This is the 2nd installment of my series about DNA & RNA! If you haven't read my first post yet, I will put the link right here! Last time, we talked about the structure of DNA, the complementary base-pairing, and how we can denature as well as reanneal DNA under different conditions. For this post, we will focus on the process of DNA replication and how we can repair DNA!
A. THE BASIS OF CHROMATIN ORGANIZATION
In our body, DNA is divided up into 46 chromosomes. About 200 base pairs of DNA wound themselves around histone proteins to form a nucleosome. The five histone proteins that are found in eukaryotic cells are H2A, H2B, H3, H4, and H1. The former four are part of the histone core that DNA wraps around. H1, on the other hand, is the last histone that helps seal off DNA and leaves the nucleosome to add stability to the structure.
In order to prevent DNA from losing information after it replicates, this process actually doesn't occur towards the end of a chromosome. Instead, a telomere - a simple repeating unit of TTAGGG - is formed and an enzyme called telomerase can help replace some of the sequences that are lost after each round of DNA replication. Telomeres are important because they can: 1) Help replace some of the sequences after each round of DNA replication; and 2) Their high G-C (triple bond) can prevent DNA from unraveling! Found in the center of chromosomes are a region of DNA called centromeres. They are composed of heterochromatin and contain high levels of G-C content. This is useful because two sister chromatids will not be separated until anaphase.
B. DNA REPLICATION
During replication, DNA serves as parental strands or "templates" for the new daughter strands. This process is considered to be semiconservative because the daughter strands still have one parental strand retained. The process of replication started when DNA unwinds at the origins of replication (specific locations on the DNA strand) and create replication forks on both sides. Note: The double stranded DNA in eukaryotic chromosome have multiple origins of replication.
Helicase, an enzyme that is responsible for unwinding DNA, is the first enzyme that arrives at the origin of replication. One it binds to the DNA strand, it will generate two single strands of DNA. Single-stranded DNA-binding proteins will bind to these strands to prevent the free purines and pyrimidines from binding to one another. After the strands are separated by helicase, however, the DNA helix is pushed further towards the telomeres and it starts to wrap onto itself - further straining the entire structure and cause the DNA strands to supercoil. To alleviate the stress and prevent it from breaking, DNA topoisomerases nick one or both strands - allowing the pressure to decrease - and reseal the cut strands.
Replisome helps assist DNA polymerases to read the parental strand that is oriented in the same direction, or "DNA template", to synthesize the new daughter strand. These enzymes read the template in a 3' to 5' direction and "continuously" synthesize the complementary/daughter strand in a 5' to 3' direction. The strand that is synthesized continuously is called the leading strand. DNA polymerases accomplish this by adding nucleotides to the growing DNA chain at the 3' end and create a complementary DNA strand to the parental strand (see figure 2). However, how do DNA polymerases add these nucleotides at the replication fork? The answer is lies in an enzyme called "primase". Primase helps synthesize a short RNA primer that is complementary to the template strand. Once the RNA primer binds to the DNA strand at the 3' end, DNA polymerase can then synthesize a new strand that is complementary to the template or "parental" strand.
As discussed above, we already know that DNA polymerases help synthesize the new strand from a 5' to 3' direction. However, what happens to the other strand, or the "lagging strand", that is antiparallel to the template strand? How do DNA polymerases read the lagging strand? In this case, the DNA polymerase has to come off the leading strand and latch itself onto the lagging strand to make a complementary strand for it. This means that the new "non-continuously" synthesized strand or "lagging strand" is consisted of small fragments called Okazaki fragments.
C. ONCOGENES AND ANTIONCOGENES
Contrary to what some might think, DNA can easily be damaged by radiation or chemicals. If it's not fixed, the damaged/mutated DNA can be replicated and passed onto daughter cells. There are genes in our body that when mutated, can lead to cancer. Genes that have been mutated and cause cancer are called oncogenes. Fun fact: When they haven't be mutated yet, we call them proto-oncogenes! These genes can be suppressed by tumor suppressor genes or antioncogenes (ie: p53 or Rb). If these genes are mutated, then they can't suppress tumor activity, promote cell cycle, and cause tumor growth.
D. DNA REPAIR
When a DNA strand is being synthesized, it will need to be inspected by DNA polymerase so that it can be proofread. If the newly synthesized strand have incorrectly paired bases, then that portion of the structure will be unstable. When DNA polymerase detects this instability, the incorrect base is excised and a correct one will take its place. The 3 different types of DNA repair are: Mismatch repair, nucleotide excision repair, and base excision repair.
In cases where there are mismatched repair, cells in the G2 phase have the necessary machinery to help fix this. Genes called MSH2 and MLH1 help detect and remove those errors. If DNA is exposed to ultraviolet light, it can induce the formation of thymine dimers. This can cause interferences in DNA replication and distort the shape of DNA helices. This can create a "bulging shape" in the middle of a strand. In this case, we can use nucleotide excision repair to remove the thymine dimers. An enzyme called excision endonuclease can then come in to nick the phosphodiester linkage in the backbone and remove the defective base pair. Once the incorrect based pair is replaced with the correct one, DNA polymerase can synthesize a new DNA and DNA ligase can then seal the nick in the strand (as seen in figure 3).
In cases where cytosine deamination (cytosine loses an amino group because DNA absorbs thermal energy) occurs, cytosine is converted to uracil. Because uracil is normally found in RNA and not DNA, we can use base excision repair to fix this error. The defected base is removed by glycosylase enzyme, AP endonuclease removes the damaged sequence at the apurinic/apyrimidinic (AP) site, DNA polymerase can fill in the gap, and DNA ligase can then seal the strand.
The 2 different types of macromolecules that we will focus on this post are DNA (deoxyribonucleic acid) and RNA (ribonucleic acid). Organic macromolecules, like DNA and RNA, are biopolymers in living organisms that help store and transfer biological information from one generation to the next. They are great at storing nitrogen in their aromatic rings because both purines and pyrimidines contain nitrogen in their aromatic rings. They therefore have exceptional stability due to the fact that the delocalized electrons can travel throughout the entire compound using available molecular orbitals!
A. DNA Structure: DNA, a polydeoxyribonucleotide, is composed of multiple nucleotide monomers called deoxyribonucleotides. The helix in DNA makes a turn every 3.4 nm, has about 10 bases within that span, and have major and minor grooves between the interlocking strands.
1. Nucleosides and Nucleotides:
Nucleosides are composed of 5-carbon sugar (pentose) that is linked to a nitrogenous base. Nucleotides, on the other hand, are similar to nucleosides. The only difference is that they also have one or more phosphate groups attached to C-5 (5th carbon) as well.
a) Pentose (five-carbon sugar group): Nucleic acids can either have a ribose or a deoxyribose as their pentose. A ribose has an -OH group at C-2 while a deoxyribose has -H (as seen in figure 1). If the five-carbon sugar is a ribose, the nucleic acid is called RNA. If it has a deoxyribose, on the other hand, then the nucleic acid is called DNA.
b) Sugar Phosphate Group: If you take a look at the DNA structure on the right (see figure 3), you will see that the backbone of DNA is consisted of sugar and phosphate groups. The nucleotides (or bases) are joined by 3'-'5 phosphodiester linkages. This means that the phosphate group links the 3rd carbon on the pentose to the 5th carbon on the phosphate group.
These two DNA strands run antiparallel to one another and each strand of DNA has distinct 5' to 3' ends. The two nucleotide chains wound together in a spiral orientation to create a double helix with sugar-phosphate groups directed toward the outside and nitrogenous bases are directed toward the inside.
B. Purines and Pyrimidines: The 2 different families of nitrogen-containing (nitrogenous) bases that we see in nucleotides are purines and pyrimidines. Adenine (A) and guanine (G) are purines and both are found in DNA and RNA. Cytosine (C), thymine (T) and uracil (U) are pyrimidines and instead of having 2 rings in their structures, they only have 1. While cytosine is found in both DNA and RNA, uracil is only found in RNA and thymine is only found in DNA.
These bases are special because they help pair one DNA strand to another. In order for two strands to wound together and form a helix structure, we need these bases to be complimentary to each together, and that's how the term complementary base-pairing comes about. Adenine is always paired with a thymine using 2 hydrogen bonds and a guanine always pair with cytosine via 3 hydrogen bonds. Because these bases have specific pairing, the amount of adenine equals the amount of thymine and the amount of G equals the amount of C. This is known as Chargaff's rules.
C. DNA Denaturation, Reannealing, Hybridization: DNA structure is normally stabilized by hydrogen bonding between base pairs along the length of the molecule. High temperature, high pH, and denaturing agents like urea can disrupt hydrogen bonds between the base pairings and cause the DNA helix to denature. Denatured DNA, though, can be brought back together (reannealed) if we reverse the conditions (ie: cooler temperature, lower pH). Singled-stranded DNA can bind to another strand of DNA if the pairs of bases match each other. This process is called hybridization.
Enzymes are biological catalysts that play a HUGE role in the chemical reactions that happen around us! The reason why they are so important is because without them, the metabolic processes in our cells won't occur at rates that are fast enough to sustain life! According to Dr. Richard Wolfenden from the University of North Carolina at Chapel Hill, most chemical reactions that occur in nature do not happen without the presence of enzymes because the amount of activation energy that the reactants need to overcome is too great! Without these biological catalysts, reactions that are required to create the building blocks of DNA and RNA will take approximately 78 million years. This means that without enzymes, there would be no life at all!
A. Enzymes are classified into 6 different categories:
These enzymes are normally globular proteins and the sequences of the amino acids ultimately determine the catalytic activity of the enzymes themselves. If you take a look at Figure 2, you will notice that in order for reactants to become products, they need to overcome a high energy transition state. The reason why enzymes are so important is because an uncatalyzed reaction requires a higher activation energy than a catalyzed reaction. Because enzymes can lower the activation energy, reactants won't have to overcome such a high activation energy to become products and thus the rate of the reaction will increases.
If enzymes are so great, you would think that we can just add any kind of enzymes to a substrate and they would bind right? Nope. That's not the case at all. The reason why enzymes won't just bind to any substrates that are being thrown at them is because of their specificity - which makes them particularly picky and won't just bind to any substrates that are dilly-dallying around. ;)
B. SUBSTRATE BINDING: Now, in order to catalyze (speed up the rate of) a reaction, substrates need to bind to enzymes at the active site - a location in the enzyme in which substrates can attached to during chemical reactions. We call this enzyme/substrate compound the enzyme-substrate complex (as seen in Figure 3). When substrates find the active site, ionic interactions, covalent bonds, and hydrogen bonding help stabilizes the spatial arrangement between the enzyme and its substrate. Once the substrate attaches to the active site, the enzyme helps converts it into a product and the product is then released from the active site.
The two different theories that explain the interaction between substrates and enzymes are: The lock and key theory and the induced fit model. In a lock and key theory, enzymes don't have to alter their structure in order for the substrate to bind to its active site. In the induced fit model, however, the binding of substrates to enzymes cause a small amount of free energy to be released. The enzyme starts to alter their conformational structure so that it will increase its affinity to the substrate. In a way, we can say that the binding of the substrate to the active site induces a conformational change in the enzyme so that the enzyme's affinity to the substrate increases (as seen in Figure 4).
C. MECHANISMS OF CATALYSIS: For the most part, enzymes require non-protein molecules called cofactors or coenzymes in order to work properly. Both of them tend to be small in size and are kept at low concentrations so that they can be recruited only when needed. The differences between cofactors and coenzymes are that: 1) Cofactors are inorganic molecules or ions (ie: Fe2+ and Mg2+). Enzymes without cofactors are inactive and are called apoenzymes; 2) Coenzymes include organic groups like NAD+, FAD, Coenzyme A, as well as water-soluble and fat-soluble vitamins. Enzymes with cofactors and coenzymes help create conjugated enzymes called holoenzymes. In the real world, deficiencies in vitamin cofactors can result in diseases that can be life-threatening. For instance, a deficiency in thiamine - a cofactor that is involved in cellular communication and metabolism - can result in Wernicke-Korsakoff syndrome. Patients who suffer from this disorder can have neurological problems and in severe cases, are unable to form new memories.
D. CONTROL OF ENZYME ACTIVITY: The concept of enzyme kinetics can be explained using the Michaelis-Menten Equation, where v is the rate of the reaction, [E] is the concentration of both of the enzymes, [S[ is the concentration of the substrate, [P] is the concentration of the product, and Km is a constant that measures the affinity of the enzyme to its substrate. It is important to remember that changes in enzyme concentration does not affect Km. Higher concentrations of enzymes will result in the reaction to reach the 1/2 Vmax at lower concentration of substrates; while lower concentrations of enzymes will require high concentrations of substrates to reach 1/2 Vmax. If we are comparing 2 different enzymes, just remember that enzymes with lower Km values will require a lower concentration of substrates to be half-saturated while those with higher Km values will require a higher concentrations of substrates to be half-saturated. This means that enzymes with lower Km values have a higher affinity for enzymes and those with higher Km values have a lower affinity for their substrates!
1) COOPERATIVITY: There are certain enzymes, however, that show an S-shaped (sigmoidal) instead of a normal hyperbola. These enzymes are considered to be "cooperative" or "allosteric" enzymes because they have multiple subunits and active sites. Binding of a substrate can cause it to transition from a low-affinity tense state (T) to a high-affinity relaxed state (R). On the other hand, the loss of substrates can cause the enzymes to convert from a relaxed state (R) to a tense state (T). Due to the fact that these enzymes show cooperative kinetics, they often act as regulatory enzymes and are subjected to activation and inhibition.
2) FEEDBACK REGULATION: Enzymes are normally subjected by products in a process called feedback regulation. While it is true that some enzymes may be regulated by feed-forward regulation, most enzymes are regulated by a process called negative feedback (feedback inhibition). These can either increase enzyme-substrate affinity or reduce/inactivate the activity at the active site. Now, you may ask yourself, "Why do we need negative feedback? Isn't the whole point of catalytic reactions is to create more products instead of inhibiting more from being created?" The answer is simple. Imagine this, our body is constantly trying to maintain homeostasis (maintain equilibrium). If we have enough of one product, we want to turn off the pathway so that it will prevent more from being created.
There are 4 types of reversible inhibition: Competitive, noncompetitive, mixed, and uncompetitive.
a. Competitive Inhibition: This is pretty straightforward. If you don't want a certain substrate to bind to the active site, all you need to do is add a competitive substrate (inhibitor) with a similar structure so that it will bind to the active site instead. This inhibition can be overcome if we add more substrate so that the concentration of substrates is higher than the concentration of inhibitors. Because the addition of tons of competitive inhibitors can outcompete the inhibitor, the Vmax value is not affected and the Km value increases. ***Remember that enzymes with higher Km values have lower affinity to their substrates because we require higher concentration of substrates to reach 1/2 Vmax in the presence of the inhibitor.
b. Noncompetitive Inhibition: This is different from competitive inhibition because the inhibitor binds to the allosteric site instead of the active site. Because the inhibitor binds to a non-catalytic region, the two molecules are not competing for the same site and the inhibition is thus considered as noncompetitive. Once the inhibitor binds to the allosteric site, the active site of the enzyme will go through a conformational change that will prevent new substrates from binding to the enzyme. Therefore, we cannot add more substrates to overcome this type of inhibition! Even though noncompetitive inhibitors don't affect the Km value, they do decrease the value of Vmax because less enzymes are available to bind to substrates. Takeaway from this: Enzymes that go through conformation changes due to competitive inhibitors will decrease the number of enzymes that are available to bind to substrates.
c. Mixed Inhibition: Similar to noncompetitive inhibition, mixed inhibitors bind to the allosteric sites instead of the active sites. Interestingly, they have the ability to bind to an enzyme or to an enzyme-substrate complex. If the inhibitor binds to an enzyme, it increases the Km value. If it binds to a complex, however, it lowers the Km value by increasing the enzyme's ability to bind to the substrate.
d. Uncompetitive Inhibition: Uncompetitive inhibitors are unique because they only bind to allosteric sites on enzyme-substrate complexes. This lowers the Km and Vmax values, which means that the enzyme's affinity to its substrate will increase and decrease the number of enzymes that are available to react.
e. Irreversible Inhibition: Unlike reversible inhibitions, in irreversible inhibitions, the active site is either unavailable for a long time or the enzyme is permanently altered and substrates can no longer bind to the active sites. The only way to overcome this is to synthesize more enzymes through transcription and translation.
3) REGULATED ENZYMES: These enzymes are often parts of biological pathways like glycolysis and the Kreb Cycle. There are 3 different types of regulated enzymes: Allosteric enzymes, covalently modified enzymes, and zymogens.
a. Allosteric Enzymes: These enzymes have multiple binding sites and alternate between an active and inactive form.
b. Covalently Modified Enzymes: Enzymes that can be activated or deactivated by phosphorylation, dephosphorylation, or glycosylation.
c. Zymogens: Enzymes that are secreted in its inactive form and are activated by cleavage. An example of trypsin, which is an enzyme that is released from the pancreas. It is secreted as trypsiniogen because without being controlled, this enzyme will digest the organ itself.
B. IMMUNE SYSTEM: The high degree of protein variability allows for a key feature of the adaptive (or acquired) immune system, the production of antibodies. An antibody is a type of protein that has a unique and very specific binding site that will readily bind its target, called an antigen, such that its target is inactivated or tagged for immune response.
C. MOTORS: A motor protein can perform mechanical work by coupling exergonic (energy releasing) ATP hydrolysis to a conformational change that allows for interaction with the protein's target substrate. Muscle contraction, for example, is achieved through a process of the motor protein myosin binding and releasing its microfilament (actin) substrate. Myosin also acts on microfilaments of the cytoskeleton to generate cellular movement.
Two other types of motor proteins, kinesins and dyneins, act on microtubules and play a role in transport within the cell. Kinesin walks microtubule "tracks" to deliver cellular cargo (e.g. chromosomes during mitosis, vesicles), generally in an antegrade direction (center to periphery). Dynein is used in retrograde cargo transport in the axons of neurons, and is capable of sliding microtubules in relation to one another, generating the movement of cilia and flagella.
Let's do a quick recap! Basically, we have learned that enzymes:
I'm Linh - a science geek who loves experimenting and tinkering with recipes! I hope that this blog brings more ideas into your kitchens! Happy eating folks! XOXO