DNA replication is the fundamental process by which a cell makes an exact copy of its entire genetic material before dividing. This complex mechanism involves unwinding the double helix and synthesizing two new, complementary strands of DNA. The structure of DNA creates a challenge for the replication machinery, forcing one of the new strands to be built in short, separate pieces. These short segments are known as Okazaki fragments, which must be connected to form a complete, continuous strand. The process of joining these fragments is the final step in synthesizing one half of the new DNA molecule.
The Constraints That Create Okazaki Fragments
Okazaki fragments exist because of the antiparallel nature of the DNA double helix. One strand runs in the 5′ to 3′ direction, while its complementary partner runs in the opposite, 3′ to 5′, direction. DNA polymerase, the enzyme responsible for building the new DNA strand, can only synthesize in one direction: the 5′ to 3′ direction.
As the DNA helix unwinds at the replication fork, one template strand is oriented perfectly for continuous synthesis. This is called the leading strand, and its new partner is built seamlessly in the direction of the moving replication fork. The other template strand is oriented in the opposite direction, forcing the polymerase to work discontinuously and away from the moving fork.
The discontinuously synthesized new strand is the lagging strand, where Okazaki fragments are created. Each fragment is a short stretch of newly synthesized DNA, typically around 100 to 200 nucleotides long in human cells. The polymerase must repeatedly stop, detach, and re-start closer to the replication fork, resulting in a series of short segments.
Preparing the Fragments for Ligation
Before the segments can be joined, a clean-up process must convert the Okazaki fragments into a form that can be sealed. DNA polymerase cannot begin synthesis on a bare template, so each Okazaki fragment starts with a small stretch of ribonucleic acid (RNA) called a primer. This temporary RNA segment provides the necessary starting point for the DNA polymerase to begin adding deoxyribonucleotides.
The RNA primers must be removed entirely because they are an incorrect molecule within the finished DNA strand. In bacterial cells, DNA Polymerase I handles this task by using its 5′ to 3′ exonuclease activity to chew away the RNA nucleotides one by one. Concurrently, this same enzyme fills the resulting gap with the correct DNA nucleotides.
In human cells, enzymes like the flap endonuclease FEN1 remove the RNA primer after it is displaced by a DNA polymerase. The end result of this preparatory phase is a newly synthesized DNA stretch with a small break, or nick, remaining. This nick is the final substrate, ready for the sealing enzyme, consisting of a 3′-hydroxyl group and a 5′-phosphate group on adjacent nucleotides.
The Final Seal: DNA Ligase
DNA Ligase is the enzyme that binds the Okazaki fragments together. This enzyme catalyzes the formation of the last single phosphodiester bond that permanently connects the adjacent fragments. Without this action, the lagging strand would remain a collection of disconnected segments, leading to chromosomal instability and cell death.
The ligation reaction is an energy-intensive process, which DNA Ligase powers using a cofactor molecule. In human cells and many viruses, this energy comes from the hydrolysis of Adenosine Triphosphate (ATP). Certain bacteria utilize Nicotinamide Adenine Dinucleotide (NAD+) as their energy source for the same reaction.
The mechanism involves three steps, beginning with the enzyme attaching an Adenosine Monophosphate (AMP) group to itself. The enzyme then transfers this AMP molecule to the 5′-phosphate end of the nick, activating it for the reaction. Finally, the 3′-hydroxyl group of the adjacent DNA fragment attacks the activated 5′-phosphate, successfully creating the new phosphodiester bond and releasing the AMP molecule. This covalent bond completes the sugar-phosphate backbone, transforming the discontinuous Okazaki fragments into a continuous strand of DNA.