DNA Replication in prokaryotes

DNA replication has been extremely well studied in prokaryotes primarily because of the small size of the genome and the mutants that are available. E. coli has 4.6 million base pairs in a single circular chromosome and all of it gets replicated in approximately 42 minutes, starting from a single origin of replication and proceeding around the circle in both directions. This means that approximately 1000 nucleotides are added per second. The process is quite rapid and occurs without many mistakes.

DNA replication employs a large number of proteins and enzymes, each of which plays a critical role during the process. One of the key players is the enzyme DNA polymerase, also known as DNA pol, which adds nucleotides one by one to the growing DNA chain that are complementary to the template strand. The addition of nucleotides requires energy; this energy is obtained from the nucleotides that have three phosphates attached to them, similar to ATP which has three phosphate groups attached. When the bond between the phosphates is broken, the energy released is used to form the phosphodiester bond between the incoming nucleotide and the growing chain. In prokaryotes, three main types of polymerases are known: DNA pol I, DNA pol II, and DNA pol III. It is now known that DNA pol III is the enzyme required for DNA synthesis; DNA pol I and DNA pol II are primarily required for repair.

How does the replication machinery know where to begin? It turns out that there are specific nucleotide sequences called origins of replication where replication begins. In E. coli, which has a single origin of replication on its one chromosome (as do most prokaryotes), it is approximately 245 base pairs long and is rich in AT sequences. The origin of replication is recognized by certain proteins that bind to this site.

An enzyme called helicase unwinds the DNA by breaking the hydrogen bonds between the nitrogenous base pairs. ATP hydrolysis is required for this process. As the DNA opens up, Y-shaped structures called replication forks are formed. Two replication forks (a replication bubble) are formed at the origin of replication and these get extended bi- directionally as replication proceeds. Single-strand binding proteins coat the single strands of DNA near the replication fork to prevent the single-stranded DNA from winding back into a double helix. DNA polymerase is able to add nucleotides only in the 5′ to 3′ direction (a new DNA strand can be only extended in this direction). It also requires a free 3′-OH group to which it can add nucleotides by forming a phosphodiester bond between the 3′-OH end and the 5′ phosphate of the next nucleotide. This essentially means that it cannot add nucleotides if a free 3′-OH group is not available. Then how does it add the first nucleotide? The problem is solved with the help of a primer that provides the free 3′-OH end.

Another enzyme, RNA primase, synthesizes an RNA primer that is about five to ten nucleotides long and complementary to the DNA. Because this sequence primes the DNA synthesis, it is appropriately called the primer. DNA polymerase can now extend this RNA primer, adding nucleotides one by one that are complementary to the template strand.

Figure 1. DNA replication fork and associated enxymes. (Figure by Mariana Ruiz is in the public domain).

Because DNA polymerase can only extend in the 5′ to 3′ direction, and because the DNA double helix is antiparallel, there is a challenge or obstacle at the replication fork. Since the two template DNA strands have opposing orientations: only one new DNA strand can be synthesized continuously towards the replication fork. This continuously synthesized strand is known as the leading strand. The other strand is extended away from the replication fork, and is synthesized in numerous small fragments known as Okazaki fragments, each requiring a primer to start the synthesis. New primer segments are laid down in the direction of the replication fork, but each pointing away from it. Okazaki fragments are named after the Japanese scientist who first discovered them. The strand with the Okazaki fragments is known as the lagging strand. The leading strand can be extended from a single primer, whereas the lagging strand needs a new primer for each of the short Okazaki fragments. The overall direction of the lagging strand will be 3′ to 5′, and that of the leading strand 5′ to 3′ (Figure 2).

Figure 2. Replication bubble with Okazaki fragments (Image credit: John Urban, via Researchgate).

As synthesis proceeds, the RNA primers are replaced by DNA. In E. coli, the removal of RNA nucleotides and their replacement with DNA nucleotides are both performed by the enzyme DNA polymerase I. The nick that remains between the newly synthesized DNA (that replaced the RNA primer) and the previously synthesized DNA are sealed by the enzyme DNA ligase, which catalyzes the formation of the phosphodiester linkage between the 3′-OH end of one nucleotide and the 5′ phosphate end of the other fragment.

After the chromosome has been completely replicated, the two DNA copies will be moved into two different cells during cell division.

DNA Replication is Semi-Conservative

During DNA replication, each of the two strands that make up the double helix serves as a template from which a new strand is copied. The new strands will be complementary to the parental or “old” strands. When two daughter DNA copies are formed, they have the same sequence and are divided equally into the two daughter cells.

Figure 3. DNA Replication is semiconservative. Each old strand of DNA is used as a template for a new strand. Thus, each DNA molecule that results from DNA replication has one old strand and one new strand of DNA. (DNA replication by Madeleine Price Ball is used under a Creative Commons Zero license).

Eukaryotic DNA Replication

Eukaryotic genomes are large and complex, and replication of them required multiple origins of replication. In addition, DNA replication is much slower than in prokaryotes and involves several specialized DNA polymerases to complete the process. Because of how eukaryotic chromosomes are packaged inside the nucleus as chromatin, this structure must be loosened to allow access. However, DNA replication follows the same basic steps in eukaryotes as it does in prokaryotes: unwinding DNA, primer synthesis, strand elongation, and ligation.

One major difference between prokaryotes and eukaryotes is that eukaryotic chromosomes are linear. This means that their ends, which are called telomeres, cannot be fully replicated on the lagging strand. Telomeres are made of repeating sequences that protect genes closest to the end of the chromosome from being lost or damaged. The enzyme telomerase, active in germ and stem cells, extends these ends using an RNA template. Interestingly, telomere shortening is linked to aging; telomerase reactivation in mice has shown potential to reverse age-related decline. In contrast, telomerase is much more active in cancer cells than normal cells. Cancer is characterized by uncontrolled cell division of abnormal cells. The cells accumulate mutations, proliferate uncontrollably, and can migrate to different parts of the body through a process called metastasis. Scientists have observed that cancerous cells have considerably shortened telomeres. Only after the telomeres were shortened in the cancer cells does telomerase become active. If the action of telomerase in these cells can be inhibited by drugs during cancer therapy, then the cancerous cells could potentially be stopped from further division.

Here is an animated explanation of DNA replication.