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Molecular Biology

DNA

DNA Polymerases are the master builders of the DNA world. These enzymes are absolutely central to molecular biology and are indispensable tools – biochemical reagents – in the clinical molecular laboratory

Think of DNA polymerases as highly skilled, specialized molecular machines whose primary job is to synthesize DNA molecules from their building blocks (deoxyribonucleotides). They are essential for life (DNA replication) and essential for many of the techniques we perform daily in the lab

Core Function: DNA Synthesis

The fundamental reaction catalyzed by ALL DNA polymerases is the addition of a deoxyribonucleotide (dNTP) to the 3’-hydroxyl (-OH) end of a pre-existing polynucleotide strand (a primer). This reaction forms a phosphodiester bond:

  1. Template Reading The polymerase reads a template DNA strand to determine which nucleotide to add next (following Watson-Crick base pairing rules: A with T, G with C)
  2. Nucleotide Selection It selects the complementary dNTP (dATP, dGTP, dCTP, or dTTP) from the surrounding environment
  3. Bond Formation It catalyzes a nucleophilic attack by the 3’-OH group of the primer strand on the alpha-phosphate of the incoming dNTP. This forms a phosphodiester bond and releases pyrophosphate (PPi)
  4. Directionality Synthesis ALWAYS proceeds in the 5’ → 3’ direction. The polymerase moves along the template strand reading it in the 3’ → 5’ direction

Essential Requirements for DNA Polymerase Activity

For a DNA polymerase to work, it needs several key components:

  • Template DNA: A single-stranded DNA sequence to be copied
  • Primer: A short nucleic acid strand (can be DNA or RNA) with a free 3’-hydroxyl group that is complementary to the template. Crucially, DNA polymerases cannot initiate synthesis de novo (from scratch); they can only extend an existing strand
  • Deoxyribonucleoside Triphosphates (dNTPs): The four building blocks (dATP, dGTP, dCTP, dTTP) providing both the bases and the energy (via hydrolysis of the high-energy phosphate bonds) for the reaction
  • Divalent Cations: Typically Magnesium ions (Mg²⁺), which act as essential cofactors, stabilizing the dNTP in the active site and facilitating catalysis. Manganese (Mn²⁺) can sometimes substitute but may decrease fidelity
  • Appropriate Buffer: To maintain optimal pH and ionic strength for enzyme activity

Key Enzymatic Activities Beyond Synthesis

Many DNA polymerases possess additional enzymatic activities crucial for their function in vivo and useful in vitro:

  • 5’ → 3’ Polymerase Activity: The primary chain elongation function (ALL DNA polymerases have this)
  • 3’ → 5’ Exonuclease Activity (Proofreading): The ability to “backspace” and remove a mismatched nucleotide immediately after it has been incorporated at the 3’ end. This significantly increases the fidelity (accuracy) of DNA synthesis
  • 5’ → 3’ Exonuclease Activity: The ability to remove nucleotides from the 5’ end of a DNA strand encountered ahead of the enzyme while it synthesizes. This is important in vivo for removing RNA primers during replication (e.g., by E. coli DNA Pol I) and DNA repair. It’s also exploited in techniques like nick translation and certain probe-based PCR assays (like TaqMan)

Types of DNA Polymerases

There’s a vast diversity of DNA polymerases across different life forms:

1. Prokaryotic DNA Polymerases (e.g., E. coli)

  • DNA Pol I: Involved primarily in DNA repair and processing Okazaki fragments (removing RNA primers via its 5’→3’ exo activity and filling the gaps with DNA). Possesses all three activities (5’→3’ pol, 3’→5’ exo, 5’→3’ exo)
  • DNA Pol II: Involved in DNA repair. Has 5’→3’ pol and 3’→5’ exo activities
  • DNA Pol III: The main replicative enzyme in E. coli. Highly processive (adds many nucleotides before dissociating), has high fidelity (due to 3’→5’ exo proofreading). A complex multi-subunit enzyme
  • DNA Pol IV & V: Involved in translesion synthesis (TLS) – replicating past damaged DNA, often with lower fidelity

2. Eukaryotic DNA Polymerases

More numerous and specialized, designated by Greek letters:

  • Pol α (alpha): Associated with primase; synthesizes RNA/DNA primers to initiate replication on both leading and lagging strands. Low processivity, lacks proofreading
  • Pol δ (delta): Thought to be the main polymerase for lagging strand synthesis. Highly processive (with PCNA clamp), has 3’→5’ exo proofreading
  • Pol ε (epsilon): Thought to be the main polymerase for leading strand synthesis. Highly processive, has 3’→5’ exo proofreading
  • Pol γ (gamma): Replicates mitochondrial DNA. Has proofreading
  • Others (β, ζ, η, θ, ι, κ, λ, μ, Rev1): Primarily involved in various DNA repair pathways and translesion synthesis

3. Thermostable DNA Polymerases (Crucial Lab Reagents!)

These are isolated from thermophilic bacteria or archaea that live in high-temperature environments (like hot springs). Their ability to withstand high temperatures (up to 95°C or more) without denaturation makes them essential for the Polymerase Chain Reaction (PCR)

  • Taq Polymerase: From Thermus aquaticus. The classic PCR enzyme
    • Optimal temperature ~72°C
    • Lacks 3’→5’ proofreading activity: Relatively low fidelity (error rate ~1 in 10⁴-10⁵ bases). Suitable for routine diagnostic PCR where absolute sequence accuracy isn’t paramount
    • Possesses terminal transferase activity Adds a non-template single Adenine (‘A’ overhang) to the 3’ end of PCR products, useful for TA cloning
    • Relatively high processivity and extension rate
  • Pfu Polymerase: From Pyrococcus furiosus
    • Possesses 3’→5’ proofreading activity: High fidelity (error rate ~1 in 10⁶ bases). Preferred for applications requiring accurate sequences (cloning, sequencing)
    • Produces blunt-ended PCR products
    • Generally slower extension rate than Taq
  • Vent, Deep Vent Polymerases: From Thermococcus litoralis and Pyrococcus species, respectively. Also high-fidelity, proofreading enzymes
  • Blends and Engineered Polymerases: Many commercial polymerases are mixtures (e.g., Taq + a small amount of a proofreading enzyme) or genetically engineered versions designed to enhance specific properties:
    • Higher Fidelity: Improved proofreading
    • Higher Processivity/Speed: Faster extension rates
    • Longer PCR products: Ability to amplify large fragments
    • Inhibitor Tolerance: Resistance to substances in crude samples (blood, soil) that inhibit PCR
    • “Hot Start” Versions: Modified enzymes (e.g., antibody-bound, chemically modified) that are inactive at room temperature and only become active after an initial high-temperature denaturation step. This prevents non-specific amplification and primer-dimer formation during reaction setup

4. Reverse Transcriptases (RTs)

These are technically RNA-dependent DNA polymerases. They synthesize a DNA strand (cDNA) using an RNA template. While distinct, they are often discussed alongside DNA polymerases and are crucial reagents (Covered separately under Reverse Transcriptase)

Key Properties for Lab Applications

When choosing a DNA polymerase reagent for the lab, consider:

  • Thermostability: Essential for PCR
  • Fidelity: Accuracy; crucial for sequencing, cloning, mutation detection
  • Processivity: Number of bases added per binding event; important for amplifying long targets
  • Extension Rate: Speed of synthesis
  • Exonuclease Activities: Presence/absence of 3’→5’ (proofreading) or 5’→3’ activities
  • End Product: Blunt ends vs. 3’-A overhangs

Clinical Laboratory Relevance & Applications

DNA polymerases are the heart of many molecular diagnostic techniques:

  • Polymerase Chain Reaction (PCR): Amplifying specific DNA sequences for detection of pathogens, genetic mutations, cancer markers, etc. The choice of polymerase impacts sensitivity, specificity, and suitability for downstream applications
  • DNA Sequencing: Both Sanger sequencing (using DNA Pol for chain termination reactions) and Next-Generation Sequencing (NGS) platforms (often involving polymerase-mediated incorporation of labeled nucleotides) rely heavily on DNA polymerases
  • Cloning and Molecular Biology: Amplifying genes of interest for research or diagnostic reagent production
  • Site-Directed Mutagenesis: Creating specific DNA sequence changes using PCR with specialized primers and polymerases
  • Probe Labeling: Incorporating labeled dNTPs into DNA probes for hybridization assays
  • Isothermal Amplification: Techniques like LAMP use polymerases with specific properties (e.g., strand displacement)

Key Terms

  • DNA Polymerase: Enzyme that synthesizes DNA from a DNA template
  • Phosphodiester Bond: Covalent bond linking nucleotides in a nucleic acid chain
  • Template: The nucleic acid strand being read by the polymerase
  • Primer: A short nucleic acid strand with a free 3’-OH, required to initiate synthesis
  • dNTPs (deoxyribonucleoside triphosphates): The building blocks of DNA (dATP, dGTP, dCTP, dTTP)
  • 5’ → 3’ Synthesis: The direction in which new DNA strands are synthesized
  • Exonuclease: Enzyme that removes nucleotides from the ends of a nucleic acid strand
  • 3’ → 5’ Exonuclease Activity (Proofreading): Removes mismatched nucleotides from the 3’ growing end, increasing fidelity
  • 5’ → 3’ Exonuclease Activity: Removes nucleotides from the 5’ end of a downstream strand
  • Fidelity: The accuracy of DNA synthesis (low error rate)
  • Processivity: The number of nucleotides incorporated before the polymerase dissociates from the template
  • Thermostable: Able to withstand high temperatures without losing function
  • Taq Polymerase: Common thermostable DNA polymerase used in PCR; lacks proofreading
  • Pfu Polymerase: High-fidelity thermostable DNA polymerase used in PCR; has proofreading
  • Hot Start: Modification preventing polymerase activity until high temperature activation, improving PCR specificity