DNA polymerases
DNA polymerases are
essential enzymes in the process of DNA replication, repair, and some
specialized forms of recombination. They are responsible for synthesizing DNA
molecules from deoxyribonucleotides, the building blocks of DNA. Here is an
in-depth look at DNA polymerases, their types, mechanisms, and functions:
1. Core Function of DNA Polymerases
Polymerization Reaction: DNA polymerases catalyze the addition of nucleotides
to the 3'-OH end of a growing DNA strand. They use a template strand to
determine the correct nucleotide to add, ensuring base pairing (A-T, C-G)
fidelity.
Directionality: DNA polymerases synthesize new DNA in the 5' to 3' direction by
adding nucleotides to the 3'-OH group of the previous nucleotide.
Requirement of a Primer: Most DNA polymerases cannot initiate the synthesis of
a new DNA strand de novo; they require a primer (usually RNA in vivo) with a
free 3'-OH group to start adding nucleotides.
2. Mechanism of DNA Polymerization
Step 1: Nucleotide Binding: A deoxyribonucleotide triphosphate (dNTP)
binds to the active site of the polymerase.
Step 2: Base Pairing: The polymerase ensures that the nucleotide is correctly
base-paired with the complementary base on the template strand.
Step 3: Catalysis: DNA polymerase catalyzes the formation of a
phosphodiester bond between the 3'-OH group of the primer and the 5'-phosphate
group of the incoming dNTP. This releases pyrophosphate (PPi).
Step 4: Translocation: The polymerase moves to the next nucleotide on
the template to continue the process of adding nucleotides.
3. Proofreading and Fidelity
3’ to 5’ Exonuclease Activity: Many DNA polymerases possess proofreading
ability. If an incorrect nucleotide is incorporated, the polymerase detects the
mismatch and removes the incorrect nucleotide using its 3’ to 5’ exonuclease
activity, then continues DNA synthesis.
Fidelity of Replication: This proofreading activity greatly enhances the
fidelity of DNA replication. For example, in E. coli, DNA polymerase III, with
proofreading, has an error rate of around 1 mistake per 10^7 nucleotides.
4. Types of DNA Polymerases
DNA polymerases are divided into several families based on sequence homology
and structure:
A. Prokaryotic DNA Polymerases
DNA Polymerase I: Involved in DNA repair and removal of RNA primers from
Okazaki fragments during lagging-strand synthesis. It has both 5' to 3'
polymerase activity and 5' to 3' exonuclease activity (to remove primers).
DNA Polymerase II: Primarily involved in DNA repair; it has proofreading
activity but is not the primary replicative enzyme.
DNA Polymerase III: The primary replicative enzyme in E. coli. It
synthesizes both the leading and lagging strands during DNA replication. It has
high processivity due to the sliding clamp and 3' to 5' exonuclease
proofreading.
B. Eukaryotic DNA Polymerases
Eukaryotic cells have several specialized DNA polymerases, including:
DNA Polymerase α: Involved in the initiation of DNA replication by
synthesizing short RNA-DNA primers during lagging-strand synthesis.
DNA Polymerase δ: Involved in lagging-strand synthesis; it has high
fidelity and proofreading capability.
DNA Polymerase ε: Mainly responsible for leading-strand synthesis in
eukaryotes.
DNA Polymerase γ: Exclusively involved in mitochondrial DNA replication.
Specialized Polymerases (e.g., Pol ζ, Pol η, Pol ι): These are involved in DNA
repair and translesion synthesis (TLS), where they bypass DNA lesions or damage
that stall normal polymerases.
5. Specialized Functions
DNA Repair: Some DNA polymerases (e.g., Pol β in eukaryotes) are
involved in DNA base excision repair, where they fill in short single-stranded
gaps after the removal of damaged bases.
Translesion Synthesis (TLS): Specialized polymerases can replicate past
DNA lesions or damage that would otherwise stall replication. For example, Pol
η can bypass UV-induced thymine dimers.
Telomerase Activity: In eukaryotes, telomerase is a reverse transcriptase
enzyme that synthesizes the repetitive sequences at the ends of chromosomes
(telomeres) using an RNA template. Telomerase is important for maintaining
chromosome stability.
6. Structural Aspects
DNA polymerases typically have a structure that can be likened to a human hand,
with domains referred to as the "palm," "fingers," and
"thumb":
Palm Domain: Contains the catalytic active site where nucleotide
addition takes place.
Fingers Domain: Helps position the incoming dNTP for base pairing with
the template.
Thumb Domain: Stabilizes the DNA template and helps with processivity by
holding the DNA in place.
7. Processivity and the Sliding Clamp
DNA polymerases are highly processive enzymes, meaning they can add many
nucleotides without dissociating from the DNA template. In prokaryotic cells,
DNA polymerase III achieves high processivity through interaction with the
sliding clamp (the β-clamp in bacteria). In eukaryotes, the sliding clamp is
known as proliferating cell nuclear antigen (PCNA).
8. Applications of DNA Polymerases in Biotechnology
DNA polymerases are invaluable in modern molecular biology techniques:
Polymerase Chain Reaction (PCR): DNA polymerases like Taq polymerase
(from Thermus aquaticus, a heat-stable polymerase) are essential for amplifying
DNA in PCR. Heat stability is crucial for PCR, which involves repeated cycles
of denaturation and synthesis.
DNA Sequencing: DNA polymerases are used in Sanger sequencing to synthesize DNA
fragments that terminate at specific nucleotides, allowing for sequence
determination.
Cloning and Mutagenesis: DNA polymerases are used in techniques such as
site-directed mutagenesis to introduce specific mutations into DNA sequences.
9. Inhibitors and Clinical Relevance
Inhibitors of DNA polymerases have therapeutic relevance:
Antiviral Drugs: Drugs like acyclovir and ganciclovir are nucleoside analogs
that inhibit viral DNA polymerases, preventing the replication of viruses such
as herpes simplex.
Anticancer Drugs: Some chemotherapy agents target DNA polymerases to inhibit
the replication of rapidly dividing cancer cells.
10. Challenges and Future Directions
Despite significant understanding, challenges remain in fully elucidating the
roles of specialized polymerases, especially in DNA repair pathways and cancer
biology. New discoveries in polymerase functions and inhibitors continue to be
crucial in the fields of medicine and biotechnology.
DNA polymerases are, thus, central to the maintenance and propagation of
genetic information across generations, safeguarding genetic integrity, while
also playing critical roles in cellular responses to damage.