DNA synthesis, also known as replication, is the fundamental biological process by which a cell duplicates its entire genetic code. This duplication is a necessary step before cell division, occurring during the S (synthesis) phase of the cell cycle. The outcome is the creation of two complete, identical copies of the DNA molecule. The process follows a semi-conservative model, meaning each newly formed DNA double helix consists of one original strand and one newly synthesized strand. This method ensures that genetic information is passed down with high fidelity from the parent cell to the two resulting daughter cells.
Initiating Replication: The Machinery and the Fork
Replication begins at specific points called origins of replication, recognized by initiator proteins. At these origins, a specialized protein machine must first separate the two strands of the double helix to expose the nucleotide templates. The enzyme helicase performs the unwinding, breaking the hydrogen bonds that hold the complementary base pairs together. This unwinding action creates a Y-shaped structure known as the replication fork.
Once the strands are separated, single-strand binding proteins (SSBPs) coat the exposed single strands. These proteins prevent the separated strands from snapping back together, thereby keeping the DNA template accessible for the replication machinery. The two original strands are antiparallel: one runs 5′ to 3′, while the other runs 3′ to 5′. This inherent antiparallel orientation dictates the different methods used to synthesize the two new strands.
The Continuous Process: Synthesis of the Leading Strand
The core rule governing DNA duplication is that DNA Polymerase can only add new nucleotides to the free hydroxyl group found at the 3′ end of a growing strand. Therefore, new DNA can only be built in the 5′ to 3′ direction. Since DNA Polymerase cannot start a new strand from scratch, the enzyme primase must first synthesize a short RNA segment called a primer.
The template strand running 3′ to 5′ toward the replication fork is perfectly oriented for continuous synthesis. Once primase places a single RNA primer, DNA Polymerase attaches and continuously adds nucleotides as the helicase unwinds the double helix. This uninterrupted new strand, synthesized in one long piece in the same direction the fork is opening, is called the leading strand. This entire synthesis process requires only one priming event to complete the copy.
The Discontinuous Challenge: Building the Lagging Strand
The second template strand runs in the opposite 5′ to 3′ direction, which presents a mechanical problem for DNA Polymerase. Because the polymerase can only synthesize 5′ to 3′, it is forced to move away from the advancing replication fork. As the fork opens up new template space, the polymerase must repeatedly detach and reattach closer to the fork opening.
This necessity results in the discontinuous synthesis of the lagging strand. The strand is constructed in a series of short segments called Okazaki fragments. Each fragment requires its own individual RNA primer to provide a starting point for DNA Polymerase. In eukaryotes, these fragments are typically short, ranging from 100 to 200 nucleotides in length.
The discontinuous mechanism requires primase to periodically synthesize a new RNA primer on the lagging strand template. DNA Polymerase then extends this primer, synthesizing a fragment backward until it reaches the primer of the previously synthesized fragment. This repeated cycle of priming, synthesis, and detachment ensures that the entire 5′ to 3′ template strand is eventually copied. This fragmented approach is significantly more complicated than the continuous process occurring on the leading strand.
Final Steps: Ligation and Error Correction
After the Okazaki fragments are synthesized, the replication process enters a clean-up phase to convert the fragmented, RNA-containing molecules into a complete, continuous DNA strand. Specialized enzymes, such as DNA Polymerase I in bacteria or other exonucleases in eukaryotes, remove all temporary RNA primers. As the RNA is excised, the gap is immediately filled in with the correct DNA nucleotides.
This replacement action leaves small breaks, or nicks, in the sugar-phosphate backbone of the newly synthesized lagging strand. The enzyme DNA ligase then finalizes the process by forming a phosphodiester bond, sealing these nicks and connecting the Okazaki fragments into a single, seamless strand of DNA.
Proofreading and Fidelity
The ability of DNA Polymerase to check its own work, called proofreading, is active throughout the entire replication process. This proofreading function uses a 3′ to 5′ exonuclease activity, allowing the enzyme to move backward to remove any incorrectly added nucleotide from the 3′ end of the growing strand. By excising the mismatched base and replacing it with the correct one, the polymerase significantly lowers the error rate of replication. This robust mechanism ensures the integrity of the genetic material, minimizing mutations and maintaining the fidelity of the duplicated genome.