During DNA replication, new DNA is made. Part of this process involves creating short, newly synthesized DNA segments called Okazaki fragments, which form on one of the two DNA strands. They were first identified in the 1960s by Japanese molecular biologists Reiji and Tsuneko Okazaki. Their work with bacteria changed the understanding of how DNA is duplicated, as it was previously believed to be a continuous process.
The Antiparallel Puzzle of DNA Replication
The structure of DNA presents a challenge for replication. DNA is a double helix with two long, coiled strands that are antiparallel, meaning they run in opposite directions. This directionality gives each strand a 5′ (five prime) end and a 3′ (three prime) end. One strand runs from 5′ to 3′, while its partner runs from 3′ to 5′.
The antiparallel arrangement is a problem for the enzyme that builds new DNA, DNA polymerase. This enzyme has a strict rule: it can only add new nucleotides to the 3′ end of a growing strand. This means it can only synthesize DNA in the 5′ to 3′ direction. As the cell prepares to replicate, an enzyme called helicase unwinds the double helix, creating a Y-shaped replication fork.
At this fork, the parent strands separate to serve as templates. For the template strand with 3′ to 5′ directionality, DNA polymerase works without interruption. It follows the helicase, continuously building a new 5′ to 3′ strand. This smoothly synthesized strand is called the leading strand.
The other template strand, the lagging strand, runs in the 5′ to 3′ direction. To build a new strand in the required 5′ to 3′ direction, DNA polymerase must move backward, away from the replication fork. This orientation prevents continuous synthesis and requires a fragmented approach.
Synthesizing the Fragments on the Lagging Strand
The cell synthesizes the lagging strand in small, discontinuous pieces. This process begins when an enzyme called primase creates a short, temporary RNA sequence known as a primer. This primer attaches to the lagging strand template, providing a starting point—a free 3′ end—for DNA polymerase to begin its work.
Once the RNA primer is in place, DNA polymerase attaches and synthesizes a new stretch of DNA. It moves away from the replication fork, adding nucleotides until it encounters the primer of the previously created fragment. The short segment of newly formed DNA between two primers is an Okazaki fragment.
This sequence repeats as the replication fork continues to open. As more of the lagging strand template is exposed, primase lays down another RNA primer further upstream. DNA polymerase then synthesizes another Okazaki fragment, working backward from that new primer. This results in a series of disconnected DNA fragments. In eukaryotic cells, these fragments are around 100-200 nucleotides long.
Joining the Fragments into a Continuous Strand
After the lagging strand is synthesized into a series of Okazaki fragments, the cell performs an editing and sealing process. The first step is removing the RNA primers that served as starting blocks for DNA polymerase. These temporary primers are replaced with DNA nucleotides to ensure the final strand is composed entirely of DNA. A specialized DNA polymerase or an enzyme with exonuclease activity handles this removal and replacement.
With the RNA primers gone and the gaps filled with DNA, small breaks or “nicks” remain in the sugar-phosphate backbone. These nicks exist between the end of one fragment and the beginning of the next. The cell uses an enzyme called DNA ligase to resolve this.
DNA ligase creates a phosphodiester bond, the chemical link that forms the DNA backbone, which seals the gap between adjacent fragments. By joining all the Okazaki fragments, DNA ligase transforms the discontinuous pieces into a single, unbroken DNA strand. This final step ensures the lagging strand becomes a continuous copy of its template.
Significance in Cellular Life and Research
The formation and joining of Okazaki fragments are processes for the accurate duplication of a cell’s genome. This mechanism ensures that both strands of the DNA double helix are copied completely before a cell divides. Without discontinuous synthesis on the lagging strand, cells would be unable to replicate the full length of their chromosomes, leading to the loss of genetic information.
Precision in this multi-step process is important for maintaining genomic stability. Errors in synthesizing or joining the fragments can lead to mutations or breaks in the DNA strand. Such damage can have serious consequences, potentially contributing to uncontrolled cell growth or cell death. Understanding this replication machinery is a focus in molecular biology and genetics.
The study of Okazaki fragments has provided insights into the mechanisms that govern life at the molecular level. This knowledge is applied in research, particularly in the study of genetic disorders and cancer. Many cancer therapies, for example, are designed to disrupt DNA replication in rapidly dividing tumor cells, and understanding Okazaki fragment maturation can inform the development of more effective treatments.