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Can a strand not be synthesised in 5' -> 3' direction?

Can a strand not be synthesised in 5' -> 3' direction?


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I've been solving some biology questions, and according to one of them ( I have the responses too) the following phrase is false:
"Both strands are always synthesised in the 5' to 3' direction."
How can this be? From what I know DNA is always read from 3' to 5', and synthesised from 5' to 3'.
When is DNA not synthesized from 5' to 3'?


It sounds like your guess was right and the exception is the lagging strand. The statement is phrased poorly, but it seems like they were going for the lagging strand. Either they meant for you to think that
-the lagging strand is growing 3' to 5'
or, more likely,
-the polymerase is only synthesizing on one (leading) strand at a time

While technically the book was correct, it was a trick question and phrased unfairly.

You are completely right that DNA is synthesized from 5' to 3'. I don't know of any exceptions to this, but hesitate to say anything overly definitive because there are so many rare exceptions in science. I can say with some confidence that DNA polymerase cannot ever synthesize in the 3' -> 5' direction. If you haven't already, I highly recommend checking out the Khan Academy article, Molecular mechanism of DNA replication! Nature Education also has a pretty good article on the topic, Major Molecular Events of DNA Replication.


What is the leading and lagging strand in DNA replication?

DNA strands are antiparallel. DNA polymerase can work continuously toward the replication fork only on one strand (the leading strand) while on the other strand (the lagging strand) it must proceed away from the replication fork. The lagging strand does so discontinuously in segments called Okazaki fragments.

what is leading strand in DNA replication? When replication begins, the two parent DNA strands are separated. One of these is called the leading strand, and it is replicated continuously in the 3' to 5' direction. The other strand is the lagging strand, and it is replicated discontinuously in short sections.

Just so, what is the difference between the leading strand and the lagging strand in DNA replication quizlet?

The leading strand is correctly oriented for DNA polymerase III to add nucleotides in the 5' - 3' direction towards the replication fork in a continuous strand whereas the lagging strand runs the opposite direction (3' - 5') and must be replicated backwards, away from the replication fork.

Why does DNA replication occur in the 5 to 3 direction?

These fragments are processed by the replication machinery to produce a continuous strand of DNA and hence a complete daughter DNA helix. DNA replication goes in the 5' to 3' direction because DNA polymerase acts on the 3'-OH of the existing strand for adding free nucleotides.


Telomerase and Aging

Cells 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. [1] 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.

In Summary: Telomeres

The ends of the chromosomes pose a problem during DNA replication as polymerase is unable to extend them without a primer. Telomerase, an enzyme with a built-in RNA template, extends the ends by copying the RNA template and extending one end of the chromosome. DNA polymerase can then extend the DNA using the primer. In this way, the ends of the chromosomes are protected. This is important as evidence indicates telomere length may play a role in regulating cell division and the process of aging.


Both DNA and RNA Chains Are Produced by Copying of Template DNA Strands

The regular pairing of bases in the double-helical DNA structure suggested to Watson and Crick a mechanism of DNA synthesis. Their proposal that new strands of DNA are synthesized by copying of parental strands of DNA has proved to be correct.

The DNA strand that is copied to form a new strand is called a template. The information in the template is preserved: although the first copy has a complementary sequence, not an identical one, a copy of the copy produces the original (template) sequence again. In the replication of a double-stranded, or duplex, DNA molecule, both original (parental) DNA strands are copied. When copying is finished, the two new duplexes, each consisting of one of the two original strands plus its copy, separate from each other. In some viruses, single-stranded RNA molecules function as templates for synthesis of complementary RNA or DNA chains (Chapter 7). However, the vast majority of RNA and DNA in cells is synthesized from preexisting duplex DNA.


Biology 171

By the end of this section, you will be able to do the following:

  • Explain the process of DNA replication in prokaryotes
  • Discuss the role of different enzymes and proteins in supporting this process

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. 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 site along the chromosome and proceeding around the circle in both directions. This means that approximately 1000 nucleotides are added per second. Thus, the process is quite rapid and occurs without many mistakes.

DNA replication employs a large number of structural 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 is complementary to the template strand. The addition of nucleotides requires energy this energy is obtained from the nucleoside triphosphates ATP, GTP, TTP and CTP. Like ATP, the other NTPs (nucleoside triphosphates) are high-energy molecules that can serve both as the source of DNA nucleotides and the source of energy to drive the polymerization. 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 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 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 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 polymerase can now extend this RNA primer, adding nucleotides one-by-one that are complementary to the template strand ((Figure)).


Question: You isolate a cell strain in which the joining of Okazaki fragments is impaired and suspect that a mutation has occurred in an enzyme found at the replication fork. Which enzyme is most likely to be mutated?

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 polymerase can only extend in the 5′ to 3′ direction, and because the DNA double helix is antiparallel, 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. (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′. 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. 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 can be summarized as follows:

  1. DNA unwinds at the origin of replication.
  2. Helicase opens up the DNA-forming replication forks these are extended bidirectionally.
  3. Single-strand binding proteins coat the DNA around the replication fork to prevent rewinding of the DNA.
  4. Topoisomerase binds at the region ahead of the replication fork to prevent supercoiling.
  5. Primase synthesizes RNA primers complementary to the DNA strand.
  6. DNA polymerase III starts adding nucleotides to the 3′-OH end of the primer.
  7. Elongation of both the lagging and the leading strand continues.
  8. RNA primers are removed by exonuclease activity.
  9. Gaps are filled by DNA pol I by adding dNTPs.
  10. The gap between the two DNA fragments is sealed by DNA ligase, which helps in the formation of phosphodiester bonds.

(Figure) summarizes the enzymes involved in prokaryotic DNA replication and the functions of each.

Prokaryotic DNA Replication: Enzymes and Their Function
Enzyme/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 hydrogen bonds between the nitrogenous bases
Ligase Seals the gaps between the Okazaki fragments to create one continuous DNA strand
Primase Synthesizes RNA primers needed to start replication
Sliding Clamp Helps to hold the DNA polymerase 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.

Watch DNA replication (video) to view the full process.

Section Summary

Replication in prokaryotes starts from a sequence found on the chromosome called the origin of replication—the point at which the DNA opens up. Helicase opens up the DNA double helix, resulting in the formation of the replication fork. Single-strand binding proteins bind to the single-stranded DNA near the replication fork to keep the fork open. Primase synthesizes an RNA primer to initiate synthesis by DNA polymerase, which can add nucleotides only to the 3′ end of a previously synthesized primer strand. Both new DNA strands grow according to their respective 5′-3′ directions. One strand is synthesized continuously in the direction of the replication fork this is called the leading strand. The other strand is synthesized in a direction away from the replication fork, in short stretches of DNA known as Okazaki fragments. This strand is known as the lagging strand. Once replication is completed, the RNA primers are replaced by DNA nucleotides and the DNA is sealed with DNA ligase, which creates phosphodiester bonds between the 3′-OH of one end and the 5′ phosphate of the other strand.

Art Connections

(Figure) You isolate a cell strain in which the joining of Okazaki fragments is impaired and suspect that a mutation has occurred in an enzyme found at the replication fork. Which enzyme is most likely to be mutated?

(Figure) DNA ligase, as this enzyme joins together Okazaki fragments.

Free Response

DNA replication is bidirectional and discontinuous explain your understanding of those concepts.

At an origin of replication, two replication forks are formed that are extended in two directions. On the lagging strand, Okazaki fragments are formed in a discontinuous manner.

What are Okazaki fragments and how they are formed?

Short DNA fragments are formed on the lagging strand synthesized in a direction away from the replication fork. These are synthesized by DNA pol.

If the rate of replication in a particular prokaryote is 900 nucleotides per second, how long would it take 1.2 million base pair genomes to make two copies?

1333 seconds or 22.2 minutes.

Explain the events taking place at the replication fork. If the gene for helicase is mutated, what part of replication will be affected?

At the replication fork, the events taking place are helicase action, binding of single-strand binding proteins, primer synthesis, and synthesis of new strands. If there is a mutated helicase gene, the replication fork will not be extended.

What is the role of a primer in DNA replication? What would happen if you forgot to add a primer in a tube containing the reaction mix for a DNA sequencing reaction?

Primer provides a 3′-OH group for DNA pol to start adding nucleotides. There would be no reaction in the tube without a primer, and no bands would be visible on the electrophoresis.

Quinolone antibiotics treat bacterial infections by blocking the activity of topoisomerase. Why does this treatment work? Explain what occurs at the molecular level.

Bacteria treated with quinolones will no longer be able to replicate their DNA. Topoisomerase relieves the excess DNA supercoiling that occurs ahead of the replication fork as DNA is unwound for replication. If topoisomerase is inhibited, DNA helicase will only be able to unwind the DNA for a short stretch before the supercoiling becomes too overwound for replication to continue.

Glossary


DNA Replication

The human body produces billions of new cells every day. But before undergoing cell division, a cell must first copy the genetic information contained in the cell nucleus &ndash its DNA. In just 6-8 hours a cell is able to copy its entire genome. To accomplish this, DNA replication begins at multiple locations along each chromosome. As the two DNA strands are pulled apart, copying begins at the rate of about 50 nucleotides per second!

DNA encodes the information needed to build proteins, to regulate physiological processes and maintain homeostasis in our bodies. The basic chemical components of DNA are phosphate, deoxyribose (a sugar) and 4 nitrogenous bases: adenine (A), guanine (G), cytosine (C) and thymine (T).

DNA is a double stranded molecule with the two strands lying antiparallel to each other (they lie parallel to each other but run in opposite directions). The two strands of DNA wind together to form a double helix &ndash a structure that looks similar to a spiral staircase.

Figure (PageIndex<1>). the chemical structure of DNA (CC BY-NC-SA Madprime)

Figure (PageIndex<2>). semi-conservative replication (CC BY-NC-SA Madprime)

The strands of DNA are also complementary to each other due to specific base pairing between the two strands: adenine on one strand will always pair with thymine on the opposite strand and cytosine will always bind with guanine.

Figure (PageIndex<3>). DNA replication (CC BY-NC-SA LadyofHats)

During the process of DNA replication, the parental DNA unwinds and since nucleotides have exclusive partners, each DNA can act as its own template for replication. The two new DNA molecules each consist of one parental strand and one newly made strand. This is known as semiconservative DNA replication.

Figure (PageIndex<4>). DNA double helix (CC BY-NC-SA Forluvoft)

Important Players in DNA Replication

DNA Helicase: breaks the hydrogen bonds between DNA strands (unwinds DNA)

Topoisomerase: alleviates positive supercoiling (twisting of DNA) ahead of the replication fork

Single-stranded binding proteins (SSBPs): keeps the parental strands apart

Primase: synthesizes an RNA primer (gets synthesis of new strand started)

DNA Polymerase III: synthesizes a daughter strand of DNA

DNA Polymerase I: excises the RNA primers and fills in with DNA

DNA ligase: covalently links the Okazaki fragments together

Mechanism of DNA Replication

Getting replication started

The replication of DNA begins at special sites called origins of replication. Bacteria, which have relatively small circular chromosomes, contain just a single origin of replication. Eukaryotic chromosomes, which are long, linear strands of DNA contain many origins of replication along the DNA strands (see below). Proteins that recognize the origins of replication bind to these sequences and separate the two strands, opening up a replication &ldquobubble&rdquo. Replication then proceeds in both directions until the entire molecule is copied.

Figure (PageIndex<5>). replication bubbles (CC BY-NC-SA Boumphreyfr)

At the end of each replication bubble is a replication fork, a Y shaped region where the parental strands of DNA are unwound so that the replication machinery can copy the DNA. Helicases are enzymes that are responsible for untwisting the double helix at the replication forks, separating the two strands and making them available to serve as templates for DNA replication. The untwisting of the double helix by helicase causes additional twisting of the DNA molecule ahead of the replication fork. Topoisomerase is the enzyme that helps to relieve this strain by breaking, untwisting and rejoining the DNA strands. After the parental strands have been separated by helicase, single stranded binding proteins bind to the parental strands to keep them from reannealing to each other.

The unwound parental strands are now ready to be copied, but DNA Polymerase, the enzyme responsible for copying DNA, cannot initiate the synthesis of DNA, it can only add nucleotides onto an existing chain of nucleotides. To solve this problem, RNA primase puts down a short sequence of RNA complementary to the template DNA, called a primer that can be used to get DNA replication started. DNA polymerase is then able to synthesize new DNA by adding nucleotides to this preexisting chain.

Making a new DNA strand

As mentioned above, DNA polymerases catalyze the synthesis of new DNA by adding nucleotides to the RNA primer that was laid down by RNA Primase. It should be mentioned that in E.coli there are several different DNA polymerases, but two appear to play major roles: DNA Pol III and DNA Pol I. In eukaryotes, the situation is more complex, with at least 11 different DNA polymerases discovered so far, but the general principles we will discuss in this tutorial are applicable to both systems.

In E.coli, DNA Pol III adds a DNA nucleotide to the 3' end of the RNA primer and then continues adding DNA nucleotides complementary to the template strand. As was mentioned previously, the two ends of a DNA strand are different, in that they are antiparallel to each other. This directionality is important for the synthesis of DNA, because DNA polymerase can only add nucleotides to the 3&rsquo end of a growing DNA strand (not the 5&rsquo end). Thus, a new DNA strand can only be made in the 5&rsquo to 3&rsquo direction.

Leading and Lagging Strands

If we look at the replication fork, we see that this 5&rsquo to 3&rsquo directionality poses a problem. Along one of the template strands, DNA Pol III can synthesize a complementary strand continuously by elongating the new DNA in the 5&rsquo to 3&rsquo direction. This is called the leading strand and it requires only one RNA primer to be made to start replication. However, to elongate the other strand in the 5&rsquo to 3&rsquo direction, DNA Pol III must elongate the new DNA strand in a direction opposite to the movement of the replication fork. This strand is known as the lagging strand. As the replication form proceeds and exposes a new section of the lagging strand template, RNA Primase must put down a new primer for DNA Pol III to elongate. In this way, the lagging strand is completed in a discontinuous manner. The synthesized DNA fragments on the lagging strand are called Okazaki fragments. Another DNA polymerase, DNA Pol I is responsible for removing the RNA primers and replacing them with DNA. DNA ligase then seals the gaps between the Okazaki fragments to complete the newly synthesized lagging strand.

Figure (PageIndex<8>). (CC BY-NC-SA)

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DNA Replication Tutorial by Dr. Katherine Harris is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.


Basics of DNA Replication

Figure 1. The three suggested models of DNA replication. Grey indicates the original DNA strands, and blue indicates newly synthesized DNA.

The elucidation of the structure of the double helix provided a hint as to how DNA divides and makes copies of itself. This model 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. What was not clear was how the replication took place. There were three models suggested: conservative, semi-conservative, and dispersive (see Figure 1).

In conservative replication, the parental DNA remains together, and the newly formed daughter strands are together. The semi-conservative method suggests that each of the two parental DNA strands act as a template for new DNA to be synthesized after replication, each double-stranded DNA includes one parental or &ldquoold&rdquo strand and one &ldquonew&rdquo strand. In the dispersive model, both copies of DNA have double-stranded segments of parental DNA and newly synthesized DNA interspersed.

Meselson and Stahl were interested in understanding how DNA replicates. They grew E. coli for several generations in a medium containing a &ldquoheavy&rdquo isotope of nitrogen ( 15 N) that gets incorporated into nitrogenous bases, and eventually into the DNA (Figure 2).

Figure 2. Meselson and Stahl experimented with E. coli grown first in heavy nitrogen ( 15 N) then in 14 N. DNA grown in 15 N (red band) is heavier than DNA grown in 14 N (orange band), and sediments to a lower level in cesium chloride solution in an ultracentrifuge. When DNA grown in 15 N is switched to media containing 14 N, after one round of cell division the DNA sediments halfway between the 15 N and 14 N levels, indicating that it now contains fifty percent 14 N. In subsequent cell divisions, an increasing amount of DNA contains 14 N only. This data supports the semi-conservative replication model. (credit: modification of work by Mariana Ruiz Villareal)

The E. coli culture was then shifted into medium containing 14 N and allowed to grow for one generation. The cells were harvested and the DNA was isolated. The DNA was centrifuged at high speeds in an ultracentrifuge. Some cells were allowed to grow for one more life cycle in 14 N and spun again. During the density gradient centrifugation, the DNA is loaded into a gradient (typically a salt such as cesium chloride or sucrose) and spun at high speeds of 50,000 to 60,000 rpm. Under these circumstances, the DNA will form a band according to its density in the gradient. DNA grown in 15 N will band at a higher density position than that grown in 14 N. Meselson and Stahl noted that after one generation of growth in 14 N after they had been shifted from 15 N, the single band observed was intermediate in position in between DNA of cells grown exclusively in 15 N and 14 N. This suggested either a semi-conservative or dispersive mode of replication. The DNA harvested from cells grown for two generations in 14 N formed two bands: one DNA band was at the intermediate position between 15 N and 14 N, and the other corresponded to the band of 14 N DNA. These results could only be explained if DNA replicates in a semi-conservative manner. Therefore, the other two modes were ruled out.

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

The model for DNA replication 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. In conservative replication, the parental DNA is conserved, and the daughter DNA is newly synthesized. The semi-conservative method suggests that each of the two parental DNA strands acts as template for new DNA to be synthesized after replication, each double-stranded DNA includes one parental or &ldquoold&rdquo strand and one &ldquonew&rdquo strand. The dispersive mode suggested that the two copies of the DNA would have segments of parental DNA and newly synthesized DNA. Experimental evidence showed DNA replication is semi-conservative.


Post-Replication DNA Mismatch, Damage and Repair

Although DNA polymerase has a very low error rate, in part due to its proofreading activity, some mismatched base pairs do escape correction during replication. In addition, DNA can be damaged in other ways that alter the DNA sequence. These DNA mismatches and damages, if not corrected or repaired, will result in a mutation(a permanent change in the DNA) in the next round of replication, which may be detrimental to the cell. For example, the genetic disorder sickle cell anemia results from a single nucleotide change (adenine changed to thymine) in the gene encoding one of the two globin proteins that comprise hemoglobin. This nucleotide change causes a change in the amino acid sequence and consequently a change in the shape of the mutant protein in the red blood cells. These alterations to the protein cause it to aggregate, forming large complexes that distort the shape of the red blood cells. These so-called "sickle cells" are more fragile than normal red blood cells and are more likely to break in the bloodstream, resulting in fewer red blood cells (anemia).

Fortunately, cells possess mechanisms for restoring mismatched base pairs and repairing DNA damage. The DNA mismatch repair system corrects 99% of the mismatched base pairs that were not removed by DNA polymerase during replication. Mismatch repair proteins recognize and bind to the mismatched base pair, nucleotides from the newly synthesized strand of DNA are excised, a repair DNA polymerase fills in the gap, and then DNA ligase rejoins this stretch of nucleotides to the rest of the DNA strand. It is important for the DNA mismatch repair system to distinguish between the template strand of DNA and the newly synthesized strand of DNA, however, the complete mechanism for this is not entirely understood.

Mutations can arise in DNA through damage that alters the chemical structure of the nucleotide. This can occur spontaneously, or in response to some environmental factor. The two most common types of damage that arise from spontaneous reactions are depurination (the loss of the base from the purine nucleotides adenine or guanine) and deamination(typically the conversion of cytosine to uracil). Depurination results in a nucleotide having no base consequently, DNA polymerase will skip this nucleotide when synthesizing the complementary strand during replication. This leads to a nucleotide loss in the next round of replication. In some cases the depurinated nucleotide is base-paired with a mismatched nucleotide on the other strand, resulting in a base pair change. Deamination results in a change of nucleotide sequence in the next round of replication, from a C-G base pair to an A-T base pair. Another common type of damage to DNA is thymine dimerformation. This is most often induced by exposure to ultraviolet light and consists of a covalent bond between the bases of adjacent thymines in one strand of DNA. This causes a block in DNA replication and can result in mutations. The rate of these types of damage is very high (e.g. depurination occurs 500 times per cell per day), however, there are numerous pathways for repairing the damaged DNA, and, as a result, the heritable mutation rate is not that high. Repair generally occurs in three steps: the damaged DNA is recognized and removed a repair DNA polymerase fills the gap and DNA ligase reseals the DNA strand. DNA repair is a complex pathway, involving many different proteins and activities that are not completely understood by biologists. However, the importance of these proteins is highlighted in several human disorders that have defective DNA repair proteins and that exhibit a much higher incidence of cancer due to the inability of the cells to efficiently repair damaged or mismatched DNA. Most cancers arise through mutations in key growth-regulating genes that accumulate throughout the lifetime of the individual. Individuals with defective DNA repair have a much greater chance of mutations arising in the cancer-causing genes.


Telomeres and Cellular Aging

Telomeres are important so their steady shrinking with each mitosis might impose a finite life span on cells. This, in fact, is the case. Normal (non-cancerous) cells do not grow indefinitely when placed in culture.

See Cancer Cells in Culture for a discussion of the differences between normal and cancerous cells grown in culture.

Cells removed from a newborn infant and placed in culture will go on to divide almost 100 times. Well before the end, however, their rate of mitosis declines (to less than once every two weeks). Were my cells to be cultured (I am 81 years old), they would manage only a couple of dozen mitoses before they ceased dividing and died out.

This phenomenon is called replicative senescence [More]. Could shrinkage of telomeres be a clock that determines the longevity of a cell lineage and thus is responsible for replicative senescence?

Evidence:

It turns out that these cells are able to maintain the length of their telomeres. They do so with the aid of an enzyme telomerase.


Replication in prokaryotes starts from a sequence found on the chromosome called the origin of replication—the point at which the DNA opens up. Helicase opens up the DNA double helix, resulting in the formation of the replication fork. Single-strand binding proteins bind to the single-stranded DNA near the replication fork to keep the fork open. Primase synthesizes an RNA primer to initiate synthesis by DNA polymerase, which can add nucleotides only to the 3' end of a previously synthesized primer strand. Both new DNA strands grow according to their respective 5'-3' directions. One strand is synthesized continuously in the direction of the replication fork this is called the leading strand. The other strand is synthesized in a direction away from the replication fork, in short stretches of DNA known as Okazaki fragments. This strand is known as the lagging strand. Once replication is completed, the RNA primers are replaced by DNA nucleotides and the DNA is sealed with DNA ligase, which creates phosphodiester bonds between the 3'-OH of one end and the 5' phosphate of the other strand.

Figure You isolate a cell strain in which the joining of Okazaki fragments is impaired and suspect that a mutation has occurred in an enzyme found at the replication fork. Which enzyme is most likely to be mutated?

Figure DNA ligase, as this enzyme joins together Okazaki fragments.



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