DNA replication involves the separation of the two strands of the double helix, with each strand serving as a template from which the new complementary strand is copied. After replication, each double-stranded DNA includes one parental or “old” strand and one “new” strand. This is known as semiconservative replication. The resulting DNA molecules have the same sequence and are divided equally into the two daughter cells.
Replication in Prokaryotes
DNA replication uses a large number of proteins and enzymes. The enzyme helicase separates the two strands of DNA. As helicase moves along the DNA, it separates the two strands to form a Y-shaped structure known as the replication fork. Following this, the enzyme primase adds short stretches of RNA known as primers to initiate the synthesis of DNA by DNA polymerase, the enzyme responsible for DNA synthesis. In bacteria, three main types of DNA polymerases are known: DNA pol I, DNA pol II, and DNA pol III. DNA pol III is the enzyme required for DNA synthesis; DNA pol I and DNA pol II are primarily required for repair. DNA pol III adds deoxyribonucleotides, each complementary to a nucleotide on the template strand, one by one to the 3’-OH group of the growing DNA chain. DNA polymerase III can only extend in the 5’ to 3’ direction. The addition of these nucleotides requires energy. This energy is present in the bonds of three phosphate groups attached to each nucleotide, similar to how energy is stored in the phosphate bonds of adenosine triphosphate (ATP). When the bond between the phosphates is broken and diphosphate is released, the energy released allows for the formation of a covalent phosphodiester bond by dehydration synthesis.
The DNA double helix is antiparallel; that is, one strand is oriented in the 5’ to 3’ direction and the other is oriented in the 3’ to 5’ direction. During replication, one strand, which is complementary to the 3’ to 5’ parental DNA strand, is synthesized continuously toward the replication fork because polymerase can add nucleotides in this direction. This continuously synthesized strand is known as the leading strand. The other strand, complementary to the 5’ to 3’ parental DNA, grows away from the replication fork, so the polymerase must move back toward the replication fork to begin adding bases to a new primer, again in the direction away from the replication fork. It does so until it bumps into the previously synthesized strand, and then it moves back again. These steps produce small DNA sequence fragments known as Okazaki fragments, each separated by RNA primer. The strand with the Okazaki fragments is known as the lagging strand, and its synthesis is said to be discontinuous.
After the synthesis by DNA polymerase III, the primers are removed by the exonuclease activity of DNA polymerase I, and the gaps are filled in. 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 that catalyzes the formation of a covalent phosphodiester linkage between the 3’-OH end of one DNA fragment and the 5’ phosphate end of the other fragment, stabilizing the sugar-phosphate backbone of the DNA molecule.
Replication in Eukaryotes
Eukaryotic genomes are much more complex and larger than prokaryotic genomes and are typically composed of multiple linear chromosomes. The human genome, for example, has 3 billion base pairs per haploid set of chromosomes, and 6 billion base pairs are inserted during replication. There are multiple origins of replication on each eukaryotic chromosome; the human genome has 30,000 to 50,000 origins of replication. The rate of replication is approximately 100 nucleotides per second—10 times slower than prokaryotic replication.
The essential steps of replication in eukaryotes are the same as in prokaryotes. Before replication can start, the DNA has to be made available as a template. Eukaryotic DNA is highly supercoiled and packaged, which is facilitated by many proteins, including histones. Following initiation of replication, in a process similar to that found in prokaryotes, elongation is facilitated by eukaryotic DNA polymerases. The leading strand is continuously synthesized by the eukaryotic polymerase enzyme pol δ, while the lagging strand is synthesized by pol ε. The enzyme ribonuclease H (RNase H), instead of a DNA polymerase as in bacteria, removes the RNA primer, which is then replaced with DNA nucleotides. The gaps that remain are sealed by DNA ligase.
As in prokaryotes, the eukaryotic DNA polymerase can add nucleotides only in the 5’ to 3’ direction. In the leading strand, synthesis continues until it reaches either the end of the chromosome or another replication fork progressing in the opposite direction. On the lagging strand, DNA is synthesized in short stretches, each of which is initiated by a separate primer. When the replication fork reaches the end of the linear chromosome, there is no place to make a primer for the DNA fragment to be copied at the end of the chromosome. These ends thus remain unpaired, and, over time, they may get progressively shorter as cells continue to divide.
This text is adapted from Openstax, Microbiology, Chapter 11.2: DNA Replication.
