DNA polymerase
The DNA polymerases are enzymes (cellular or viral) that are involved in the DNA replication process. They carry out the synthesis of the new DNA strand by pairing deoxyribonucleotide triphosphates (dNTPs) with the corresponding complementary deoxyribonucleotides of the template DNA. dNTPs used in DNA replication contain three phosphates attached to the 5' hydroxyl group. deoxyribose and depending on the nitrogenous base will be dATP, dTTP, dCTP or dGTP. The fundamental reaction is a transfer of a phosphate group in which the 3'-OH group acts as a nucleophile at the 3' end. of the growing chain. The nucleophilic attack occurs on the α phosphate (closest to deoxyribose) of the 5' deoxyribonucleoside. triphosphate that enters, releasing inorganic pyrophosphate and elongating the DNA (by forming a new phosphodiester bond). Unlike most biological processes that occur in the cell in which only one phosphate group (Pi) is separated, during replication the last two phosphate groups are separated, in the form of a pyrophosphate group. (PPi)
This process can be summarized in a chemical equation:
(DNA)n + dNTP ♦ (DNA)n+1 + PPi
Although DNA polymerase only has one active site to pair the four different dNTPs, the correct binding of A:T, C:G base pairs is possible based on their geometry: if the binding is incorrectly, a displacement of the α-phosphate occurs, making it more difficult for it to bind to the 3'-OH end and thus slowing down the rate of catalysis, which gives rise to DNA polymerase preferentially adding the correct bases.
DNA polymerases can add up to 1,000 nucleotides per second. This is due to their nature, that is, the number of nucleotides that they are capable of adding each time they associate with the DNA template that they are going to copy. Since the addition of nucleotides is a process that lasts a few milliseconds, the speed of catalysis will depend on the time that the DNA polymerase remains attached to the DNA, that is, on its processivity.
Chain growth occurs in the 5' → 3', since a free 3'-OH group is required for the start of the synthesis since this is the one that carries out the nucleophilic attack on the α phosphate of the dNTP, so that DNA polymerases require of a 3'-OH primer (which can be DNA or RNA) called a primer that is synthesized by RNA primase. The 3' of the primer is called the primer end.
DNA polymerases also perform other functions during the replication process. In addition to participating in elongation, they perform a corrective and repair function thanks to their 3' exonuclease activity, which gives them the ability to degrade DNA starting from one end of it. It is important that these correction mechanisms exist, since otherwise the errors produced during DNA copying would give rise to mutations.
Functions
DNA polymerases can add free nucleotides only to the 3' end. of the forming DNA strand. This causes the elongation of the chain being formed to take place in the 5'-3' direction. No known DNA polymerase can start a DNA strand again, that is, it cannot put the first nucleotide on the strand, then join the second, then the third, and so on. All they can do is lengthen an existing chain at its 3'-OH end, so there must always be an initial fragment already formed. This is the reason why DNA polymerase needs a pre-formed primer to which to add nucleotides. Primers can be made of RNA or DNA. In DNA replication, the first two bases are always RNA and are synthesized by another enzyme called primase. An enzyme called helicase is also required to untangle the DNA and undo its double-stranded structure in that region and form the structure of two bifurcated single-strands (this region is the replication fork), thus facilitating the replication of each of the two strands. strands following the semiconservative model of DNA replication.
Another property that some, but not all, DNA polymerases have is error correction. This process corrects the errors produced during the formation of the newly synthesized DNA. When a misplaced base pair is recognized, the DNA polymerase reverses the direction of its movement by delaying a base pair. This 3'-5' of the enzyme allows the elimination of the incorrect base pair to be later replaced by the correct one, the polymerase itself that will continue the replication. This activity is called proofreading. DNA polymerases are widely used in molecular biology experiments.
DNA polymerases have a highly conserved structure, which means that their catalytic subunits vary very little from one species to another. Conserved structures generally perform important and irreplaceable functions in the cell, the maintenance of which yields a number of advantages.
Structure
Known DNA polymerases have a highly conserved structure, which means that their overall catalytic subunits vary very little from species to species, regardless of their domain structures. Conserved structures often indicate important and irreplaceable functions of the cell, the maintenance of which provides evolutionary advantages. The shape can be described as a right hand with thumb, finger, and palm domains. The palm domain appears to function by catalyzing the transfer of phosphoryl groups in the phosphoryl transfer reaction. The DNA binds to the palm when the enzyme is active. This reaction is believed to be catalyzed by a two-metal ion mechanism. The finger domain functions to bind the nucleoside triphosphates with the template base. The thumb domain plays a potential role in DNA processivity, translocation, and positioning.
Processivity
The rapid catalysis of DNA polymerase is due to its processive nature. Processivity is a characteristic of enzymes that function on polymeric substrates. In the case of DNA polymerase, the degree of processivity refers to the average number of nucleotides added each time the enzyme binds to a template. The average DNA polymerase requires about a second to locate and bind a primer/template junction. Once bound, a non-processing DNA polymerase adds nucleotides at a rate of one nucleotide per second. However, processive DNA polymerases add multiple nucleotides per second, dramatically increasing the rate of DNA synthesis. The degree of processivity is directly proportional to the rate of DNA synthesis. The rate of DNA synthesis in a living cell was first determined as the elongation rate of T4 phage DNA in bacteriophage-infected bacteria. During the period of exponential increase of DNA at 37 °C, the rate was 749 nucleotides per second.
The ability of DNA polymerase to glide along the DNA template allows for greater processivity. There is a dramatic increase in processivity at the replication fork. This increase is facilitated by the association of DNA polymerase with proteins known as the DNA slip clamp. The clamps are multiple protein subunits associated in a ring. Using ATP hydrolysis, a class of proteins known as slip-clamp cargo proteins open the ring structure of DNA slip-clamps allowing the binding and release of the DNA strand. Protein-protein interaction with the clamp prevents the DNA polymerase from diffusing from the DNA template, ensuring that the enzyme binds to the same primer/template junction and continues replication. DNA polymerase changes conformation, increasing affinity for the clamp when associated with it and decreasing affinity when it completes replication of a stretch of DNA to allow release of the clamp.
Polymerase chain reaction
The property of DNA polymerases to replicate DNA strands is used for the polymerase chain reaction, known as PCR, to obtain a large number of copies of a particular DNA fragment, amplifying it to research purposes. In PCR processes, the use of thermostable polymerases is required, such as Taq polymerase, because it is necessary to apply high temperatures to denature the DNA molecule.
DNA polymerase families
Based on the homology of their structure and the sequence of amino acids, DNA polymerases can be classified into 6 families. The DNA polymerases of DNA viruses are difficult to classify into the following groups due to the high divergence of viral sequences.
- Family A: Includes γ polymerase DNA, θ, ν, procariot I and viral T7.
- Family B: Includes eukaryotic polymerase DNA, α, δ, ε and procariot II. The inclusion of some viral polymerase DNA has also been proposed.
- Family D: Includes procariot D polymerase DNA and has also proposed the inclusion of some viral polymerase DNA.
- Family X: Includes β, σ, λ, μ, TDT and procariot III polymerase DNA.
- Family and: Includes eukaryotic polymerates ι, κ, η and procariot IV and V.
- Family RT: Includes the exclusive telomerase of eucariots and viral reverse transcribases, eucariots and procarotes.
Prokaryotic DNA polymerases
The DNA polymerases of prokaryotes (archaea and bacteria) are: Pol I, II, III, IV, V, B, D, and RT, each of which is specialized in one or more of these functions, depending on which one it is. its role in replication. Prokaryotic polymerases exist in two forms: core polymerase and holoenzyme. The core polymerase synthesizes DNA from the DNA template, but cannot initiate the synthesis alone or accurately. The holoenzyme initiates the synthesis precisely.
Pol I
Prokaryotic family A polymerases include the enzyme DNA polymerase I (Pol I), which is encoded by the polA gene and is ubiquitous among prokaryotes. This repair polymerase is involved in excision repair with 3'-5' and 5'-3' exonuclease activity. and in processing Okazaki fragments generated during lagging strand synthesis. Pol I is the most abundant polymerase and represents > 95% polymerase activity in E. coli; however, cells have been found to lack Pol I, suggesting that Pol I activity may be replaced by the other four polymerases. Pol I adds ~15-20 nucleotides per second, thus showing poor processivity. Instead, Pol I begins to add nucleotides at the RNA primer:template junction known as the origin of replication (ori). Approximately 400 bp downstream of the origin, the Pol III holoenzyme assembles and takes over replication at a highly processive nature and rate.
Taq polymerase is a heat-stable enzyme in this family that lacks proofreading capabilities.
Pol II
DNA polymerase II is a B-family polymerase encoded by the polB gene. Pol II has 3'-5' exonuclease activity and is involved in DNA repair, replication restart to prevent injury, and its cellular presence can go from ~30-50 copies per cell to ~200- 300 during SOS induction. Pol II is also thought to be a backup of Pol III, as it can interact with holoenzyme proteins and assume a high level of processivity. The main role of Pol II is thought to be the ability to direct polymerase activity at the replication fork, and it helped to stop the terminal shunt mismatches of Pol III.
Pol III
DNA polymerase III holoenzyme is the major enzyme involved in DNA replication in prokaryotes and belongs to the C polymerase family. It consists of three sets: core pol III, clamp processivity factor beta slider and the clamp charge complex. The core consists of three subunits: α, the center of polymerase activity, ɛ, an exonucleolytic proofreader, and θ, which can act as a stabilizer for ɛ. The beta sliding clamp processivity factor is also present in duplicate, one for each nucleus, to create a clamp that encloses the DNA, allowing for high processivity. The third set is a complex seven-subunit clamp loader (τ2γδδ′χψ).
The old "trombone model" Most textbooks describe an elongation complex with two equivalents of the core enzyme at each replication fork (RF), one for each strand, the leading and the lagging. However, recent evidence from single-molecule studies indicates an average of three stoichiometric equivalents of core enzyme in each RF for both Pol III and its B. subtilis counterpart, PolC. Fluorescence microscopy in the cell has revealed that backbone synthesis may not be fully continuous, and Pol III* (i.e., the α, ε, τ, δ, and χ subunits of the holoenzyme without the ß2 sliding clamp) have a high frequency of dissociation from the active RFs. In these studies, the turnover rate of the Replication fork was approximately 10 s for Pol III*, 47 s for the β2 sliding clamp, and 15 μm for the DnaB helicase. This suggests that the DnaB helicase may remain stably associated in the RFs and serve as a nucleation point for the competent holoenzyme. In vitro single molecule studies have shown that Pol III* has a high RF turnover rate when in excess, but remains stably associated with replication forks when concentration is limiting. Another single molecule study showed that BDNA helicase activity and strand elongation can proceed with uncoupled stochastic kinetics.
Pol IV
DNA polymerase IV (Pol IV) is an error-prone DNA polymerase that is involved in non-directed mutagenesis. Pol IV is a family Y polymerase expressed by the dinB gene that is activated by induction of SOS caused by polymerases stuck at the replication fork. During SOS induction, Pol IV production increases tenfold and one of the functions during this time is to interfere with the processivity of the Pol III holoenzyme. This creates a checkpoint, halts replication, and allows time for DNA damage to be repaired via the appropriate repair pathway. Another function of Pol IV is to perform the synthesis of translesions at the stalled replication fork, such as, bypass N2-deoxyguanine adducts at a faster rate than bypassing undamaged DNA. Cells lacking the dinB gene have a higher rate of mutagenesis caused by DNA-damaging agents.
Pol V
DNA polymerase V (Pol V) is a Y-family DNA polymerase that is involved in the SOS response and the DNA repair mechanisms of translesion synthesis. Pol V transcription across genes umuDC is highly regulated to only produce Pol V when damaged DNA is present in the cell and generates an SOS response. The stalled polymerases cause RecA to bind to ssDNA, causing the LexA protein to self-digest. LexA then loses its ability to repress the transcription of the umuDC operon. The same RecA-ssDNA nucleoprotein post-translationally modifies the UmuD protein into 'UmuD' protein. UmuD and UmuD 'form a heterodimer that interacts with UmuC, which in turn activates the catalytic activity of umuC polymerase in damaged DNA. A 'toolbelt' model has been proposed; of polymerase to switch pol III to pol IV in an arrested replication fork, where both polymerases bind simultaneously to the β-clamp. However, the involvement of more than one TLS polymerase working in succession to prevent injury is not yet has been shown in E. coli. Furthermore, Pol IV can catalyze both insertion and extension with high efficiency, while pol V is considered the main SOS TLS polymerase. An example is the derivation of the intrastrand guanine thymine crosslink where it was shown, based on the difference in the mutational signatures of the two polymerases, that pol IV and pol V compete for TLS of the intrastrand crosslink.
Family D
In 1998, the D family of DNA polymerases was discovered. The PolD complex is a heterodimer of two strands, each encoded by DP1 (small correction) and DP2 (large catalytic). Unlike other DNA polymerases, the structure and mechanism of the DP2 catalytic core resemble those of multisubunit RNA polymerases. The DP1-DP2 interface resembles that of the eukaryotic class B polymerase zinc finger and its small subunit. DP1, an exonuclease similar to Mre11, is likely the precursor of a small Pol α and ε subunit, providing proofreading capabilities now lost in eukaryotes. Its N-terminal HSH domain is similar to AAA proteins, especially the δ and RuvB subunit of Pol III in structure. DP2 has a class II KH domain. PolD is more heat stable and more precise than Taq polymerase but has not yet been commercialized. It has been suggested that the D family DNA polymerase was the first to evolve in cellular organisms and that the last universal common ancestor (LUCA) replicative polymerase belonged to the D family.
Reverse Transcriptase (RT)
It is an RNA-dependent DNA polymerase (RdDp) that synthesizes DNA from an RNA template. The reverse transcriptase family contains both DNA polymerase functionality and RNase H functionality, which degrades DNA-paired RNA. Reverse transcriptase is commonly used in RNA amplification for research purposes. Using an RNA template, PCR can use reverse transcriptase, creating a DNA template. This new DNA template can be used for typical PCR amplification. The products of such an experiment are therefore PCR products amplified from RNA.
Reverse transcription is accompanied by a template switch between the two copies of the genome (copy choice recombination). From 5 to 14 recombination events per genome occur in each replication cycle. Template switching (recombination) appears to be necessary to maintain genome integrity and as a repair mechanism to salvage damaged genomes.
DNA polymerase in eukaryotes
There are several types of DNA polymerase in eukaryotes: they are Pol ζ, α, δ, γ, θ, ν, ε, β, σ, λ, μ, ι, κ, η, TDT and RT. Primase (which is part of the DNA polymerase α molecule) synthesizes RNA primers and also begins DNA elongation of the two strands. Then a polymerase switch occurs and DNA polymerase σ enters, which continues the synthesis.
β, λ, σ, μ (beta, lambda, sigma, mu) and TdT polymerases
Family X polymerases contain the well-known eukaryotic pol β (beta) polymerase, as well as other eukaryotic polymerases such as Pol σ (sigma), Pol λ (lambda), Pol μ (mu), and terminal deoxynucleotidyl transferase (TdT). Family X polymerases are found primarily in vertebrates and some are found in plants and fungi. These polymerases have highly conserved regions that include two helix-hairpin-helix motifs that are imperative in DNA-polymerase interactions. One motif is located in the 8 kDa domain that interacts with downstream DNA and one motif is located in the thumb domain that interacts with the primer strand. Pol β, encoded by the POLB gene, is required for short-patch base excision repair, a DNA repair pathway that is essential for repairing alkylated or oxidized bases as well as abasic sites. Pol λ and Pol μ, encoded by the POLL and POLM genes respectively, are involved in non-homologous end splicing, a mechanism for rejoining DNA double-strand breaks due to hydrogen peroxide and ionizing radiation, respectively. 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.
α, δ and ε (alpha, delta and epsilon) polymerases
Pol α (alpha) Pol δ (delta) and Pol ε (epsilon) are members of the B family polymerases and are the major polymerases involved in nuclear DNA replication. The Pol α complex (pol α-DNA primase complex) consists of four subunits: the catalytic POLA1 subunit, the POLA2 regulatory subunit, and the small and large primase subunits PRIM1 and PRIM2, respectively. Once the RNA primer has been created by primase, Pol α begins replication by elongating the primer by ~20 nucleotides. Due to its high processivity, Pol δ takes over the synthesis of the leading and lagging chains of Pol α. Pol δ is expressed by genes POLD1 creating the catalytic subunit, POLD2 POLD3 and POLD4 creating the other subunits that interact with proliferating cell nuclear antigen (PCNA), which is a DNA clamp that allows Pol δ to possess processivity. Pol ε is encoded by the POLE1 gene, the catalytic subunit, POLE2 and POLE3. The function of Pol ε has been reported to be to extend the leading strand during replication, while Pol δ mainly replicates the lagging strand; however, recent evidence suggested that Pol δ might also have a role in backbone DNA replication. The C-terminal region of Pol ε "polymerase relic", despite being unnecessary for polymerase activity, is believed to be essential for cell vitality. The C-terminal region is believed to provide a checkpoint prior to entering anaphase, provide stability to the holoenzyme, and add proteins to the holoenzyme necessary for initiation of replication. Pol ε has a "palm" larger that provides high processivity regardless of PCNA.
Compared to other B-family polymerases, the DEDD family of exonucleases responsible for proofreading is inactivated in Pol α. Pol ε is unique in that it has two zinc finger domains and an inactive copy of another B-family polymerase at its C-terminus. The presence of this zinc finger has implications for the origins of Eukaryota, which in this case is placed in the Asgard group with archaeal polymerase B3.
η, ι and κ (eta, iota and kappa) polymerases
Pol η (eta) Pol ι (iota) and Pol κ (kappa) are DNA polymerases of the Y family involved in DNA repair by translesion synthesis and encoded by the POLH, POLI and POLK genes, respectively. Members of Family Y have five common motifs to help bind the substrate and primer end and all include the typical thumb, palm, and right-hand finger domains with added domains such as little finger (LF), the polymerase-associated domain (PAD) or doll. The active site, however, differs among family members due to the different lesions that are repaired. The Y-family polymerases are low-fidelity polymerases, but they have been shown to do more good than harm, as mutations affecting the polymerase can cause various diseases, including skin cancer and the Xeroderma Pigmentosum (XPS) variant.. The importance of these polymerases is evidenced by the fact that the gene encoding DNA polymerase η is known as XPV, because loss of this gene results in the Xeroderma Pigmentosum variant of the disease. Pol η is particularly important in allowing accurate translesion synthesis of DNA damage resulting from ultraviolet radiation. The functionality of Pol κ is not fully understood, but researchers have found two likely functions. Pol κ is believed to act as a base-specific extender or inserter in certain DNA lesions. All three translesion-synthesizing polymerases, along with Rev1, are recruited into damaged lesions via stalled replicative DNA polymerases. There are two pathways of damage repair leading researchers to conclude that the pathway chosen depends on which strand contains the damage, the leading strand or the laggard.
Rev1 and ζ (zeta) polymerases
Pol ζ, another polymerase of the B family, is formed by two subunits Rev3, the catalytic subunit, and Rev7 (MAD2L2), which increases the catalytic function of the polymerase and participates in the synthesis of translesions. Pol ζ lacks 3' to 5' exonuclease activity, it is unique in that it can extend primers with terminal mismatches. Rev1 has three regions of interest in the BRCT domain, ubiquitin-binding domain, and C-terminal domain and has dCMP transferase capacity, which adds opposing deoxycytidine lesions that would arrest the Pol δ and Pol ε replicative polymerases. These stalled polymerases activate ubiquitin complexes which in turn dissociate replication polymerases and recruit Pol ζ and Rev1. Together, Pol ζ and Rev1 add deoxycytidine and Pol ζ extends beyond the lesion. Through a still undetermined process, Pol ζ dissociates and replication polymerases reassociate and continue replication. Pol ζ and Rev1 are not required for replication, but loss of the REV3 gene in budding yeast may cause increased sensitivity to DNA-damaging agents due to collapse of replication forks where replication polymerases have stalled.
Polymerases γ, θ and ν (gamma, theta and nu)
Pol γ (gamma), Pol θ (theta) and Pol ν (nu) are family A polymerases. Pol γ, encoded by the POLG gene, was long thought to be the only mitochondrial polymerase. However, recent research shows that at least Pol β (beta), a Family X polymerase, is also present in mitochondria. Any mutation that produces limited or non-functioning Pol γ has a significant effect on mtDNA and is the most common cause of autosomal inherited mitochondrial disorders. Pol γ contains a C-terminal polymerase domain and a 3'-5' N-terminal exonuclease domain that are connected via the linker region, which binds to the accessory subunit. The accessory subunit binds to DNA and is required for Pol γ processivity. The A467T point mutation in the linker region is responsible for more than one third of all Pol γ-associated mitochondrial disorders. While many Pol θ homologues, encoded by the POLQgen, are found in eukaryotes, their function is not clearly understood. The amino acid sequence at the C-terminus is what classifies Pol θ as an A-family polymerase, although the error rate of Pol θ is more closely related to Y-family polymerases. Pol θ extends primer ends than they do not match and you can bypass abasic sites by adding a nucleotide. It also has deoxyribophosphodiesterase (dRPase) activity in the polymerase domain and can display ATPase activity in close proximity to ssDNA. Pol ν(nu) is considered the least efficient of the polymerase enzymes. However, DNA polymerase nu plays an active role in homology repair during cellular responses to cross-linking.
Reverse Transcriptase (RT)
It is an RNA-dependent DNA polymerase (RdDp) that synthesizes DNA from an RNA template. The reverse transcriptase family contains both DNA polymerase functionality and RNase H functionality, which degrades DNA-paired RNA. Reverse transcriptase is commonly used in RNA amplification for research purposes. Using an RNA template, PCR can use reverse transcriptase, creating a DNA template. This new DNA template can be used for typical PCR amplification. The products of such an experiment are therefore PCR products amplified from RNA.
Reverse transcription is accompanied by a template switch between the two copies of the genome (copy choice recombination). From 5 to 14 recombination events per genome occur in each replication cycle. Template switching (recombination) appears to be necessary to maintain genome integrity and as a repair mechanism to salvage damaged genomes.
Telomerase
Telomerase is an enzyme that works to replicate the ends of linear chromosomes, as normal DNA polymerase cannot replicate the ends or telomeres. The single-stranded 3' overhang of the double-stranded chromosome with the sequence 5'-TTAGGG-3' recruit telomerase. Telomerase acts like other DNA polymerases by extending the 3' end, but unlike other DNA polymerases, telomerase does not require a template. The TERT subunit, an example of reverse transcriptase, uses the RNA subunit to form the primer-template junction that allows telomerase to extend the 3' end of the chromosome ends. The gradual decrease in telomere size as a result of many replications throughout life is thought to be associated with the effects of aging.
Viral DNA polymerases
DNA viruses synthesize a wide variety of DNA polymerases, most of which are not related to cellular DNA polymerases or those of other DNA viruses. Retrotranscribed viruses such as HIV are characterized by synthesizing reverse transcriptases, which are related to each other and also to cellular reverse transcriptases, especially eukaryotic ones.
The phage Φ29 synthesizes its own DNA polymerase. This enzyme is widely used in molecular biology for multiple DNA amplification displacement procedures, and has a number of characteristics that make it particularly suitable for this application.