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DNA Polymerases: Types, Roles and Significance in Biotechnology

BY: ILA RUCHIE | Category: DNA | Submitted: 2016-01-19 06:57:26
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Article Summary: "DNA molecules are the troves of genetic information of an organism. DNA is the basis of life and is transferred from parent to offspring's. The DNA content of the parent is doubled by means of replication mechanism aided by a specific enzyme, DNA polymerases. DNA polymerase plays a central role in process of life and carries a w.."


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INTRODUCTION TO DNA POLYMERASES
 
DNA molecules are the troves of genetic information of an organism. DNA is the basis of life and is transferred from parent to offspring’s. The DNA content of the parent is doubled by means of replication mechanism aided by a specific enzyme, DNA polymerases. DNA polymerase plays a central role in process of life and carries a weighty responsibility of making an accurate copy of the cell’s genome. The DNA polymerases are enzymes that create DNA molecules by assembling nucleotides, the building blocks of DNA. The first evidence of the existence of an enzymatic activity capable of synthesizing DNA came in 1958 with the discovery of E. coli Pol I by A. Kornberg and colleagues. DNA polymerase moves along the old strand in the 3'-5' direction, creating a new strand having a 5'-3' direction.

CLASSIFICATION OF DNA POLYMERASES
Based on sequence homology and the comparison of the features of their primary sequence DNA polymerases are classified into seven families. A, B, C, D, X, Y, and RT.

Family

Types of DNA polymerase

Species

Examples

A

Replicative and Repair Polymerases

Eukaryotic and Prokaryotic

T7 DNA polymerase, Pol I, and DNA Polymerase γ

B

Replicative and Repair Polymerases

Eukaryotic and Prokaryotic

Pol II, Pol B, Pol ζ, Pol α, δ, and ε

C

Replicative Polymerases

Prokaryotic

Pol III

D

Replicative Polymerases

Euryarchaeota

Not well-characterized

X

Replicative and Repair Polymerases

Eukaryotic

Pol β, Pol σ, Pol λ, Pol μ, and Terminal deoxynucleotidyl transferase

Y

Replicative and Repair Polymerases

Eukaryotic and Prokaryotic

Pol ι (iota), Pol κ (kappa), Pol IV, and Pol V

RT

Replicative and Repair Polymerases

Viruses, Retroviruses, and Eukaryotic

Telomerase, Hepatitis B virus

 
 
TYPES AND ROLES OF DNA POLYMERASES (REPLICATIVE AND REPAIRATIVE)

1.      Prokaryotic DNA polymerase


Prokaryotes contain five different types of DNA polymerase. These are described below.
Pol 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 accounts for more than 95% of polymerase activity in  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.
  • This repair polymerase is involved in excision repair with 3'-5' and 5'-3' exonuclease activity and processing ofOkazaki fragments generated during lagging strand synthesis. 

Pol II
  • DNA polymerase II, a Family B polymerase. Polymerase II is a DNA repair enzyme with a 3’ to 5’ exonuclease activity.
  • 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.
  • Pol II has 3'-5' exonuclease activity and participates in DNA repair, replication restart to bypass lesions, and its cell presence can jump from ~30-50 copies per cell to ~200-300 during SOS induction.

Pol III
  • This holoenzyme is the main polymerase in coliDNA replication and is one of the family C polymerases.
  • Polymerase III is the exonucleolytic proofreader, and can process of both the leading and lagging DNA strands.

Pol 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.

Pol V
  • Pol V also belongs to the Y family of polymerases and allows DNA damage to be bypassed in order for replication to continue.
  • It is involved inSOS response and translesion synthesis DNA repair mechanisms. 

 

2.     Eukaryotic DNA polymerase


 
POL α
 
  • POL α is a members of Family B Polymerases and are the main polymerases involved with nuclear DNA replication.
  • This unique enzyme has two distinct polymerase activities: a 5’- 3’ DNA-dependent DNA polymerase, and a 5’- 3’
  • DNA-dependent RNA polymerase. The RNA polymerase activity is a primase. Because of this, the enzyme is often referred to as Pol a:primase. It is the only enzyme known to have both DNA polymerase and primase activities, and the only one capable of selfprimed DNA synthesis on a previously unprimed ssDNA.
  • Pol α does not have an intrinsic 3'- 5' exonuclease activity and also lacks a 5'- 3' exonuclease activity. In vivo, the primary function of Pol a:primase is to make short RNA/DNA primers for replicative DNA synthesis.

 
DNA polymerase ?
 
  • DNA polymerase ? has 5’- 3’ DNA polymerase activity and an intrinsic 3'- 5' exonuclease activity.
  • Pol ? requires an associated 30 kDa protein, called proliferating cell nuclear antigen (PCNA), for full polymerase activity and processivity. In the presence of PCNA, Pol ? is highly processive, and it is PCNA that acts as a processivity factor. Biochemical and genetic studies in yeast and mammalian cells suggest that Pol ? is also involved in some types of DNA repair synthesis.
  • Due to its high processivity, Pol δ takes over the leading and lagging strand synthesis from Pol α.

 
DNA polymerase ?
 
  • Belongs to Family B Polymerases and are the main polymerases involved with nuclear DNA replication.
  • Pol ? has 3'-5' exonuclease activity.
  • Pol e is a reasonably processive enzyme that also associates with PCNA at a primer terminus. This suggests that it is a replicative polymerase. Because of its high processivity and proofreading activity, it has been further suggested that Pol ? is involved in primer elongation, in addition to Pol ?.
  • Pol ε participates in repairing errors made in the leading strand during Pol δ replication in conjunction with DNA mismatch repair machinery.

 
DNA polymerase β
 
  • Belongs to family X polymerases are found mainly in vertebrates, and a few are found in plants and fungi.
  • Pol β 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.
  • This is the smallest and simplest of the classical eukaryotic polymerases; it is composed of a single ~40-48 kDa protein.
  • Pol β is not highly active and is not very processive. It has no intrinsic exonuclease activities.
  • Its preferred template is duplex DNA with short gaps, although it can bind a nicked duplex and is capable of some limited displacement synthesis. Pol β is primarily involved in DNA repair.

 

Polymerases λ, σ and μ (lambda, sigma, and mu)

Polymerases η, ι and κ (eta, iota, and kappa)

  • Pol η (eta), Pol ι (iota), and Pol κ (kappa), are Family Y DNA polymerases involved in the DNA repair by translesion synthesis.
  • Polymerases in Family Y are low-fidelity polymerases, but have been proven to do more good than harm as mutations that affect the polymerase can cause various diseases, such asskin cancer and Xeroderma Pigmentosum Variant (XPS).
  • Pol η is particularly important for allowing accurate translesion synthesis of DNA damage resulting fromultraviolet radiation.
  • Pol κ is thought to act as an extender or an inserter of a specific base at certain DNA lesions.

Polymerases ζ (zeta)

  • Pol ζ another B family polymerase, is involved in translesion synthesis.
  • Pol ζ lacks 3' to 5' exonuclease activity, is unique in that it can extend primers with terminal mismatches.

Polymerases γ and θ (gamma and theta)

  • Pol γ (gamma) and Pol θ (theta) are Family A polymerases.
  • Pol γ, is the only mtDNApolymerase and therefore replicates, repairs, and has proofreading 3'-5' exonuclease.
  • Any mutation that leads to limited or non-functioning Pol γ has a significant effect on mtDNA and is the most common cause of autosomal inherited mitochondrial disorders.
  • Pol θ, found in eukaryotes, its function is not clearly understood. Pol θ belongs to Family A polymerase.
  • Pol θ extends mismatched primer termini and can bypass abasic sites by adding a nucleotide.

 
  1. Reverse transcriptase

Retrovirus reverse transcriptase
  • Retroviruses package their genomes as ssRNA but replicate this RNA through a dsDNA intermediate. To do this, they employ an RNA dependent DNA polymerase (reverse transcriptase).
  • RT is a typical DNA polymerase in the 5'- 3' direction of synthesis, requirement for a template primer with a 3'-OH terminus, and requirements for dNTPs and Mg2+.
  • RT does not have a detectable DNA specific exonuclease activity, and therefore has no proofreading function. As a result, its error rate is relatively high. This is reflected in a high mutation rate; virus variants are generated at high frequency. This is an important aspect of pathogenesis: retroviruses are adept at evading host immunosurveillance because of high mutation frequency.
  • RT is unique among DNA polymerases in at least two respects: • It can use primed, natural ssRNAs as template. It can also use a primed ssDNA as template. • It has intrinsic RNase H activity. RNase H is a processive exonuclease that specifically degrades the RNA strand of a DNA-RNA hybrid beginning from either the 5' or 3' end. It can also act as an endonuclease. RNase H hydrolyzes phosphodiester bonds to leave products with 3' hydroxyl and 5' phosphate ends.
  • The enzyme is relatively processive and can replicate the 8 kb retrovirus genome without a processivity factor.

 

Telomerase

  • Telomeraseis a ribonucleoprotein recruited to replicate ends of linear chromosomes because normal DNA polymerase cannot replicate the ends, or telomere. Telomerase acts like other DNA polymerases by extending the 3’ end, but, unlike other DNA polymerases, telomerase does not require a template.
  • The protein and RNA components make up an active enzyme of ~200 kDa. The RNA component (~1.3 kb in yeast) contains the template sequence that is used for DNA synthesis. (So telomerase carries its own template.) In vitro, telomerase from a given species synthesizes the G-rich strand sequence characteristic of the species.
  • However, telomerase activity is present in actively dividing cells, including immortalized (transformed) cells in culture, and in most cancer cells. So telomerase may be useful for cancer diagnostics, and is a possible target for therapeutics.

 
 
SIGNIFICANCE OF DNA POLYMERASES IN BIOTECHNOLOGY
DNA polymerases also play central roles in modern molecular biology and biotechnology, enabling techniques including DNA cloning, the polymerase chain reaction (PCR), DNA sequencing, single nucleotide polymorphism (SNP) detection, whole genome amplification (WGA), synthetic biology, and molecular diagnostics.
THERMOSTABLE DNA POLYMERASE
Taq DNA polymerase. The original report of this enzyme, purified from the hot springs bacterium Thermus aquaticus, was published in 1976.  Later, the polymerase chain reaction was developed and shortly thereafter "Taq" became a household word in molecular biology circles.
The thermophilic DNA polymerases, like other DNA polymerases, catalyze template-directed synthesis of DNA from nucleotide triphosphates. A primer having a free 3' hydroxyl is required to initiate synthesis and magnesium ion is necessary. In general, they have maximal catalytic activity at 75 to 80C, and substantially reduced activites at lower temperatures. At 37C, Taq polymerase has only about 10% of its maximal activity.
In addition to Taq DNA polymerase, several other thermostable DNA polymerases have been isolated and expressed from cloned genes. Three of the most-used polymerases are described in the following table:
 
 

Polymerase

3'->5'
Exonuclease

Source and Properties

Taq

No

From Thermus aquaticus. Halflife at 95C is 1.6 hours.

Pfu

Yes

From Pyrococcus furiosus. Appears to have the lowest error rate of known thermophilic DNA polymerases.

Vent

Yes

From Thermococcus litoralis; also known as Tli polymerase. Halflife at 95 C is approximately 7 hours.

 
Future Challenges
Engineered DNA polymerases will continue to play important roles in biotechnology and the delivery of health care. Over the next several years, molecular methods that are easier, cheaper, and faster will emerge. At the same time, molecular biology will move toward analysis of low concentration biomolecules (i.e., a single set of chromosomes). Novel amplification techniques are also required to profile genetic variations among single cells because the quantity of genomic DNA from a single cell is insufficient to sequence directly. Therefore, DNA must first be amplified prior to further analysis. Furthermore, DNA polymerases with very low error rates are needed to ensure that long, amplified DNA are exact copies of the starting material.
Therefore, novel DNA amplification systems are needed to accelerate progress in emerging technologies and to make high-fidelity in vitro genome analysis and manipulation routine. Engineered DNA polymerases or cellular replication machineries capable of amplifying large DNA fragments have the potential to enable single cell genomics, genome synthesis, and manipulation. This issue summarizes the known properties of various DNA polymerase systems and how DNA polymerases are currently being manipulated to meet these growing demands.

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Comments on this article: (1 comments so far)

Comment By Comment
ila
2016-01-19 10:35:36 1026
There has been a mistake in publishing some symbols..please note..After pol alpha, the next is pol delta..next to delta is pol epsilon.. Thanks & regards

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