DNA replication is a fundamental process where a cell duplicates its DNA, ensuring that genetic information is passed on during cell division. This operation involves a host of molecular machines working in concert. Due to the inherent structure of the DNA molecule, the two intertwined strands are not copied in a continuous fashion. This necessitates two distinct modes of synthesis, one of which is known as lagging strand synthesis.
The DNA Directionality Problem
The structure of a DNA double helix is antiparallel, meaning the two strands run in opposite directions. One strand is oriented in a 5′ to 3′ direction, while its partner strand runs from 3′ to 5′. This chemical directionality is based on the numbering of carbon atoms in the DNA’s sugar backbone.
This antiparallel arrangement presents a challenge for the primary DNA-building enzyme, DNA polymerase. DNA polymerase has a strict operational rule: it can only add new nucleotides to the 3′ end of a growing DNA strand. Consequently, it can only synthesize new DNA in the 5′ to 3′ direction.
This limitation is not an issue for the leading strand, which is oriented from 3′ to 5′, allowing the new, complementary strand to be built continuously in the 5′ to 3′ direction. However, the other template strand, the lagging strand, runs from 5′ to 3′. This orientation means that to synthesize a new strand in the required 5′ to 3′ direction, the polymerase must move away from the replication fork.
Key Enzymes and Proteins in Synthesis
The synthesis of the lagging strand requires a coordinated effort from a specialized cast of enzymes and proteins.
- Helicase: This enzyme functions like a zipper, unwinding the DNA double helix and separating the two parental strands at the replication fork.
- Single-Strand Binding (SSB) Proteins: To prevent the separated single strands from rejoining, these proteins coat the exposed DNA, keeping them stable.
- Primase: Because DNA polymerase cannot start a new chain from scratch, primase synthesizes a short RNA primer which provides the necessary 3′ end starting point.
- DNA Polymerase III: This is the primary builder, adding DNA nucleotides one by one to extend from the RNA primer and create a new DNA segment.
- DNA Polymerase I: This enzyme acts to excise the RNA primers from the synthesized fragments and replace them with the correct DNA nucleotides.
- DNA Ligase: After primers are replaced, small gaps or “nicks” remain in the backbone. DNA ligase acts as a molecular glue, forming phosphodiester bonds to seal these gaps and join the fragments.
Synthesizing in Fragments
The solution to the directionality problem on the lagging strand is to synthesize the new DNA in a discontinuous, back-stitching manner. As the helicase enzyme unwinds the DNA at the replication fork, it continuously exposes more of the 5′ to 3′ template strand. On this lagging strand, the primase enzyme synthesizes a short RNA primer, providing a starting block for replication.
Following the placement of the primer, DNA polymerase III attaches and begins synthesizing a short, new piece of DNA. It builds this fragment in the required 5′ to 3′ direction, which means it moves away from the advancing replication fork. These short, discontinuous segments of newly synthesized DNA are known as Okazaki fragments, named after their discoverers. In eukaryotic cells, these fragments are around 100 to 200 nucleotides long.
As the replication fork continues to open, exposing more of the lagging strand template, the process repeats. Primase lays down a new RNA primer further upstream, and DNA polymerase III synthesizes another Okazaki fragment. This creates a series of separate DNA pieces, each starting with an RNA primer.
Once an Okazaki fragment is complete, the machinery prepares it for integration. DNA polymerase I removes the RNA primer from the beginning of each fragment and fills the resulting gap with DNA. The final step is performed by DNA ligase, which seals the nick between the end of one Okazaki fragment and the start of the next.
The End Replication Problem
Lagging strand synthesis creates a unique issue at the very ends of linear chromosomes, a phenomenon known as the end replication problem. When replication reaches the end of the chromosome on the lagging strand, a final RNA primer must be removed. Once this last primer is excised, there is no upstream 3′ end for DNA polymerase to add nucleotides to, leaving a small gap of unreplicated single-stranded DNA. This results in the chromosome becoming slightly shorter with each successive round of cell division.
To prevent the loss of genetic information from this progressive shortening, the ends of eukaryotic chromosomes are capped with protective structures called telomeres. Telomeres are long, repetitive sequences of non-coding DNA that act as a buffer, absorbing the loss of genetic material during replication without affecting functional genes.
In certain types of cells, such as stem cells and germ cells, an enzyme called telomerase is active. Telomerase is a specialized reverse transcriptase that carries its own RNA template, which it uses to extend the telomeres. By adding more repetitive sequences to the chromosome ends, telomerase counteracts the shortening that occurs during replication, maintaining the integrity of the chromosome over many cell cycles.