assists the two β subunits in forming a unit and binding to DNA. The γ unit comprises five subunits, including three γ subunits, one δ subunit (holA), and one δ’ subunit (holB). The δ subunit is specifically involved in copying the lagging strand. Furthermore, the complex involves Χ (holC) and Ψ (holD) subunits, which form a 1:1 complex and bind to γ or τ. The Χ subunit has an additional role in facilitating the switch from RNA primer to DNA during replication.
• In summary, DNA Polymerase III is a crucial component of the replisome and the primary enzyme complex responsible for prokaryotic DNA replication. Its proofreading capabilities and interaction with other subunits ensure accurate and efficient replication, contributing to the faithful transmission of genetic information.

4. DNA Polymerase IV

• DNA Polymerase IV, also known as Pol IV, is a prokaryotic polymerase that plays a crucial role in mutagenesis. Encoded by the dinB gene, Pol IV lacks 3’→5′ exonuclease activity, making it error-prone. In E. coli, Pol IV is involved in non-targeted mutagenesis. It belongs to Family Y polymerases and is activated through SOS induction triggered by stalled polymerases at the replication fork.
• During SOS induction, the production of Pol IV is significantly increased, serving multiple functions. One of its roles is to interfere with the processivity of the Pol III holoenzyme, creating a checkpoint that halts replication and allows time for DNA lesions to be repaired through the appropriate repair pathway. Additionally, Pol IV performs translesion synthesis at the stalled replication fork. For example, it can bypass N2-deoxyguanine adducts at a faster rate compared to traversing undamaged DNA. Cells lacking the dinB gene experience a higher rate of mutagenesis caused by DNA damaging agents.
• Reactive oxygen species, which are generated during normal metabolic processes, can damage DNA. DNA Polymerase IV is capable of catalyzing translesion synthesis across various DNA damages, including 8-oxoguanine, a major oxidative damage with high mutagenic potential. In the absence of repair, unrepaired 8-oxoguanine tends to mispair with adenine (A) during chromosome duplication by replicative polymerases. This results in a G:C to T:A transversion mutation during the next round of replication. However, when DNA Polymerase IV bypasses the damage, it preferentially incorporates the correct nucleotide cytidine triphosphate (CTP) opposite 8-oxoguanine with high efficiency, thereby preventing potential mutations.
• In summary, DNA Polymerase IV is an error-prone polymerase involved in mutagenesis. Its activation during SOS induction and its ability to perform translesion synthesis contribute to DNA repair and prevent the occurrence of mutations caused by DNA damage, particularly oxidative damage like 8-oxoguanine.

5. DNA Polymerase V

• DNA Polymerase V (Pol V) is a crucial enzyme involved in DNA repair mechanisms in bacteria, specifically in Escherichia coli. Composed of a UmuD’ homodimer and a UmuC monomer, Pol V forms the UmuD’2C protein complex. It belongs to the Y-family of DNA polymerases, which are specialized in translesion synthesis (TLS). Translesion polymerases like Pol V play a vital role in bypassing DNA damage lesions during DNA replication to prevent replication fork stalling and cell death.
• Initially, when the UmuC and UmuD’ proteins were discovered in E. coli, they were believed to inhibit accurate DNA replication, resulting in high mutation rates after exposure to UV-light. However, it was not until the late 1990s that the polymerase function of Pol V was uncovered through experiments that unequivocally proved UmuD’2C to be a polymerase. This discovery led to the identification of many Pol V orthologs and the recognition of the Y-family of polymerases.
• In E. coli, Pol V functions as a TLS polymerase as part of the SOS response to DNA damage. When DNA is damaged, regular DNA synthesis polymerases, such as DNA Polymerase III (Pol III), are unable to add nucleotides to the newly synthesized strand. This puts the replication fork at risk of collapsing, leading to cell death. Pol V relies on its association with other elements of the SOS response, particularly the formation of RecA nucleoprotein filaments, to carry out its translesion activity effectively.
• Pol V can perform TLS on lesions that block replication or miscoding lesions, which alter the bases and result in incorrect base pairing. However, it cannot bypass 5′ → 3′ backbone nick errors. Notably, Pol V lacks exonuclease activity, making it unable to proofread its synthesis and rendering it error-prone.
• The SOS response in E. coli is a protective mechanism that aims to mitigate the effects of DNA damage. Pol V’s role in the SOS response triggered by UV radiation involves several steps, including the stalling of Pol III at the lesion site, expansion of the replication fork by the DNA replication helicase DnaB, stabilization of single-stranded DNA (ssDNA) segments by ssDNA binding proteins (SSBs), recruitment and loading of RecA onto ssDNA, formation of RecA nucleoprotein filaments (RecA*), activation of Pol V through mediator proteins, access of Pol V to the 3′-OH of the nascent DNA strand, and ultimately the resumption of elongation by Pol III.
• In summary, DNA Polymerase V (Pol V) is an essential DNA polymerase involved in DNA repair mechanisms, specifically in translesion synthesis (TLS), in bacteria such as E. coli. It plays a critical role in bypassing DNA damage lesions during replication and is an integral part of the SOS response triggered by DNA damage.

6. Taq DNA polymerase

• Taq DNA polymerase, also known as Taq pol, is a thermostable DNA polymerase derived from the bacterium Thermus aquaticus. It was initially isolated in 1976 and has become a key component in the polymerase chain reaction (PCR), a widely used technique for amplifying short segments of DNA.
• Taq polymerase earned its name from the thermophilic nature of the organism from which it was extracted. T. aquaticus is found in hot springs and hydrothermal vents, and Taq polymerase was identified for its ability to withstand the high temperatures required during PCR, which denature proteins. It replaced the original DNA polymerase from E. coli used in PCR.
• One of the remarkable properties of Taq polymerase is its optimal activity at temperatures between 75-80 °C. It maintains its enzymatic function at high temperatures and exhibits a half-life of over 2 hours at 92.5 °C. Taq can replicate a 1000 base pair DNA strand in less than 10 seconds at 72 °C. Deviations from the optimal temperature range hinder its extension rate. At temperatures above 90 °C, Taq’s activity is significantly reduced, although the enzyme remains intact without denaturing.
• The presence of specific ions in the reaction vessel also influences Taq polymerase’s activity. Small amounts of potassium chloride (KCl) and magnesium ions (Mg2+) enhance the enzyme’s performance, while high concentrations of these ions inhibit its activity. Taq is maximally activated at 50mM KCl and an appropriate concentration of Mg2+, determined by the concentration of nucleoside triphosphates (dNTPs).
• However, Taq polymerase has a drawback in that it lacks 3′ to 5′ exonuclease proofreading activity, leading to relatively low replication fidelity. Its error rate was initially measured at approximately 1 in 9,000 nucleotides. To achieve higher fidelity amplification, other thermostable DNA polymerases with proofreading activity, such as Pfu DNA polymerase, are used either instead of or in combination with Taq.
• Taq polymerase generates DNA products with A (adenine) overhangs at their 3′ ends. This feature is valuable in TA cloning, a technique that involves using a cloning vector with a T (thymine) 3′ overhang, which complements the A overhang of the PCR product. This enables the ligation of the PCR product into the plasmid vector, facilitating cloning processes.
• In summary, Taq DNA polymerase is a thermostable enzyme derived from T. aquaticus. It plays a crucial role in PCR due to its ability to withstand high temperatures. Taq polymerase’s optimal activity, along with its ability to generate DNA products with A overhangs, makes it a valuable tool in molecular biology research and various applications like cloning and DNA amplification.

Family D

• Family D of DNA polymerases was discovered in 1998 in Pyrococcus furiosus and Methanococcus jannaschii, marking a significant advancement in our understanding of DNA replication. The PolD complex, belonging to family D, is a heterodimer composed of two chains encoded by DP1 (small proofreading) and DP2 (large catalytic) genes.
• What sets the family D DNA polymerases apart is their unique structure and mechanism. The DP2 catalytic core exhibits similarities to multi-subunit RNA polymerases, distinguishing it from other DNA polymerases. The interface between DP1 and DP2 resembles the zinc finger found in Eukaryotic Class B polymerases, as well as their small subunit. This structural resemblance suggests that DP1, which possesses a Mre11-like exonuclease domain, may be the precursor of the small subunit in Eukaryotic Pol α and ε, contributing to the lost proofreading capabilities observed in Eukaryotes.
• DP1 also features an N-terminal HSH domain that shares structural similarities with AAA proteins such as Pol III subunit δ and RuvB. On the other hand, DP2 contains a Class II KH domain, further distinguishing it within the family D DNA polymerases.
• One notable member of family D is Pyrococcus abyssi polD, which exhibits increased thermal stability and accuracy compared to the well-known Taq polymerase. However, commercialization of Pyrococcus abyssi polD has yet to be realized.
• Interestingly, family D DNA polymerase is believed to have been one of the earliest polymerases to evolve in cellular organisms. It has been proposed that the replicative polymerase of the Last Universal Cellular Ancestor (LUCA) belonged to family D, highlighting its significance in the evolution of DNA replication.
• The discovery and study of family D DNA polymerases have contributed to our knowledge of the diverse mechanisms and structures employed by these enzymes. Their unique characteristics provide insights into the evolutionary history of DNA replication in cellular organisms.

Mechanism of DNA polymerase – How does DNA Polymerase work?

DNA polymerases work by catalyzing the synthesis of new DNA strands through a reaction known as phosphoryl group transfer. The growing DNA strand, with a 3′-OH group, acts as a nucleophile and attacks the incoming deoxyribonucleoside triphosphate (dNTP) at the 𝜶-phosphorus, forming a phosphodiester bond and releasing inorganic phosphate (Pi).

The active site of DNA polymerase requires the presence of two magnesium ions. It’s crucial to understand that DNA polymerase can only add nucleotides to the 3′ end of the growing strand, which is why replication always proceeds in the 5’→3′ direction. DNA polymerases cannot initiate the formation of new DNA strands.

In order to carry out their function, DNA polymerases rely on a template strand that guides the polymerization reaction. They also require a primer, as they can only add nucleotides to the 3′ OH group. The primer can be a short segment of RNA, DNA, or a combination of both. Typically, in living systems, the primer is an RNA oligonucleotide.

Once a nucleotide is added, DNA polymerase can either dissociate from the DNA strand or continue moving along to add more nucleotides. The processivity of DNA polymerase, which varies among different types of polymerases, determines its ability to remain associated with the DNA template.

DNA replication is a highly accurate process, as even a single nucleotide change can result in a mutation. DNA polymerases employ two mechanisms to ensure fidelity.

Firstly, the geometry of the active sites of DNA polymerase allows only correct nucleotide base pairs to fit together, reducing the likelihood of incorrect nucleotide insertion. However, this alone is not sufficient, as studies have shown that DNA polymerases can occasionally add an incorrect nucleotide after correctly incorporating 104 to 105 nucleotides.

To address these errors, DNA polymerases possess 3’→5′ exonuclease activity, which allows them to proofread the newly synthesized DNA. DNA polymerase actively checks each added nucleotide and, if a mismatch is detected, removes the incorrect nucleotide. This process of error correction is known as proofreading. In DNA polymerase I, different active sites are dedicated to polymerization and proofreading functions.

Overall, DNA polymerases play a crucial role in faithfully replicating DNA, ensuring accurate transmission of genetic information from one generation to the next.

DNA Polymerases for DNA Repair

DNA polymerases play a crucial role in DNA repair processes, ensuring the maintenance of genomic integrity. The genome is susceptible to various types of damage, including mutations arising from replication errors and exposure to damaging agents like reactive oxygen species (ROS) and UV light. To counteract these threats, multiple DNA repair mechanisms have evolved, such as nucleotide excision repair (NER), mismatch repair (MMR), base excision repair (BER), DNA double-strand break repair (DSBR), and translesion synthesis (TLS).

Repairing DNA lesions requires the replacement of damaged or lost DNA sequences with accurate copies, often derived from the intact complementary DNA strand. DNA polymerases are uniquely capable of duplicating the genetic information stored in DNA, making them essential for both replication and repair processes. Different DNA polymerases are involved in specific repair pathways, depending on the type of damage, the phase of the cellular cycle, the nature of the repair reaction, and tissue specificity.

The complexity and specificity of DNA repair pathways necessitate the involvement of various DNA polymerases in human cells. Some of the DNA polymerases utilized for DNA repair include DNA pols β, ζ ,  λ, σ, μ, δ, and n. These polymerases exhibit distinct properties and functions, allowing them to participate in specific repair mechanisms. Additionally, replicative enzymes like DNA pols d, e, and g are frequently involved in DNA repair reactions, contributing to the overall repair capacity of the cell.

Through their ability to accurately synthesize DNA, DNA polymerases ensure the fidelity of DNA repair processes, enabling the restoration of the original genetic information. The coordinated actions of these polymerases, together with other repair proteins, form an intricate network that safeguards the integrity of the genome and helps maintain the stability of the cell’s genetic material.

Functions of DNA Polymerase

The functions of DNA polymerase are crucial for various processes related to DNA replication and maintenance of genetic integrity. Here are the key functions of DNA polymerase:

1. DNA Synthesis: DNA polymerase is responsible for synthesizing new DNA strands during replication. It uses deoxyribonucleotides as building blocks to create complementary copies of the original DNA molecule. By pairing nucleotides in specific combinations (cytosine with guanine and thymine with adenine), DNA polymerase generates accurate replicas of the genetic material.
2. Repairing DNA: DNA polymerase plays a vital role in DNA repair mechanisms. It helps rectify errors and lesions in the DNA structure to maintain genomic stability. One of its important functions is proofreading, where it acts as a “molecular editor” by removing incorrect nucleotide pairs from the newly synthesized DNA strand. This proofreading activity helps prevent mutations and ensures the fidelity of DNA replication.
3. Directionality and Elongation: DNA polymerase exhibits a specific directionality during DNA synthesis. It can only add free nucleotides to the 3′ end of the growing DNA strand. As a result, DNA polymerase elongates the new strand in a 5′ to 3′ direction. This directionality is opposite to the movement of DNA polymerase along the template strand, enabling the formation of antiparallel DNA strands.
4. Error Correction: Although DNA polymerase strives for accuracy, it can occasionally make mistakes during replication. However, many DNA polymerases possess an exonuclease domain, allowing them to detect and correct errors. The exonuclease activity of DNA polymerase enables it to remove incorrect nucleotides and replace them with the correct ones, enhancing the fidelity of DNA replication.
5. Fidelity and Mismatch Detection: Fidelity is crucial in DNA replication to prevent errors that can lead to dysfunctional proteins and diseases such as cancer. DNA polymerases employ various mechanisms to detect base pair mismatches. Hydrogen bonds and shape complementarity play significant roles in recognizing correct base pairs. When a mismatch occurs, DNA polymerase can shift from the polymerization site to the exonuclease site, excise the incorrect nucleotide, and replace it with the appropriate one.

What is RNA dependent DNA polymerase?

• RNA-dependent DNA polymerase, also known as reverse transcriptase, is an enzyme that catalyzes the synthesis of DNA from an RNA template. It is a key enzyme in the process of reverse transcription, which is the conversion of RNA into DNA. Reverse transcription is a characteristic feature of retroviruses, a family of viruses that includes HIV (Human Immunodeficiency Virus).
• During reverse transcription, RNA-dependent DNA polymerase uses an RNA template to synthesize a complementary DNA (cDNA) strand. This cDNA strand can then serve as a template for the synthesis of a double-stranded DNA molecule. The resulting DNA can integrate into the host genome, allowing the virus to replicate and persist in infected cells.
• Reverse transcriptase has a unique property among DNA polymerases because it can initiate DNA synthesis without a pre-existing DNA primer. It uses the RNA template as a primer and synthesizes DNA in a template-directed manner. The enzyme possesses both polymerase activity, which incorporates deoxyribonucleotides into the growing DNA chain, and ribonuclease H (RNase H) activity, which degrades the RNA strand of an RNA-DNA hybrid.
• In addition to retroviruses, reverse transcriptase activity is also found in certain other types of viruses, such as hepadnaviruses (e.g., hepatitis B virus) and retrotransposons, which are genetic elements that can move within a genome via an RNA intermediate.
• The discovery and characterization of reverse transcriptase have had significant implications in molecular biology and medical research, leading to advances in techniques like reverse transcription-polymerase chain reaction (RT-PCR) and the development of antiretroviral drugs for the treatment of HIV infections.

DNA vs RNA polymerase

FAQ

References

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• https://www.thermofisher.com/in/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/pcr-education/pcr-reagents-enzymes/dna-polymerase-characteristics.html
• https://www.news-medical.net/life-sciences/What-is-DNA-Polymerase.aspx