Compare the number of replication forks in prokaryotic and eukaryotic DNA during replication
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Unlike prokaryotic chromosomes, eukaryotic chromosomes are linear. As you’ve learned, the enzyme DNA pol can add nucleotides only in the 5' to 3' direction. In the leading strand, synthesis continues until the end of the chromosome is reached. 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 way to replace the primer on the 5’ end of the lagging strand. The DNA at the ends of the chromosome thus remains unpaired, and over time these ends, called telomeres, may get progressively shorter as cells continue to divide. Show Telomeres comprise repetitive sequences that code for no particular gene. In humans, a six-base-pair sequence, TTAGGG, is repeated 100 to 1000 times in the telomere regions. In a way, these telomeres protect the genes from getting deleted as cells continue to divide. The telomeres are added to the ends of chromosomes by a separate enzyme, telomerase (Figure), whose discovery helped in the understanding of how these repetitive chromosome ends are maintained. The telomerase enzyme contains a catalytic part and a built-in RNA template. It attaches to the end of the chromosome, and DNA nucleotides complementary to the RNA template are added on the 3' end of the DNA strand. Once the 3' end of the lagging strand template is sufficiently elongated, DNA polymerase can add the nucleotides complementary to the ends of the chromosomes. Thus, the ends of the chromosomes are replicated. Telomerase is typically active in germ cells and adult stem cells. It is not active in adult somatic cells. For their discovery of telomerase and its action, Elizabeth Blackburn, Carol W. Greider, and Jack W. Szostak (Figure) received the Nobel Prize for Medicine and Physiology in 2009. Telomerase and AgingCells that undergo cell division continue to have their telomeres shortened because most somatic cells do not make telomerase. This essentially means that telomere shortening is associated with aging. With the advent of modern medicine, preventative health care, and healthier lifestyles, the human life span has increased, and there is an increasing demand for people to look younger and have a better quality of life as they grow older. In 2010, scientists found that telomerase can reverse some age-related conditions in mice. This may have potential in regenerative medicine.Jaskelioff et al., “Telomerase reactivation reverses tissue degeneration in aged telomerase-deficient mice,” Nature 469 (2011): 102-7. Telomerase-deficient mice were used in these studies; these mice have tissue atrophy, stem cell depletion, organ system failure, and impaired tissue injury responses. Telomerase reactivation in these mice caused extension of telomeres, reduced DNA damage, reversed neurodegeneration, and improved the function of the testes, spleen, and intestines. Thus, telomere reactivation may have potential for treating age-related diseases in humans. 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 and that telomerase is active in these cells. Interestingly, only after the telomeres were shortened in the cancer cells did the 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. The prokaryotic chromosome is a circular molecule with a less extensive coiling structure than eukaryotic chromosomes. The eukaryotic chromosome is linear and highly coiled around proteins. While there are many similarities in the DNA replication process, these structural differences necessitate some differences in the DNA replication process in these two life forms. DNA replication in prokaryotes has been extensively studied, so we will learn the basic process of prokaryotic DNA replication, then focus on the differences between prokaryotes and eukaryotes. How does the replication machinery know where to start? It turns out that there are specific nucleotide sequences called origins of replication where replication begins. E. coli has a single origin of replication on its one chromosome, as do most prokaryotes (Figure 1). The origin of replication is approximately 245 base pairs long and is rich in AT sequences. This sequence of base pairs 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 because it requires energy. As the DNA opens up, Y-shaped structures called replication forks are formed (Figure 1). Two replication forks are formed at the origin of replication and these get extended bi-directionally as replication proceeds. Single-strand binding proteins (Figure 2) coat the single strands of DNA near the replication fork to prevent the single-stranded DNA from winding back into a double helix. The next important enzyme is DNA polymerase III, also known as DNA pol III, which adds nucleotides one by one to the growing DNA chain (Figure 2). The addition of nucleotides requires energy; this energy is obtained from the nucleotides that have three phosphates attached to them. ATP structurally is an adenine nucleotide which has three phosphate groups attached; breaking off the third phosphate releases energy. In addition to ATP, there are also TTP, CTP, and GTP. Each of these is made up of the corresponding nucleotide with three phosphates 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 existing chain. In prokaryotes, three main types of 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 is used later in the process and DNA pol II is used primarily required for repair (this is another irritating example of naming that was done based on the order of discovery rather than an order that makes sense). 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 requires a free 3′-OH group (located on the sugar) to which it can add the next nucleotide 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. RNA primase does not require a free 3′-OH group. 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 2). The replication fork moves at the rate of 1000 nucleotides per second. DNA polymerase can only extend in the 5′ to 3′ direction, which poses a slight problem at the replication fork. As we know, the DNA double helix is anti-parallel; that is, one strand is in the 5′ to 3′ direction and the other is oriented in the 3′ to 5′ direction. One strand, which is complementary to the 3′ to 5′ parental DNA strand, is synthesized continuously towards the replication fork because the 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, is extended away from the replication fork, in small fragments known as Okazaki fragments, each requiring a primer to start the synthesis. 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 by one primer alone, 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 polymerase 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. 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. As synthesis proceeds, the RNA primers are replaced by DNA pol I, which breaks down the RNA and fills the gaps with 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 that catalyzes the formation of phosphodiester linkage between the 3′-OH end of one nucleotide and the 5′ phosphate end of the other fragment. (Lisa’s note: I think this process is almost impossible to visualize from reading text. I strongly recommend that you watch a couple of animations / videos like the one available here. There are additional links in Blackboard) Once the chromosome has been completely replicated, the two DNA copies move into two different cells during cell division. The process of DNA replication can be summarized as follows:
Table 1: The enzymes involved in prokaryotic DNA replication and the functions of each.
DNA replication has been extremely well-studied in prokaryotes, primarily because of the small size of the genome and large number of variants 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 origin of replication and proceeding around the chromosome in both directions. This means that approximately 1000 nucleotides are added per second. The process is much more rapid than in eukaryotes. ReferencesUnless otherwise noted, images on this page are licensed under CC-BY 4.0 by OpenStax. OpenStax, Concepts of Biology. OpenStax CNX. May 18, 2016 http://cnx.org/contents/:2ousESf0@5/DNA-Replication How does the number of replication forks differ between prokaryotic and eukaryotic DNA replication?Several replication forks are formed in multiple replication bubbles. The prokaryotic chromosome has one replicon. The eukaryotic chromosome has over 50,000 replicons.
Does eukaryotic DNA has more replication forks than prokaryotic DNA?Eukaryotic DNA replication requires multiple replication forks, while prokaryotic replication uses a single origin to rapidly replicate the entire genome. DNA replication always occurs in the nucleus. Eukaryotic DNA replication involves more polymerases than prokaryotic replication.
How many replication forks are used in eukaryotic DNA replication?Key Points. During initiation, proteins bind to the origin of replication while helicase unwinds the DNA helix and two replication forks are formed at the origin of replication. During elongation, a primer sequence is added with complementary RNA nucleotides, which are then replaced by DNA nucleotides.
How many replication forks are in prokaryotes?Two replication forks are formed at the origin of replication and these get extended bi- directionally as replication proceeds.
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