What is DNA polymerase and its Types, structure, function?

The DNA polymerases are enzymes that responsible for creating DNA molecules by assembling deoxyribonucleotides, the building blocks of DNA.

What is DNA polymerase?

The discovery of the first DNA polymerase (DNA pol), along with the demonstration of its ability to synthesize a DNA chain, using deoxynucleotide triphosphates (dNTPs) as the building blocks and the complementary strand as a template, is due to Arthur Kornberg (1918–2007), who in 1956 isolated DNA pol I from the bacterium Escherichia coli, gaining the Nobel Prize in Physiology and Medicine in 1959. Since then, DNA pols have been isolated from all living organisms.

These enzymes are essential to DNA replication and usually work in pairs to create two identical DNA strands from one original DNA molecule. During this process, DNA polymerase “reads” the existing DNA strands to create two new strands that match the existing ones.

Every time a cell divides, DNA polymerase is required to help duplicate the cell’s DNA, so that a copy of the original DNA molecule can be passed to each of the daughter cells. In this way, genetic information is transmitted from generation to generation.

Before replication can take place, an enzyme called helicase unwinds the DNA molecule from its tightly woven form. This opens up or “unzips” the double stranded DNA to give two single strands of DNA that can be used as templates for replication.

DNA polymerase adds new free nucleotides to the 3’ end of the newly-forming strand, elongating it in a 5’ to 3’ direction. However, DNA polymerase cannot begin the formation of this new chain on its own and can only add nucleotides to a pre-existing 3′-OH group. A primer is therefore needed, at which nucleotides can be added. Primers are usually composed of RNA and DNA bases and the first two bases are always RNA. These primers are made by another enzyme called primase.

Although the function of DNA polymerase is highly accurate, a mistake is made for about one in every billion base pairs copied. The DNA is therefore “proofread” by DNA polymerase after it has been copied so that misplaced base pairs can be corrected. This preserves the integrity of the original DNA strand that is passed onto the daughter cells

The Basic Structure of DNA Polymerase

The crystallographic resolution of DNA pols from several organisms belonging to all seven families has revealed a common overall architectural, which has been intuitively linked to a half-open human right hand.

Based on this analogy, the catalytic subunits of all DNA pols known so far are composed of three domains, all contained within a single polypeptide chain: thumb, palm and fingers The thumb binds the double-stranded part of the nucleic acid template, the fingers interact with the incoming dNTP and the single-stranded strand.

The palm contains a cleft able to accommodate the nucleic acid substrate and contains the catalytic amino acids responsible for the interaction with the dNTP and its incorporation into the growing DNA chain.

Binding of the nucleic acid template and dNTP causes a conformational change, where the fingers rotate toward the palm, effectively “closing” the active site and positioning the dNTP and nucleic acid substrates in the proper reciprocal orientation for the catalytic step to occur.

The palm domain is extremely conserved in most families, while the fingers and thumb domains are much more variable. Deviations from this common architecture are observed in some members of the Y family, that contain much shorter thumb and fingers domains, as well as an extra domain called “wrist” connected to the palm.

The X-family DNA polymerase of the African swine virus, represents the most notable exception to this general architecture. This enzyme is the smallest known DNA polymerase (174 aa) and it is formed uniquely by a palm domain and a short C-terminal extension. It is characterized by a very low fidelity.

What Are the Families of DNA Polymerase?

The DNA polymerases are divided into seven families based on their sequence homology and crystal structure analysis. These include families A, B, C, D, X, Y and RT.

Some of the characteristics of the different families include:

Family A

Polymerases in this family are classed as either replicative polymerases or repair polymerases. When DNA is replicated, replicative polymerases match free nucleotides to sequences in template DNA. This coupling of nucleotide bases always occurs in certain combinations, with cytosine always paired to guanine and adenine always paired to thymine.

The repair polymerases “proofread” the new strands created and rectify any mistakes in the base pairing. In this way, the integrity of the original DNA strand that is passed onto daughter cells is preserved.

The replicative members of family A include the T7 DNA polymerase as well as the eukaryotic mitochondrial DNA polymerase γ. The repair polymerases include DNA pol I from E. coli, pol I from Thermus aquaticus and pol I from Bacillus stearothermophilus.

Family B

This family mainly contains replicative polymerases that are involved in processing DNA replication during cell division. Many of the polymerases in this family are present in fungi, plants and some are present in bacteriophages. These enzymes contribute to synthesis of both the leading and lagging DNA strands during replication. Family B polymerases are highly accurate in their function and perform 3′-5′ proofreading of newly synthesized DNA in order to correct any errors that occur during DNA replication.

Family C

Family C polymerases are the major replicative polymerases in bacteria. DNA polymerase III is the main family C polymerase involved in E. coli DNA replication. Polymerase III is made up of the clamp-loading complex, the beta sliding clamp processivity factor and the pol III core. The core comprises three subunits – the α subunit which is the polymerase activity hub, the δ subunit which is the exonucleolytic proofreader, and the θ subunit which may stabilize δ. The core and the beta sliding clamp are present in duplicate, to allow for processing of both the leading and lagging DNA strands.

Family D

These polymerases are present in Euryarchaeota, a subdomain of archaea, and are mainly replicative. This family of polymerases is not clearly defined but studies of Pyrococcus furiosus DNA polymerase II suggest this enzyme is a replicative polymerase.

Family X

This family includes the eukaryotic polymerase pol β, along with others such as pol μ, pol λ, pol σ and terminal deoxynucleotidyl transferase. Polymerase β performs short patch repair of damaged DNA by fixing alkylated, oxidized or a basic site that have formed due to DNA damage.

Pol λ and pol μ are involved in the rejoining of breaks that have occurred in double strands of DNA due to hydrogen peroxide (in the case of pol λ) and ionizing radiation (in the case of pol μ). Terminal deoxynucleotidyl transferase is only found in lymphoid tissue and adds non-templated nucleotides at V(D)J junctions, to provide diversity.

Family Y

These polymerases have a low fidelity for intact DNA strands and are capable of replicating damaged DNA.

One example of a family Y polymerase is pol IV, an error-prone polymerase that has no 3’ to 5’ proofreading activity and is involved in mutagenesis. The enzyme is expressed by a gene (dinB) that is switched on when polymerases stall at the replication fork. This interferes with the processivity of pol III which acts as a checkpoint, stopping replication and allowing time for DNA to be repaired. Cells that lack dinB are at an increased risk of developing mutations caused by agents that damage DNA.

Pol V also belongs to the Y family of polymerases and allows DNA damage to be bypassed in order for replication to continue.

Types of DNA polymerase.

Basically, the types of DNA polymerase are also divided depending on the organism that possess themi.e. eukaryotic and prokaryotic DNA polymerases. These types of DNA polymerase are classified based on their characteristics including structural sequences, and functions.

Prokaryotic DNA Polymerases

Prokaryotes contain five different types of DNA polymerase. These are described below.

DNA Polymerase I

Polymerase I is a DNA repair enzyme from the family A polymerases that has a 5’ to 3’ and 3’ to 5’ activity. Pol I account for more than 95% of polymerase activity in E. coli, although cells that lack this polymerase have been found and its activity can be replaced by the other four types of polymerase. This DNA polymerase has a poor processivity rate, adding around 15 to 20 nucleotides per second. Pol I begin the process of DNA elongation at a point called the “origin of replication” and about 400 base pairs downstream of this point, Pol III takes over replication, which it performs at a much higher speed.

DNA Polymerase II

Polymerase II is a DNA repair enzyme with a 3’ to 5’ exonuclease activity. Pol II is a family B polymerase and provides support to Pol III. When DNA acquires damage in the form of short gaps, which block Pol III activity, Pol II helps to remedy this problem by restarting DNA synthesis downstream of these gaps.

DNA Polymerase III

This holoenzyme is the main polymerase in E.coli DNA replication and is one of the family C polymerases. Polymerase III is made up of the clamp-loading complex, the beta sliding clamp processivity factor and the Pol III core. The core comprises three subunits – the α subunit which is the polymerase activity hub, the δ subunit which is the exonucleolytic proofreader, and the θ subunit which may stabilize δ. The core and the beta sliding clamp are present in duplicate, to allow for processing of both the leading and lagging DNA strands.

DNA Polymerase IV

This enzyme belongs to the Y family of DNA polymerases. Pol IV is an error-prone polymerase that has no 3’ to 5’ proofreading activity and is involved in mutagenesis or the altering of DNA to give rise to a mutation. The enzyme is expressed by a gene (dinB) that is switched on when polymerases stall at the replication fork. This interferes with the processivity of Pol III which acts as a checkpoint, stopping replication and allowing time for DNA to be repaired. Cells that lack dinB are at an increased risk of developing mutations caused by agents that damage DNA.

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’t proofread the synthesis of DNA replicas making it less efficient.

Eukaryotic DNA Polymerase

DNA Polymerase γ

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.

DNA Polymerase α, δ, and 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.

DNA Polymerase β, μ, and 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.

DNA Polymerases η, ι, and 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.

What is the function of DNA polymerase?

DNA replication requires unwinding of the complementary two-stranded structure of DNA. This process is mediated by the severance of the hydrogen bonds that hold the bases together; the result is the formation of two single strands.

The resultant Y-shaped appearance in this region of DNA is called the replication fork. The initiation of replication occurs at specific sites called the origin of replication (ori). Following the establishment of the replication fork is the replisome, several factors which enable replication to take place.

DNA Polymerases are one such crucial factor. They are multi-subunit enzymes that participate in the process of DNA replication in the cell. They catalyze the addition of nucleotides onto existing DNA strands. There are many families of DNA polymerase that play a role in DNA replication; there are at least 15 in humans and are required at different points during the process.

Polymerase function during DNA replication

DNA polymerase enzymes typically work in a pairwise fashion; each enzyme replicates one of the two strands that comprise the DNA double helix. These are called the leading strand and lagging strand and are named according to the relative speed at which they are replicated.

The replicated strands are synthesized using the leading and lagging strands as templates. Consequently, the two new double-stranded DNA molecules produced consist of one strand from the original helix (either the leading or lagging strand) and one new strand. This process is called semi-conservative replication and is essential as it permits genetic information to be transmitted from generation to generation.

The activities of both DNA polymerases are coordinated by two structures called the sliding clamp loader and the sliding clamp. The sliding clamp loader contacts single-stranded binding proteins that coat the separated helix as well as the sliding clamp.

Two sliding clamps encircle the two strands of DNA, and together with accessory proteins called the clamp loader complex, provide a stable binding site for the two DNA polymerases. The unwound single-stranded DNA templates move toward the complex; the behavior of the clamp loader on the leading and lagging strand differ because of a property called directionality.

This is determined by the orientation of the phosphate bond and characterized by the conventions 5’ to 3’ and 3’ to 5’. Each of the two strands of the helix necessarily possess opposite directionality; this is essential for base pairing to occur. Their pairing is also referred to as antiparallel.

DNA polymerase synthesizes only in a 5′ to 3′ direction. Consequently, the strand with the complementary 3’ to 5’ directionality, the leading strand, is synthesized as one continuous piece. Conversely, the strand with 5’ to 3’ directionality is synthesized as a series of small fragments called Okazaki fragments.

The lagging strand orientation of 5’ to 3’ is incompatible with DNA polymerase; to accommodate this requirement, the clamp loader must continually release and reattach at a new location. This requires the lagging strand to bubble out from the replisome.

Polymerases for DNA repair

Several polymerases exist in both prokaryotes and eukaryotes. They provide polymerase activity under two broad categories; normal replication and repair. Under conditions of normal replication, DNA polymerase corrects errors by 3′ → 5′ exonuclease activity.

Outside of normal replicative events, DNA repair is an ongoing process that is necessary to maintain the integrity of the genome. Both endogenous and exogenous insults result in damaged DNA; for example, single-strand and double-strand breaks, strand cross-linking, base loss, and base modification.

Multiple pathways exist to repair these DNA damage events in a selective manner. These include mismatch repair, nucleotide excision repair, base excision repair, double-strand break repair, and inter-strand cross-link repair. The biochemical difference that exists between these polymerases allows them to fulfill distinct roles under these specific conditions of repair.

comment