13Week 14 Lecture Biochemistry of Nutrition, 560B Dr.

Charles Saladino Introduction In spite of great differences in lifestyle and appearance, organisms tend to exhibit striking similarities at the molecular level. We have seen that the metabolic activities of basically all cells rely upon common groups of molecules that include amino acids, carbohydrates, and lipids, often in polymer form. Each molecular type has a signature of physiological function, chemical composition, and interactions with other biomolecules. For example, the DNA is a repository for our genetic information. We shall start by describing its structure. Nucleotides I am sure you know that DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) are what are commonly referred to as nucleic acids. Most biochemistry texts describe the metabolism, structure and function of nucleic acids and their components reasonably well. Unfortunately, your text frankly trivializes the subject of nucleic acids by its glaring omissions. With that said, we can start by noting that both of these biomolecules (DNA and RNA) are long polymers that are able to carry information in a manner that can be passed from one generation to the next, whether it is at the level of the organism, the cell, or the molecular level. Both forms of nucleic acids consist of a very large number of fundamental units, known as nucleotides. Each nucleotide is composed of a sugar, a phosphate, and a heterocyclic nitrogen base. The common backbone of DNA and RNA is the sugars linked by phosphate groups. The bases vary, as will be explained in more detail later, and they are involved in nearly every aspect of cellular life. To be a little specific, nucleotides participate in oxidation-reduction reactions, transignaling sequences, energy transformation, and biosynthesis reactions. Remarkably, nucleotides also play structural and catalytic roles in the cell. In fact, I will stick my neck out a bit and state that no other class of molecules participates in such varied functions and those essential to life as do the nucleotides. Nucleotides show structural diversity, and they are quite ubiquitous. They are also capable of having more than one phosphate attached, as in ADP and GDP or ATP and GTP, for example. I have included in a figure below the structures of the five (by far) most prominent bases that help make up nucleic acids. Notice that two, adenine (A) and guanine (G) are referred to as purines, being double ring structure. On the other hand,

thymine (T), cytosine (c), and uracil (U) are pyrimidines and are single ring structures. Lets note right here that DNA contains A, T, C, and G, whereas the various RNAs contain A, U, C. and G. In other words, the T of DNA is replaced by the U in all RNA molecules, and, thus, we normally do not find U in DNA or T in RNA. As you probably know, fluorouracil is an analogue of thymine, and is used in chemotherapy. This is because it gets substituted into the DNA of cancer cells (and unfortunately many normal cells) and interferes with normal DNA synthesis. This then blocks cell replication, although not without severe, toxic side effects. Sugars The basis upon which the names of the two classes of nucleic acids are based appear to rest with the sugar. Thus, deoxyribonucleic acid contains the sugar deoxyribose in the backbone, whereas the backbone of RNA contains the sugar ribose. Below is a figure showing the structure of the deoxyribose, a five carbon sugar manufactured (remember?) in the pentose phosphate pathway. Please note that it is termed deoxyribose, because there is no OH group at carbon number two. On the other hand, the ribose sugar does contain an OH group in the carbon number two position. Now lets link the nucleotide together in a single strand.This shows a polynucleotide segment linking by phosphodiester bonds. Notice that although there is one A, T, C, and G base, respectfully, linked to a deoxyribose sugar, there can be any arrangement of bases, for ex, TTTAGCCGT. We shall see later on that the order of the base sequence is what ultimately determines the genetic code. Please take note of what might seem trivial, but what turns out to be a very important detail. That is, notice that on one end of the polynucleotide is a 5 phosphate attached to the OH OH group on carbon # 5, whereas on the other end is a 3 hydroxyl group. This is almost always the case. The symbol behind the 5 of the phosphate and the 3 of the hydroxyl is termed prime. Thus, we have a 3 prime hydroxyl and a 5 prime phosphate end, neither of which is linked to another phosphate, and they represent their respective ends of the polynucleotide chain. This will become very important for DNA synthesis. We should note here that one characteristic of naturally-occurring DNA molecules is their length. As you probably know, the DNA structure was elucidated by Watson and Crick (for which they received a Nobel Prize). They utilized x-ray diffraction crystallography, and it was in the 1950s before the computer would have been available for such work. As I am sure you also know, the DNA structure is a double -helix, wherein two polynucleotide strands are wound around one another in a right-handed twist. To achieve this, we require a second polynucleotide strand, just like that one above. (Devlin or any on-line source can easily be found to observe this double helix.) So what holds the two polynucleotide strands

together? The short answer is hydrogen bonds. The longer answer comes now. Lets look at the figure below.It not a coincidence that in the figure above, A is shown paired to T with a double hydrogen bond; and G is shown paired to C with a triple hydrogen bond. This is the base pairing arrangement under normal conditions: A-T and G-C. Notice one pyrimidine is paired to one purine, because the spacing arrangement would not be stable with two purines or two pyrimidines paired up to each other, respectively.You will notice that it is the base pairing through hydrogen bonding that holds the two polynucleotide strands of the helix together. The fact that G pairs to C and T pairs to A is referred to as complementary base pairing, and that represents the only normal base pairing combination. To the left of the base pairing figure is the helix. The term refers to the fact that the helical twist is right handed, with the polynucleotide chains coiled around a common axis. The sugarphosphate backbones occupy the outside position, with the purine and pyrimidine bases lying on the inside of the helix. The bases are nearly perpendicular to the helical axis, and there are ten bases (on each side) per turn (360 degrees) of the helix. The double helix is stabilized by Van der Waals forces between adjacent bases on the same strand and by the hydrophobic effect - mentioned to you when I described the entropy changes in the water shell near a protein. A striking feature of DNA is its length. Even in the simplest organisms, such as the polyoma virus (which can cause cancer in some organisms), the DNA is 5100 nucleotides in length. This corresponds to 5100 x 2 = 10, 200 bits or 1275 bytes (1 byte = 8 bits. Now the E. coli genome, which is a single DNA molecule comprising two chains, is 4.6 million nucleotides (1.15 megabites of information). The human genome is approximately 3 billion nucleotides divided among the various chromosomes. Just for your interest, there is an Asiatic deer with a genome about as large as that of humans. However, it has only three sets chromosomes, with the largest chromosome containing more than 1 billion nucleotides! If this chromosome were to be stretched out, it would reach one foot in length. Amazing how all that is packed into a cell nucleus. Believe it or not, some plants contain even larger DNA molecules. Finally, please note the term kb (kilobases). So for example, if we say that a particular mammalian chromosome contains 250,000 kilo base pairs. That means 250,000 x 1000 base pairs. Now please notice one important feature in the above diagram of the base pairing. One polynucleotide strand runs in the 5 to 3 direction (on the left), whereas the complementary strand (on the right) runs 3 to 5. This arrangement of the polynucleotide strands is referred to as antiparallel. Please remember this important feature.

We should note that bacteria contain a single helix, but it is in a closed circle. The same applies to mitochondrial DNA. Yes, mitochondria contain DNA, but not enough to encode for all the mitochondrial proteins. Thus, by way of example, some of the electron transport system proteins are coded for by mitochondrial DNA, whereas others are encoded by the nuclear DNA. Reversing Melting of DNA As we shall discuss shortly, during replication of DNA and during transcription, the two strands of the DNA double helix must be separated from one another. at least in one local region of the long DNA. In the laboratory, where I once studied DNA repair, I used to disrupt the DNA helix by heating it in solution. Heating will disrupt the hydrogen bonds between the base pairs, allowing the polynucleotides to separate. One can also do this in a very alkaline solution, which ionizes the base pairs and disrupts the base pairing. This disruption of the helix is sometimes referred to as melting. The Tm is defined as the melting temperature. Because the double bonds of A-T require less energy to break than the triple bond of G-C, DNA with a higher G-C content than that of A-T will show a higher Tm value. However, once the strands separate, these complementary nucleotide strands can spontaneously reform a double helix. This renaturation process is often called reannealing. Although this sounds like test tube work, during DNA replication and during transcription of RNA, this reassociation of the two strands is crucial for the biological functions of DNA. However, inside cells, helicase enzymes are present to facilitate this ATP-requiring denaturation during DNA synthesis. Finally, DNA from two different species can be denatured and permitted to reanneal or hydridize, in the presence of each other. The hybrid DNA duplexes (with each organism contributing one DNA strand of the double helix) will form; and the degree of hybridization will be an indication of how closely related the genomes of the two organisms are. Further, similar hybridization experiments with DNA and RNA can locate genes in cellular DNA that correspond to a particular RNA (to be discussed in our next lecture).

DNA Synthesis It is intuitively obvious that without a mechanism for maintaining the fidelity of the genetic code from one generation to the next, and that

means at the cellular level too, the survival of the next cell generation, and thus the species, would never be possible. The mechanism by which DNA is replicated produces two identical and complete DNA molecules from an original DNA whose code is determined by the order of occurrence of each of the four types of nucleotide bases. With only four different bases, but with literally millions of each one occurring in various combinations and permutations, probably about 100,000 genes might be present in the genome to code for an even larger number of polypeptides (that later point to be explain in the next lecture). Thus, our genetic code simply is which of the four bases occurs in what order and how many of each there are. Remember, eventually we shall see that one gene codes for one polypeptide (not one protein, again, next week). This week, our task is to explain DNA synthesis (replication). Then we will be in a good position discuss the characteristics of the various RNA species next week. The molecular mechanism of DNA synthesis involves more than 20 proteins that interplay with one another. Among the most prominent are the DNA polymerases (properly abbreviated DNA pol - singular), which promote the formation of bonds joining new nucleotides together. Specifically, DNA pol catalyzes the step by step addition of nucleotides together, with the new DNA strand being assembled directly on a preexisting template which will be complementary to the new growing polynucleotide. Observe the figure below. This figure starts with DNA synthesis already in progress. Well will go back to the initiation of it later. Please notice several things. First, notice that the growing strand is antiparallel to the template. That is, the growing strand is running in the 5 to 3 direction, whereas its template is running 3 5. The template represents one of the two original DNA strands of the double helix, after the helix has been unwound in this area of the whole DNA by a helicase enzyme. Only one of the two original DNA strands is shown in this figure; and it is the strand on the right that starts with an AAC sequence. At some point, the opposite strand of the original DNA will act as a template for a new polynucleotide strand in the same manner. It is important to note that the DNA must unwind, in order to engage in replication. It does not open up like a zipper from one end to the other. Can you imagine the logistical nightmare of doing that???? It is far too long (much longer than the cell) for that to happen. Thus, it opens up locally at what is called a replication fork. This is what happens. A genetic signal occurs wherein an endoculease enzyme puts a break in the sugar phosphate backbone, so that the helicase can unwind the strands. The strands now separate, so that both of the original DNA

strands can act as a template, wherein proper base pairing can occur, as is shown in the figure above. Note that a G is being added, because the template is a C. Note also that what is being added is a dGTP. The d is for the deoxyribose sugar of the nucleotide, the G is obviously the purine guanine, and there are three phosphates in this nucleotide. As the nucleitode is being added by the polymerase, the two end phosphates are cleaved off, so that a nucleotide is added (one sugar, one phosphate, and one base. The removal of the two phosphates (PPi) which are subsequently split into two single phosphates (Pi + Pi) are what provide the energy for this endothermic, endergonic reaction of DNA synthesis. We should note that the replication of DNA requires DNA pol that takes it instructions from the DNA template. The synthesis of DNA thus produces two new DNA strands from one original DNA. This is called semi-conservative DNA synthesis, because each new DNA is half new and half old (the latter half from the original DNA molecule). If you understood the above material, then you will notice that new growing strands of DNA always synthesize in the 5 to 3 direction, antiparallel to their respective templates. I will now place a diagram for you to observe, and I ask you to pay particular attention to what is labeled the leading strand and then the lagging strand.

You will notice that because the leading strand runs 5 to 3, it can just keep growing until the synthesis is halted in that area of the replication fork. However, the opposite growing strand, called the lagging strand must also grow in its 5to 3 direction. That means that new pieces of growing DNA must grow from behind the previous growing strand i.e. back fill. Thus you can deduce from this, as well as by rechecking the figure above the present figure, that the polymerase requires a free 3OH group to which to add the next nucleotide. That is why one new strand of DNA is made continuously, whereas the other new strand is synthesized in fragments. DNA Synthesis Requires an RNA Primer Having just stated that the DNA pol that adds a nucleotide to a growing strand requires a free 3OH group, we can ask how the process of DNA synthesis can start at a new replication fork when there is nothing to add to in the first place. The answer is that an RNA primer must be synthesized on the template to provide that free 3OH group to initiate DNA synthesis. That RNA primer is synthesized by a special RNA pol called primase. This then synthesizes a short stretch of RNA (maybe 5

nucleotides in length) that is complementary to the DNA template strand. This seems counterproductive, as the primer will have to be removed later; but the answer lies in the fact that the DNA pol can not start polynucleotides de novo, whereas RNA pols can. You will notice in the above figure the term Okazaki fragments. These are the segments (about 200 nucleotides long) of newly growing DNA that result from back filling as lagging strand segments. Each would have to begin with an RNA primer. Now obviously, these primers do not remain as part of the newly growing DNA strand. Thus they must all be removed; and indeed they are by an exonuclease enzyme. This enzyme sequentially chops out the primer. Now a different DNA pol will fill in the gap left by the RNA primer, and all thats left to do is to seal the bond. That last step is carried out with an enzyme called DNA polynucleotide ligase, which does require ATP as an energy source. Thus, DNA synthesis in general is energy intensive. A figure showing the ligase action is presented below.

Replication Rate and Replicons DNA replication is substantially slower in eukaryotes than in prokaryotes (like bacteria). Specifically, in a eukaryotic cell, about 50 nucleotides per second are added to a growing DNA strand one tenth the rate of that of prokaryotes. This difference is undoubtedly due to the complex structure of the chromosomal material found in eukaryotes. For the last exam, look up the structure of chromatin. In spite of this relatively slow rate of DNA synthesis, eukaryotic DNA synthesis can be accomplished in a matter of hours in a given cell. That is quite a feat, considering the very large amount of DNA in the human genome, because if we take the above-described eukaryotic nucleotide addition rate of about 50 per second, it would take about a month to replicate the chromosomal DNA (about 150 million base pairs) in a given cell! So how does the cell get around this problem? Again, the design is so intelligent. The answer is multiple replicons (areas around a replication fork origins of replication) operating simultaneously to compress the replication time of their large genomes. In humans are you ready for this replication requires about 30,000 replicons, with each chromosome containing hundreds! Now think about what such a system requires. How about (for one thing) a mechanism that each sequence is replicated once and only once? In the cell cycle, DNS synthesis and cell division are coordinated

so as to ensure that the synthesis of all DNA sequences is complete before the cell can progress into the next stage of the cell cycle. This requires several checkpoints that control the progression through the cell cycle. Finally, I can not cover all the proteins involved in DNA synthesis in this lecture. It has gotten long enough already. However, to further try and stimulate great awe for the way the cells biochemistry works, I will close with this the following: Besides the various proteins we have already discussed (DNA pols, replicase, helicase), once the strands of DNA are separated by the helicase, they must be stabilized by the binding of replication protein A. This is a single-stranded DNA binding protein. Then DNA synthesis begins with the binding of DNA pol , which is the initiator pol. Now, hear this. This enzyme has primase activity, as well as DNA polymerase activity. Make sense? After about 20 deoxynucleotides have been added to the primer, another replication protein (protein replication factor C PFC) displaces DNA pol and attracts another protein associated with replication, called proliferating cell nuclear antigen (PCNA). The PCNA then binds to DNA pol . This binding probably causes an allosteric change in the DNA pol that cause this enzyme to be extremely active and quite suitable for synthesizing long stretches of new DNA. What I have described here is called polymerase switching. This is because the pol has replaced the pol . Finally, and again amazingly, pol has 3 to 5 (yes thats different than that we have discussed before) exonuclease activity. This exonculease activity can then act as a proofreader and edit the replication of DNA (meaning remove mismatched bases thus minimizing mutations). Finally, as we have already discussed, replication continues in both directions from the origin of the replication fork, until adjacent replicons meet and ligate. While all this is going on, the RNA primers are being removed, as previously described. Arent you just at least a little impressed that all this works as well as it does? I am.