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DNA replication requires three processes: the initiation of replication, the elongation of the new DNA during actual copying, and the termination of the process. Each of these consists of reactions between enzymes and the DNA macromolecule. And each has to be performed precisely to produce two identical strands of DNA.
The importance of DNA replication has led to it being extensively studied in cells, replicated as chemical reactions in experiments, and modeled at a molecular level. The basic processes for DNA replication are similar in Bacteria, Archaea, and Eukarya, although there are significant differences in the details of the processes. The text below describes the processes of initiation, elongation and termination generally, and then discusses some key differences among the three domains of life as well as for DNA in organelles (like plastids and mitochondria), plasmids, and viruses.
Let’s start, however, with two beautiful animations of the elongation process (in a eukaryotic cell) based on a very detailed molecular simulation. The first video shows the overall process, and the second video shows how the new strand of DNA is built. Each process illustrated in the videos is caused by spontaneously chemical reactions when the right molecules are present in the right places in the cells. Evolution over billions of years has produced these impressive molecular machines.
The initiation of DNA replication occurs at a specific nucleotide sequence called the origin of replication. There is at least one origin of replication on each chromosome so that it can be copied. The origin of replication provides a bonding site for the specific proteins that begin the replication process. These proteins are important for making single-stranded regions of DNA accessible for replication. Chromosomal DNA is typically wrapped around histones (in eukaryotes and archaea) or histone-like proteins (in bacteria), and is supercoiled, or extensively wrapped and twisted on itself. This packaging makes the information in the DNA molecule inaccessible. However, enzymes change the shape and supercoiling of the chromosome. An enzyme called helicase then separates the two DNA strands by breaking the hydrogen bonds between the nitrogenous base pairs. As the DNA opens up, Y-shaped structures called replication forks are formed. Two replication forks are formed at the origin of replication, one facing each direction along the chromosome. This allows the chromosome to be copied in both directions. The DNA near each replication fork is coated with single-stranded binding proteins to prevent the single-stranded DNA from rewinding into a double helix.
Once single-stranded DNA is accessible at the origin of replication, it bonds to a special RNA sequence, called a primer, which provides the right chemical environment for making the complementary DNA strands. Then DNA replication, specifically the process of elongation, can begin.
If chromosomes have more than one origin of replication, which is common in eukaryotes and archaea, initiation can occur at multiple sites, producing multiple pairs of replication forks.
The complementary DNA strand is added to the original, or template, DNA strand by an enzyme called DNA polymerase. DNA polymerase is held in place by an enzyme that acts as a sliding clamp; it keeps the DNA polymerase at the proper distance from the fork and facilitates smooth passage of the single DNA strand to the polymerase. As the DNA slides through, the DNA polymerase adds the appropriate nucleotide to complement each one on the template strand. The addition of these nucleotides requires energy. This energy is obtained by breaking bonds in the phosphate groups attached to each nucleotide (a triphosphate nucleotide). When the bond between the phosphates is broken, diphosphate is released along with energy that allows the formation of a covalent bond between the incoming nucleotide and the growing DNA strand.
DNA polymerase can only extend the complementary strand of DNA in one specific direction defined by the geometry of the pentose sugar and phosphate groups bound to the nitrogenous base. This asymmetry poses a problem at the replication fork. The DNA double helix is antiparallel; that is, one strand is oriented in one direction and the other is oriented in the opposite direction (see Structure and Function of DNA). During replication, one complementary strand can be synthesized continuously as it leaves the replication fork because polymerase can add nucleotides in this direction. This continuously synthesized strand is known as the leading strand. The other strand has to grow in the opposite direction. This requires that the polymerase copies part of the original DNA strand and then jump back toward the replication fork to begin adding bases to a new primer. It adds bases until it bumps into the previously synthesized strand, and then it moves back again (ref to video, figures). These steps produce small DNA sequence fragments, each separated by RNA primer. This segment of new DNA is called the lagging strand. The RNA primers are replaced with DNA as one of the final steps in the elongation process.
Once the complete chromosome has been replicated, termination of DNA replication must occur. Although much is known about initiation of replication, less is known about the termination process, and it is different for bacteria and eukaryotes. For organisms with circular chromosomes (most Bacteria and Archaea), elongation process stops when two replication forks encounter each other. If there is only one origin of replication on the circular chromosome, the two forks will encounter each other once full chromosome has been copied. However, the new chromosomes are interlocked, so they have to be broken to separate from each other and the ends have to be joined to reform the circle. For organisms with linear chromosomes, termination happens when either two replication forks meet or the end of the chromosome is reached. When the replication fork reaches the end of a linear chromosome, there is no place to add a primer for lagging strand to copy the end of the chromosome. Thus, the ends of the template strand remain unpaired and, over time, chromosomes may get progressively shorter as cells continue to divide.
Variations in DNA Replication
DNA replication follows a similar process in all three domains of life – Bacteria, Archaea, and Eukarya – in terms of having a genetic marker for the origin of replication, similar mechanisms for elongation, and the need for termination. However, the actual enzymes that perform the replication show some variations related to their evolutionary history. The genes that code for the enzymes composing the sliding clamp are very similar in all three domains (Bell, 2017), which suggests that the basic process for duplicating DNA evolved in the last common ancestor of all life. In contrast, most of the other genes involved in DNA replication are similar in Archaea and Eukarya, but different in Bacteria (Bell, 2017). The differences between Bacteria and Archaea/Eukarya are consistent with other evolutionary relationships between the groups that suggest Archaea and Eukarya are more closely related to each other than they are to Bacteria.
Plasmids and viruses also require DNA replication to reproduce. They lack the genes to make their own replication enzymes, so they rely on their host cells for DNA replication.
DNA replication has been well studied in bacteria primarily because of the small size of the genome and the mutants that are available. E. coli has 4.6 million base pairs (Mbp) in a single circular chromosome and all of it is 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 with few errors.
Most archaea also have a single circular chromosome, but their processes of DNA replication are less well studied. The enzymes for DNA replication in Archaea is similar to that in Eukarya, but they are in general simpler (Bell, 2017). Most archaeal chromosomes have more than one origin of replication, with four being the maximum documented to date. In general, there is substantially more research to be done to understand how archaea function as cells.
Eukarya and Organelles
Eukaryotic genomes are much more complex and larger than bacterial and archaeal genomes and are typically composed of multiple linear chromosomes. The human genome, for example, requires the insertion of 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 bacterial replication.
Eukaryotic chromosomes are linear, which means that their ends sometime contain unpaired bases. Thus, over time, they may get progressively shorter as cells continue to divide. To help prevent important information from being lost, the ends of the linear chromosomes consist of noncoding repetitive sequences called telomeres. The telomeres protect coding sequences from being lost as cells continue to divide. In humans, telomeres consist of 100 to 1000 repetitions of a six base-pair sequence, TTAGGG. There is a specific enzyme that attaches to this sequence at the end of the template DNA for the lagging strand of DNA and extends it with an RNA template. Once the lagging strand template is long enough for the DNA polymerase to shift to a new primer, the polymerase can finish adding the complementary nucleotides to the end of the chromosome.
In humans, telomerase is typically active in germ cells and adult stem cells; it is not active in adult somatic cells and may be associated with the aging of these cells. Eukaryotic microbes including fungi and protozoans also produce telomerase to maintain chromosomal integrity. For her discovery of telomerase and its action, Elizabeth Blackburn (1948–) received the Nobel Prize for Medicine or Physiology in 2009.
Eukaryotes also host DNA in their organelles. This orDNA was inherited from bacteria that merged ancient ancestors to modern eukaryotes through a process called endosymbiosis. Over hundreds of millions of years, some of the bacterial genes were transferred to the host cells, some were lost, and all evolved in response to being permanently encased within another cell. One of the key processes transferred from the ancestors to the organelles to the host cells was DNA replication. The replication of orDNA is controlled by genes in the nucleus, even though orDNA replication is not necessarily coordinated with replication of nuclear DNA. The replication process is also variable among organisms, in part due to the different evolutionary paths of different eukaryotic lineages after the endosymbiosis events. Overall, orDNA replication is less well understood by scientists than nuclear DNA replication (see https://www.frontiersin.org/articles/10.3389/fpls.2015.00883/full), even though it is critical for the function of most eukaryotes.
Plasmids and Viruses
Plasmids and DNA viruses depend on host cell DNA replication processes to replicate their DNA. Plasmids are small circular molecules of double-stranded DNA that are separate from the main chromosome(s) of their host cells. They usually contain one or two non-essential genes, like antibiotic resistance, and none of the genes necessary for DNA replication. Thus, they depend on enzymes coded in genes in the host cell chromosomes to replicate their DNA. Plasmids have their own origin of replication to initiate DNA replication, but they will only be replicated if the host cell’s enzymes recognize their origin of replication. Because origins of replication have different coding sequences in Bacteria, Archaea, and Eukarya, plasmids are usually specific to one domain of life or even a smaller subset of organisms. In addition, sometimes the replication process proceeds differently than for chromosomal DNA, initiating with a nick in one of the DNA strands and elongating the two strands separately.
Viruses also depend on their host cell’s enzymes for replication. Some viruses contain DNA whereas others have RNA. DNA viruses do not include all the genes for DNA replication enzymes. Thus, they rely on their host cell’s cellular machinery to produce more viral particles and use multiple methods for tricking host cells into replicating their DNA. RNA viruses can replicate their genetic code more easily since RNA is a reactive molecule and does not require the processes of initiation, elongation, and termination essential for DNA replication. For more information on viruses, see The Viral Life Cycle.
More Information and References
Bell, S.D., 2017. Chapter 5 - Initiation of DNA Replication in the Archaea, in H. Masai, M. Foiani (eds.), DNA Replication, Advances in Experimental Medicine and Biology 1042, https://doi.org/10.1007/978-981-10-6955-0_5