Deoxyribonucleic acid, known as DNA, carries the genetic instructions that guide the development, functioning, growth, and reproduction of all known organisms. For life to continue, this genetic information must be accurately duplicated through a process called DNA replication. This process involves numerous specialized enzymes working to ensure the faithful copying of the entire genome. Among these, DNA ligase plays an important role in completing replication.
The Process of DNA Replication
DNA replication is a highly coordinated process where a cell creates an exact copy of its DNA, ensuring that each new daughter cell receives a complete set of genetic material. This process is semi-conservative, meaning each new DNA molecule consists of one original strand and one newly synthesized strand. Replication begins at specific points on the DNA molecule called origins of replication, where the DNA double helix unwinds and separates, forming a Y-shaped structure known as a replication fork.
DNA polymerases, the enzymes responsible for synthesizing new DNA strands, can only add nucleotides in a specific direction, from the 5′ end to the 3′ end. This directional constraint leads to different synthesis mechanisms for the two DNA strands at the replication fork. One strand, termed the leading strand, is synthesized continuously in the direction of the replication fork’s movement. This continuous synthesis requires only a single RNA primer to initiate the process.
In contrast, the other strand, known as the lagging strand, is synthesized discontinuously because its template runs in the opposite direction. To accommodate this, DNA polymerase synthesizes the lagging strand in short segments called Okazaki fragments. These fragments each require a separate RNA primer to begin synthesis. After the DNA polymerase extends these primers, the RNA primers are later removed, leaving small gaps, or “nicks,” between the newly synthesized Okazaki fragments.
DNA Ligase’s Specific Action
DNA ligase seals “nicks” in the DNA backbone, which are discontinuities where a phosphodiester bond is missing between adjacent nucleotides. These nicks commonly arise during DNA replication, particularly on the lagging strand after the removal of RNA primers between Okazaki fragments. The enzyme repairs these single-strand breaks by forming a new phosphodiester bond.
This action joins the short Okazaki fragments into a single, continuous DNA strand, completing the synthesis of the lagging strand. DNA ligase specifically acts on the sugar-phosphate backbone of the DNA, not on the nitrogenous bases, ensuring the structural integrity of the newly replicated DNA molecule.
The Mechanism of DNA Ligation
DNA ligation is an enzymatic process that requires an energy source. This energy is supplied by either adenosine triphosphate (ATP) in eukaryotes and bacteriophages, or nicotinamide adenine dinucleotide (NAD+) in bacteria. The mechanism involves a three-step reaction to form the new phosphodiester bond.
First, DNA ligase becomes “adenylated” by reacting with the energy cofactor, attaching an adenosine monophosphate (AMP) molecule to a lysine residue within the enzyme’s active site. Next, this AMP group is transferred from the enzyme to the 5′-phosphate end of one of the DNA strands at the nick. This step activates the 5′ end for the subsequent reaction.
Finally, the 3′-hydroxyl group of the adjacent DNA strand at the nick attacks the activated 5′-phosphate, leading to the formation of a phosphodiester bond and the release of AMP. This series of steps closes the gap in the DNA backbone, creating a continuous strand. This coordination ensures DNA repair and replication.
Significance of DNA Ligase Activity
Beyond its role in joining Okazaki fragments during DNA replication, DNA ligase maintains the integrity of the genome. Without proper ligase function, the DNA replication process would remain incomplete, resulting in fragmented DNA strands. Such fragmentation can lead to chromosomal instability and an increased potential for mutations.
The enzyme’s function extends to various DNA repair pathways, where it seals single-strand breaks that can occur due to DNA damage or other cellular processes. For instance, it plays a part in pathways like base excision repair, where it closes gaps after damaged nucleotides are removed and replaced. This broader involvement is important for cellular health and preventing genetic abnormalities.