The autocatalytic function is how DNA reproduces itself, that is the process of replication by semiconservative mechanism with duplication of genetic information.
The model of DNA replication
When a cell divides, it is important that each daughter cell receives an identical copy of the DNA. This is accomplished by the process of DNA replication. The replication of DNA occurs during the S phase (synthesis) of the cell cycle, before the cell enters mitosis or meiosis[1].
The elucidation of the structure of the double helix provided a hint as to how DNA is copied. The Chargaff’s rule says that adenine nucleotides pair with thymine nucleotides and cytosine with guanine, otherwise the two strands of nucleotides are complementary to each other. For example, a strand of DNA with a nucleotide sequence of AGTCATGA will have a complementary strand with the sequence TCAGTACT (Figure 6.1a). Because of the complementarity of the two strands, having one strand means that it is possible to recreate the other strand.
This property suggests that the two strands of the double helix separate during replication and each strand serves as a template from which the new complementary strand is copied (Figure 6.1b). The new strand will be complementary to the parental or “old” strand. Each new double strand consists of one parental strand and one new daughter strand. This model is known as semi-conservative replication. When two DNA copies are formed, they have an identical sequence of nucleotide bases and are divided equally into two daughter cells.
Figure 6.1: a) The two strands of DNA are complementary, meaning the sequence of bases in one strand can be used to create the correct sequence of bases in the other strand; b) the semiconservative model of DNA replication is shown with gray indicating the original DNA strands and blue indicating newly synthesized DNA.
DNA replication in prokaryotes
DNA replication has been well studied in prokaryotes primarily because of the small size of the genome and because of the large variety of mutants that are available. Escherichia 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 site along the chromosome and proceeding around the circle in both directions. This means that approximately 1,000 nucleotides are added per second. Thus, the process is quite rapid and occurs without many mistakes.
DNA replication employs a large number of enzymes and structural proteins, 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 is complementary to the template strand. The addition of nucleotides requires energy; this energy is obtained from the nucleoside triphosphates (NTPs): ATP, GTP, TTP and CTP. Similarly to ATP, the other NTPs are high-energy molecules that can serve both as source of DNA nucleotides and source of energy to drive the polymerization. When the bond between the phosphates is “broken,” the energy released is used to form a 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 is an important accessory enzyme in DNA replication, and along with DNA–pol II, is 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), this origin of replication 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 are formed at the origin of replication and these get extended bidirectionally 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–pol has two important restrictions: it 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 segment that is about five to ten nucleotides long and complementary to the template DNA. Because this sequence primes the DNA synthesis, it is appropriately called the primer. DNA–pol can now extend this RNA primer, adding nucleotides one-by-one that are complementary to the template strand.
The replication fork moves at the rate of 1000 nucleotides per second. Topoisomerase prevents the over-winding of the DNA double helix ahead of the replication fork as the DNA is opening up; it does so by causing temporary nicks in the DNA helix and then resealing it. Because DNA–pol can only extend in the 5′ to 3′ direction, and because the DNA double helix is anti-parallel, there is a slight problem at the replication fork. The two template DNA strands have opposing orientations: one strand is in the 5′ to 3′ direction and the other is oriented in the 3′ to 5′ direction.
Only one new DNA strand, the one that is complementary to the 3′ to 5′ parental DNA strand, can be synthesized continuously towards the replication fork. This continuously synthesized strand is known as the leading strand. The other strand, complementary to the 5′ to 3′ parental DNA, is extended away from the replication fork, in 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. 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′.
A protein called the sliding clamp holds the DNA–pol in place as it continues to add nucleotides. The sliding clamp is a ring shaped protein that binds to the DNA and holds the polymerase in place. As synthesis proceeds, the RNA primers are replaced by DNA. The primers are removed by the exonuclease activity of DNA–pol I, which uses DNA behind the RNA as its own primer and fills-in the gaps left by removal of the RNA nucleotides by the addition of DNA nucleotides. The nicks that remain 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 phosphodiester linkages between the 3′-OH end of one nucleotide and the 5′ phosphate end of the other fragment.
Once the chromosome has been completely replicated, the two DNA copies move into two different cells during cell division.
The process of DNA replication (Figure 6.2) can be summarized as follows:
- DNA unwinds at the origin of replication.
- Helicase opens up the DNA-forming replication forks; these are extended bidirectionally.
- Single-strand binding proteins coat the DNA around the replication fork to prevent rewinding of the DNA.
- Topoisomerase binds at the region ahead of the replication fork to prevent supercoiling.
- Primase synthesizes RNA primers complementary to the DNA strand.
- DNA–pol III starts adding nucleotides to the 3′ (-OH) end of the primer.
- Elongation of both the lagging and the leading strand continues.
- RNA primers are removed by exonuclease activity.
- Gaps are filled by DNA–pol I.
- The gap between the two DNA fragments is sealed by DNA–ligase, which helps in the formation of phosphodiester bonds.
Figure 6.2: A replication fork is formed by the opening of the origin of replication and helicase separates the DNA strands. An RNA primer is synthesized and is elongated by the DNA–pol. On the leading strand DNA is synthesized continuously, whereas on the lagging strand DNA is synthesized in short stretches. The DNA fragments are joined by DNA–ligase (not shown).
| Enzyme or protein | Specific Function |
|---|---|
| DNA–pol I | Removes RNA primer and replaces it with newly synthesized DNA. |
| DNA–pol III | Main enzyme that adds nucleotides in the 5′-3′ direction. |
| Helicase | Opens the DNA helix by breaking the hydrogen bonds between nitrogenous bases. |
| Ligase | Seals the gaps between Okazaki fragments to create one continuous DNA strand. |
| Primase | Synthesizes RNA primers needed to start replication. |
| Sliding Clamp | Helps to hold DNA–pol in place when nucleotides are being added. |
| Topoisomerase | Helps relieve the strain on DNA when unwinding by causing breaks, and then resealing the DNA. |
| Single-strand binding proteins (SSB) | Binds to single-stranded DNA to prevent DNA from rewinding back. |
Table 6.1: A summary of the enzymes involved in prokaryotic DNA replication and the functions of each of them.
DNA replication in eukaryotes
Eukaryotic genomes are much more complex and larger in size than prokaryotic genomes. Eukaryotes also have a number of different linear chromosomes. The human genome has 3 billion base pairs per haploid set of chromosomes and 6 billion base pairs are replicated during the S phase of the cell cycle.
The essential steps of replication are the same as in prokaryotes: initiation, elongation, and termination. There are multiple origins of replication on each eukaryotic chromosome; humans can have up to 100,000 origins of replication across the genome. The rate of replication is approximately 100 nucleotides per second, much slower than prokaryotic replication. In yeast, which is a eukaryote, special sequences known as autonomously replicating sequences (ARS) are found on the chromosomes. These are equivalent to the origins of replication in E. coli.
The number of DNA–polymerases in eukaryotes is bigger than in prokaryotes: 14 are known, of which 5 are known to have major roles during replication and have been well studied. They are known as pol α, pol β, pol γ, pol δ, and pol ε.
Before replication can start, the DNA has to be made available as a template. Eukaryotic DNA is bound to basic proteins known as histones to form the structures called nucleosomes. Histones must be removed and then replaced during the replication process, which helps to account for the lower replication rate in eukaryotes. The chromatin (the complex between DNA and proteins) may undergo some chemical modifications, so that the DNA may be able to slide off the proteins or be accessible to the enzymes of the DNA replication machinery. At the origin of replication, a pre-replication complex is made with other initiator proteins. Helicase and other proteins are then recruited to start the replication process (Table 6.2).
A helicase using the energy from ATP hydrolysis opens up the DNA helix. Replication forks are formed at each replication origin as the DNA unwinds. The opening of the double helix causes over-winding, or supercoiling, in the DNA ahead of the replication fork. These are resolved with the action of topoisomerases. Primers are formed by the enzyme primase, and using the primer, DNA–pol can start synthesis.
Three major DNA–polymerases are then involved: α, δ and ε. DNA–pol α adds a short (20-30 nucleotides) DNA fragment to the RNA primer on both strands, and then hands off to a second polymerase. While the leading strand is continuously synthesized by the enzyme DNA–pol ε, the lagging strand is synthesized by DNA–pol δ.
A sliding clamp protein known as PCNA (proliferating cell nuclear antigen) holds the DNA–pol in place so that it does not slide off the DNA. As DNA–pol δ runs into the primer RNA on the lagging strand, it displaces it from the DNA template. The displaced primer RNA is then removed by the enzyme RNase H (flap endonuclease) and replaced with DNA nucleotides. The Okazaki fragments in the lagging strand are joined after the replacement of the RNA primers with DNA. The gaps that remain are sealed by DNA–ligase, which forms the phosphodiester bond.
| Property | Prokaryotes | Eukaryotes |
|---|---|---|
| Origin of replication | single | multiple |
| Rate of replication | 1000 nucleotides/s | 50 – 100 nucleotides/s |
| DNA–pol types | 5 | 14 |
| Telomerase | not present | present |
| RNA primer removal | DNA–pol I | RNase H |
| Strand elongation | DNA–pol III | DNA–pol α, DNA–pol δ, DNA–pol ε |
| Sliding clamp | Sliding clamp | PCNA |
Table 6.2: Differences between prokaryotic and eukaryotic replication.