DNA polymerases are molecular machines responsible for constructing new DNA strands. They play a role in copying the genetic information stored within an organism’s DNA, a process known as DNA replication. This duplication is necessary for cell division and the inheritance of traits across generations. Despite their efficiency in building DNA molecules, these enzymes operate under specific constraints that prevent them from replicating an entire DNA strand.
The Need for a Primer
DNA polymerases possess a limitation: they cannot initiate the synthesis of a new DNA strand. Instead, they require a pre-existing short segment of nucleotides to which they can add new building blocks. This segment is known as a primer, and it must provide a free 3′-hydroxyl group, which serves as the attachment point for incoming nucleotides. Without this group, the polymerase lacks the necessary anchor to begin its work.
Another enzyme, primase, creates these initial primers. Primase synthesizes short RNA primers, typically about 5 to 10 nucleotides long, directly onto the DNA template strand. Once the RNA primer is in place, DNA polymerase binds to its 3′-hydroxyl end and begins elongating the new DNA strand. This reliance on a pre-existing primer means that DNA polymerase is extending an existing chain, rather than starting a new one.
The Directional Constraint
A constraint for DNA polymerases is their strict directionality of synthesis. These enzymes can only add new nucleotides to the 3′ end of a growing DNA strand, meaning synthesis proceeds in a 5′ to 3′ direction relative to the new strand being built. This unidirectional process creates a challenge during the replication of double-stranded DNA, where the two strands run in opposite directions.
One strand, the leading strand, is synthesized continuously because its 3′ end points towards the replication fork, allowing the polymerase to move along the template. The other strand, the lagging strand, runs in the opposite orientation. Due to the 5′ to 3′ synthesis rule, this strand must be synthesized discontinuously in short segments. These discrete pieces, Okazaki fragments, each require their own RNA primer and elongation. After synthesis, RNA primers are removed and replaced with DNA, and DNA ligase seals the gaps, joining these fragments into a continuous strand.
The End Replication Dilemma
The combined requirements of a primer and unidirectional synthesis pose a problem at the ends of linear chromosomes, a phenomenon known as the end replication dilemma. Eukaryotic chromosomes have repetitive DNA sequences at their ends called telomeres, which do not code for proteins. During lagging strand synthesis, the final RNA primer near the end of the chromosome cannot be replaced with DNA because no upstream 3′-hydroxyl group is available for DNA polymerase to extend from.
When this last primer is removed, a small segment of the telomere remains unreplicated. With each round of DNA replication and cell division, the telomeres progressively shorten. This shortening limits the number of times a cell can divide, contributing to cellular aging and potentially impacting genomic stability. When telomeres become short, cells may stop dividing or undergo programmed cell death.
To counteract this shortening, some cells, particularly germ cells, stem cells, and cancer cells, employ an enzyme called telomerase. Telomerase contains an RNA template and reverse transcriptase activity, allowing it to add new repetitive DNA sequences to the ends of telomeres. This enzyme extends the telomeric DNA, preventing the loss of genetic information and maintaining chromosome length over multiple cell divisions.
Dealing with DNA Damage
DNA polymerases are accurate in copying DNA, but they can encounter difficulties when the DNA template is damaged. Environmental factors, such as ultraviolet (UV) radiation or certain chemicals, can cause lesions in the DNA structure. When a replicative DNA polymerase encounters a damaged nucleotide, its progress can be impeded, causing it to stall or detach from the DNA template.
Many DNA polymerases possess a proofreading capability that allows them to correct misincorporated nucleotides, but they are unable to accurately read through complex types of DNA damage. Synthesizing past severe damage can lead to the insertion of incorrect nucleotides, resulting in mutations. To prevent permanent errors or stalled replication forks, cells employ specialized DNA repair mechanisms. These systems can either remove the damaged section and replace it with correct nucleotides, or they can utilize specialized “bypass” DNA polymerases. These bypass polymerases synthesize across damaged regions, sometimes with lower fidelity, allowing replication to continue even when primary replicative polymerases cannot proceed.