DNA polymerase is an enzyme present in all living things that synthesizes new DNA strands by using an existing strand as a template. This process of creating new copies of DNA molecules is fundamental for cell reproduction and the transfer of genetic information.
The Role in DNA Replication
The accurate duplication of an organism’s genome is required every time a cell divides, ensuring each new cell receives identical genetic instructions. DNA polymerase is the enzyme that drives this replication process. It moves along a single strand of existing DNA, assembling the building blocks, called nucleotides, to build a new, complementary strand based on the template.
All DNA polymerases synthesize the new DNA strand in a specific direction, from 5′ (five prime) to 3′ (three prime). This directional rule is a constraint that dictates how the replication machinery operates on both strands of the DNA double helix.
Mechanism of Action
Before DNA polymerase can begin, the DNA double helix must be unwound by an enzyme called helicase. Helicase separates the two strands to expose the nucleotide bases. This creates two single-stranded templates, making the genetic code accessible for the polymerase to copy.
DNA polymerase cannot start a new DNA strand from scratch and requires a starting point. This is provided by a short piece of nucleic acid called a primer, which is synthesized by an enzyme named primase. The primer binds to the template strand, creating an exposed 3′ end for the polymerase to begin synthesis.
Once docked, the polymerase moves along the template, adding the corresponding complementary nucleotide to the new strand. It pairs Adenine (A) with Thymine (T) and Guanine (G) with Cytosine (C). This process links them together to form the new DNA chain.
Due to the 5′ to 3′ synthesis rule, one strand, the leading strand, is synthesized continuously. The other strand, the lagging strand, must be synthesized discontinuously in small sections called Okazaki fragments. Each fragment requires its own primer and they are later joined together to form a complete strand.
Proofreading and Error Correction
While DNA polymerase is accurate, it can insert an incorrect nucleotide during replication. Without a correction mechanism, these mistakes would accumulate as mutations, potentially leading to disease or cellular dysfunction.
Many DNA polymerases have a proofreading function known as 3′ to 5′ exonuclease activity. If the enzyme detects a mismatch, it can pause, reverse its direction, and remove the erroneous nucleotide.
This proofreading action is similar to the “backspace” key on a keyboard. After excising the incorrect nucleotide, the polymerase moves forward again and inserts the correct one before continuing. This immediate error correction improves the fidelity of DNA replication.
Varieties of DNA Polymerase
DNA polymerase refers to a large family of enzymes, not a single one. While all share the ability to synthesize DNA, different types have evolved to perform specialized tasks within the cell. These polymerases are categorized into families based on their amino acid sequence similarities.
Organisms are classified as prokaryotes (like bacteria) and eukaryotes (like plants and animals), and each group has its own set of DNA polymerases. In prokaryotes such as E. coli, DNA Polymerase III is the main enzyme for replication, while DNA Polymerase I is involved in removing RNA primers and repairing DNA.
Eukaryotic cells contain a larger variety of at least 15 different DNA polymerases. The primary replicative enzymes are Polymerase delta (Pol δ) and Polymerase epsilon (Pol ε), responsible for synthesizing the lagging and leading strands, respectively. Other types, like Polymerase alpha (Pol α), help initiate replication, while Polymerase beta (Pol β) specializes in DNA repair.
Applications in Biotechnology
The capabilities of DNA polymerase have been harnessed by scientists for applications in molecular biology and medicine. The enzyme’s ability to copy DNA allows researchers to analyze and manipulate genetic material in the laboratory.
The most prominent application is the Polymerase Chain Reaction (PCR), a technique used to amplify a specific segment of DNA, generating millions of copies from a small sample. This process relies on a heat-stable DNA polymerase, like Taq polymerase, isolated from the bacterium Thermus aquaticus. Its ability to withstand the high temperatures required in PCR enabled the automation of the technique.
PCR has revolutionized numerous fields, including:
- Rapid diagnosis of infectious diseases by detecting the DNA of pathogens.
- Amplification of trace amounts of DNA from crime scenes for genetic fingerprinting.
- Genetic testing for hereditary conditions.
- General scientific research to study genes.
- Creation of genetically modified organisms.