11_BL_20231030_WhisperAI_1.docx

Today I'll tell you about the structure of DNA, a little bit of history, enzymes involved in DNA replication, DNA topoisomerases, DNA polymerases, DNA ligase, DNA helicases, primases, and ultimately replication fork and a little bit about processivity of DNA polymerase, Okazaki fragments, and next h...

Today I'll tell you about the structure of DNA, a little bit of history, enzymes involved in DNA replication, DNA topoisomerases, DNA polymerases, DNA ligase, DNA helicases, primases, and ultimately replication fork and a little bit about processivity of DNA polymerase, Okazaki fragments, and next hour will be recombination and DNA repair. So with this, I will now start the lecture on DNA replication. So DNA replication is the first topic on the general topic of the central dogma of molecular biology. So I will tell you about the expression of genetic information from DNA to RNA to protein. These will be the chapters that I'll be presenting. But before we talk about how this genetic information is expressed, I would like to show you how DNA is copied. This is this replication problem and to explain this, of course, we will first have to understand what are the features of DNA molecule and what are the problems as it needs to be copied. One of the difficulties in copying DNA is the fact that it has to be done extremely accurately. Ultimately, DNA replication and preservation of genetic information from generation to generation happens with an error rate of 1 in 10 billion cases. 1 in 10 billion cases there is error rate. This is quite remarkable and this error correction happens not only through error minimization, happens not only through a very accurate DNA synthesis, but also through a range of proofreading methods and subsequent repairs. So a little bit of history of DNA. If you think of DNA, probably you might remember from your high school about the two scientists who discovered the way DNA is copied and DNA structure and that's Watson and Crick. But maybe some of you also heard about the fact that DNA was in fact discovered in Switzerland by Swiss scientist Friedrich Mischer in 1860s. What is fascinating is that Mischer used very simple methods to analyze cells that were accumulated in bandages of people who were injured. So he had access to this not so nice type of material from the local hospital and then he took this literally pus-filled bandages and analyzed the cells that are found in those samples. So what he did is he tried to separate different cellular compartments and fractions and he could separate the nucleus part of the cell from the rest and try to figure out what is inside and he used very simple methodology. He used centrifuges that probably could not spin faster than currently our laundry machines spin when they try to get rid of their water at the end of the war cycle. Basically using such primitive centrifuges and primitive tools, Mischer managed to extract nuclei from those cells and recognize that the material that is in those nuclei, the sample, the substance as you refer to it, is different in properties than proteins and up to that point everybody was aware of proteins. They were the substance that was found in muscles. Proteins were considered the essence of living beings and for a long time they were also considered as the hereditary molecule. Nevertheless, Friedrich Mischer showed that when he isolated this nuclein nuclein was in fact insoluble when he would reduce the pH using acid and today we understand that reducing the pH values led to protonation, to a change of charge of acidic molecule. Upon protonation it stopped being negatively charged and as a consequence it became less soluble. When he raised the pH the molecule became soluble again and this way he could chemically demonstrate that we are really dealing with a different type of molecule. Almost 50 years later Albert Kossel discovered that this substance, nuclein, consists of four building blocks in case of DNA. Adenine, cytosine, guanine and thymine and gave DNA its current name, deoxyribonucleic acid. Nevertheless, at the beginning of 20th century it was not clear what the function of DNA was. So gradually evidence became more and more indicative that DNA might have very important roles in the cell and that it might even be involved as a storage of genetic information. Nevertheless, it was not until 1952 when Hershey and Chase performed a very elegant experiment that this was unambiguously demonstrated. So what Hershey and Chase did is they used different chemical labeling of either protein or nucleic acid and their experimental system was a phage which is in fact a virus that infects bacterial cells which consists exclusively of DNA molecule, nucleic acid and proteins. And they could use an elegant way of labeling either one or the other radioactively. So using radioactive sulfur, which is an element that exists only in proteins and for example cysteine or methionine amino acids, they could label the protein part or if they grew phages, these viruses, on a medium that had radioactive phosphorus, they would only have the nucleic acid label because phosphorus as you'll see is part of nucleic acids. Upon infection of one or the other type of phage they would spin down the bacteria that had no more surrounding phages present and this isolated bacteria when they analyzed they realized that inside the bacteria only radioactive nucleic acid was found, only radioactive DNA and this bacteria that had no protein left could in fact be used to generate entire new phage that had not only the nucleic acid but also the protein part. This experiment clearly demonstrated that DNA is the molecule that contains the genetic information and it's launched a series of investigations into the structure of DNA molecule and how it might explain the mechanism of copying genetic information replication from generation to generation. So this race involved a number of people in international science but I would just like to mention a team in Cambridge, Watson and Crick and Linus Pauling at Caltech. Linus Pauling at that time was one of the most famous chemists known. Not only he pioneered the science of and the explanation of chemical bonds and wrote a very well-known book, The Nature of Chemical Bond, that was used for teaching chemistry everywhere in all the universities. He also was the person who discovered the fundamental structural element of proteins, alpha helix of proteins that is present in many, many proteins. He received a Nobel Prize for his research and in 1950s, he realized that DNA molecule is very important and this he decided to try to solve its structure. At the same time, a very young scientist, James Watson, was barely aware of the structure of DNA molecule. He was barely out of college. He was very young because he managed to enter college at age 15, which is even according to those standards when people had started their careers a little bit earlier than today, remarkably early. So he finished his studies and even carried out his doctoral studies before he was 23 years old. Then after studying zoology, so not really molecular science, in Chicago, he moved for his postdoctoral studies. That's often what people do to continue their education and to carry out original research. He moved to Laboratory of Molecular Biology of Medical Research Council in Cambridge and there he met with Crick. Crick had a different career track. He was in fact much older and he studied physics, but then during his PhD study, his studies were interrupted because of war and he embarked on a problem that ultimately turned out, in his own words, to be relatively dull. He was studying the viscosity of water at high temperatures and he was not too upset when his laboratory was damaged during the war. So after the war his focus turned to studying biological problems and at Laboratory of Molecular Biology he met Watson. The two of them teamed up to try to answer this emerging question of how DNA structure looks like. So Pauling was the first person to propose possible structure of DNA. Nevertheless his model of triple helix, where you will see later charged phosphates were pointing inwards and the bases of DNA were pointing outwards, turned out to be incorrect. Not only that this model required some special consideration in terms of stabilization of structure with positively charged metals in the middle, but also the structure did not explain how DNA molecule might serve as a molecule that can be copied. The structure did not explain this. So this was wrong and more information was probably necessary to be considered to really arrive at an correct DNA structure. Just before the structure of DNA was proposed Erwin Chagrath, he was an Austrian born biochemist, found that the composition of DNA and individual nucleotides varied a lot between species. So if you would compare one type of bacteria versus another he could see that in one case there were 20 percent A's and the other case maybe 50 percent of A's and so on. So it varied a lot, probably 50 percent is too much, but anyway it varied a lot the numbers of A's, relative amounts of A, C, T, G. However what Chagrath found is that in any DNA that he would analyze, any single DNA from one species, the number of A's was the same as the number of T's and the number of G's was the same as number of C's. So at that point it was not obvious what that meant, but obviously it had very important implications for thinking about how the structure of DNA can be modeled. In the neighboring university to Cambridge in London Rosalind Franklin and Maurice Wilkins carried out X-ray diffraction experiments on DNA fibers. So what they did is they would isolate DNA and then they would keep it in a hydrated form and they would kind of stretch it and put it in sealed tubes and they would shoot X-rays through this DNA and then record patterns and they obtained some of the most beautiful and clear patterns of DNA fibers. Many many DNA molecules kind of strung together in one direction and they could find out based on these patterns that DNA likely is built as a double helix. So that was in clear contradiction to this triple helix model that Pauling proposed, but Pauling didn't have access to this data. However, because Watson and Crick attended a meeting where this data was presented, they were able to use modeling. They literally cut out models of these bases out of cardboard and tried to see how they would fit together and when they would fit G and C together they would end up with three hydrogen bonds between them and A and T together they would end up with two hydrogen bonds. But the remarkable thing is that no matter which pair they would form with this type of arrangement, so-called Watson-Crick base pairing, the adjacent, the connection between the bases and the backbone of DNA, and I'll show you in a minute what is meant by that, was the same. So the distance between these attachment points was the same for both and that meant that even though the sequence varied, the DNA molecule could be described to have a very uniform double helical structure. And then in one of the most famous understated statements in the history of publishing, Watson and Crick said, it has not escaped our notice that the specific pairing that they have postulated immediately suggests a possible copying mechanism for the genetic material. And indeed, this structure told us how, if you would separate the two strands, you could recreate the missing strand just based on the sequence of G's, C's, A's and T's. So in the end, what does the structure of DNA look like? It consists of, in a splayed out form, in three dimensions, it's a double helix, but it has chemical directionality. So basically, if you look at this ribose phosphate backbone, these are phosphodiester bonds here. So if you think of this backbone, the ribose itself has chemical asymmetry. It has different types of carbon atoms. This is carbon three where hydroxy groups is attached. This is carbon five here that's sticking out of the ring of five atoms. This is carbon five. And because of this, through this connection of phosphates in between, this strand of DNA has a five prime group on one side and three prime moiety on the other side. This strand on the opposite side runs in the opposite direction. So they're anti-parallel. This one has a three prime group here and a five prime group here. And in between, besides this phosphosugar backbone, there are bases. And each base pairs according to Watson-Crick principles with either three or two hydrogen bonds. And these bases can be on either side. And this very nicely explained that whenever you have an A, you will have a T. And that's why the amounts of A and T have to be the same in any organism. Whenever we have a G, you always have a C. And then again shows very nicely Chagrav's prediction. Very, very nice explanation. But before we move on, I would like you to now see very precisely the architecture of DNA. And because this has a lot of implications for our continued discussion. So these two anti-parallel strands involve base pairs that upon every new base pair in this helix, double helix. So the reason it's called double helix is because you have one strand forming a helix and the other one next to it forming a second helix. A little bit like a spiral staircase. So between each base pair, there is a distance of 3.4 angstroms. You might remember that this 3.4 angstrom base separation is actually the distance that two atoms can approach each other before causing steric clashes. So this is so-called van der Waals type of interaction where things stack. So there's no water molecules in between. They literally stack one on top of the other. And this is about 3.5 angstroms, the distance between centers of two atoms. And that's exactly the distance between consecutive base pairs in DNA. Now, this is a very important number. You have to remember it. And the second very important number, and that one is easy to remember, is the turn for each base pair. Between one and the other, there is a 36 degree turn. This is a very nice number because it means that one full twist of DNA will happen after exactly 10 steps because that would fulfill 360 degree turn for 10 times 36 degree turn for each base pair. So in a way, you can think of the geometry or the architecture of DNA repeating itself every 10 steps, every 10 base pairs. A lot of these numbers here are expressed in angstroms. Just as a reminder, an angstrom is 10 to the minus 10th meter. So it's a very, very small molecule that we are dealing with here. This is the reason why Maurice Wilkins and Rosalind Franklin had to use x-rays. They couldn't use light microscopy to see DNA. It was so small. The x-rays have much shorter wavelengths and that's why x-rays interact with such small objects and they could give them the kind of information that I explained to you. So you see here in a space filling representation, the consecutive bases are literally stacking against each other and that contributes a lot to the stability of DNA. So this number 10 is not exactly 10, it's 10.4 bases per turn, but at least you should remember the approximate 10 bases per full turn. So there is one other consideration of this architecture of DNA as a double helix. I mentioned to you that the connection between the bases and the backbone of DNA follows the same geometry, no matter which base pair we are talking about, whether it's AT or TA or GC or CG. The position of the bond, which is called glycosidic bond, which connects the base with the sugar, ribose, is always at the same place. That means that no matter what the sequence is, the double helix will have a smooth backbone that will not deviate much in terms of structure. The only real difference is looking from the side, will be the nature of atoms that originate from different bases. But this double helical feature and the asymmetric positioning of these glycosidic bonds, so that means that these glycosidic bonds are not just pointing outwards, 180 degree, one relative to the other, they're pointing at an angle, right? And so because of this, this double helical and so because of this, the geometry of this double helix has a large groove here and a minor groove, a smaller groove here. So this generates a unique appearance of this DNA, which is very important ultimately for different proteins you'll see later that have to recognize particular sequences in a DNA, even when it's closed, when it's not opened up. And there are proteins that can recognize this and many of them will bind here to the major groove because there is more space for them to bind and more features to recognize. So last thing I would like to tell you about the DNA geometry and architecture is that what I explained to you so far is the so-called B-form DNA. That's the B-form. It has the large, shallow major groove, deep and narrow minor groove and the geometry here. However, this B-form DNA is not the only type of DNA that can be seen. So if you have lower hydration, less water around, the DNA can also form so-called A-form here and this A-form has slightly different appearance. It has a slightly more open minor groove and different number of bases until they reach a full turn. But those parameters you don't have to worry about right now. But what is important is that the A-form DNA is, in terms of how it looks, very similar to the double helical architecture of RNA molecule. That's the other type of nucleic acid that has one hydroxy group more than the deoxyribonucleic acid in the ribose sugar at the two prime position. And these RNA molecules can sometimes fold on themselves and be self-complementary and they form local double helical regions or they can also be complementary. You can have two different RNAs that are complementary to each other just like two strands of DNA and then this RNA will have this geometry. So just keep that in mind. That's important later for thinking about RNA-based processes in the cell. So the reason for these different geometries is the so-called pucker of the sugar. So five-membered ring of ribose with five atoms always will have four atoms that can be approximately put in a plane and one of the atoms will be out of the plane and in this case it's the carbon three atoms that's out of the plane and then it's called C3 prime endo because it points in the same direction as the base and when carbon two is out of the plane and the other four are in plane this is called C2 endo and this is the basis of the A-form DNA and this is the basis of the B-form DNA. Okay, so with this a question rose that took several years to be answered experimentally. How is this DNA copied? And you will see maybe it's even easier to imagine initially that the DNA would be copied such that you would simultaneously copy both strands and peel out a new DNA molecule. Another mechanism is also quite possible that the copying takes place such that you open up these two DNA strands and you synthesize a complementary molecule, the blue one here, but for a very long molecule this will be a problem as you will see in a minute. So Meselson and Stahl five years after the structure of DNA was determined performed a very elegant experiment. What they did is they used a very simple experiment. What they did is they used two different isotopes of nitrogen, nitrogen 15 and nitrogen 14. They would initially grow bacteria in the presence of nitrogen 15 salt and this bacteria would incorporate this nitrogen 15 in their DNA because DNA of course has a lot of nitrogens and oxygens and phosphates and so on and then they would transfer this bacteria, they would remove the medium, they would spin down the bacteria and then they would put the bacteria in a different medium with nitrogen 14 isotope and then after 20 minutes they analyzed what happened and it was known that bacteria divide every 20 minutes. It was known that in 20 minutes the DNA in the bacterial cell has to divide in two and then they also analyzed it after 40 minutes. What happens then? Then you would have expected to have two cell divisions and what kind of results they saw. So this is the actual data from their experiment and it's very clean and very illustrative. So basically if they would analyze these are bands that they would see in a so-called sucrose gradient. They will literally spin down the DNA that they isolated from these cells and if DNA is heavier it would sediment lower in this gradient because it would be heavier so N15 would be heavier and if it was lighter then it would sediment at a higher point and generate the band here. So in this experiment what they saw this was N15 DNA, this was N14 DNA as a control. It would sediment in two different places in the gradient. After zero generation they had only N15 labeled DNA. After one generation they had single band but the single band was between N15 and N14 and that meant that the replication is semi-conservative. That meant that the new molecules, both new molecules had one strand of the old DNA and one newly synthesized strand. After two generations as you can see here they also saw two bands equally intense. One that was mixed N15 and N14 and the other band that was N14 and this is what explains it. So basically after the second round of replication they would see two of the four molecules that were still hybrid that still had the old parent DNA and two that were completely new, nicely explaining what the experiments showed. So now I would like to just briefly explain to you a little bit the vocabulary and terminology that is used to talk about DNA. So I introduce to you now what nucleic acids are and what nucleotides are. These are monomers or building blocks of nucleic acids. Now if you have a longer DNA stretch or RNA stretch these are called polynucleotides and if you have small regions of just a few nucleotides this is usually called as oligonucleotides. So just so that you later if you hear those terms you understand them. So once you have nucleic acid molecule, polymer, partial information in this long molecule is usually responsible for... So basically the information in the long molecule is usually separated into little regions and one information unit of DNA is called gene. So there is other types of information in DNA but to oversimplify this point functional unit of genetic information is a gene. Genes are part of genetic elements and for example these bigger DNA molecules that constitute entire genomes like the entire genome of E. coli is a single circular DNA molecule that's called bacterial chromosome. We also have chromosomes but our chromosomes have linear DNA pieces and are much much larger. So just a little bit about the size of DNA molecules they're typically very large because they have to contain so much information. So a thousand base pairs we usually refer to as a kilobase pair. One million base pair is a megabase pair and this is how it's expressed that's how it's shown. So for example E. coli genome is 4.6 megabase pairs, 4.6 million base pairs. Just think of it and try to calculate a little bit what would be the length of 4.6 million base pairs if each base pair has 3.4 angstroms rise along the double helical DNA. Certainly this would be much longer than the size of the bacterial cell which is about two microns. So usually you will see the DNA has to be packaged somehow to be contained within the cell and think about it a little bit. How long would a single copy of our DNA be? If it was stretched from a single cell, if you would take all our chromosomes and string them together in a linear DNA molecule and our DNA is 6 billion base pairs large, how long would it be? Would it be millimeters? Would it be centimeters? Would it be meters? How long would DNA from all our cells be? If you would take all the chromosomes, stretch the DNA, put them one after the other on a line and then put all the chromosomes, all the DNA from the next cell and from the next cell, I think you would be surprised to see what the answer is. So semi-conservative mechanism of copying genetic information causes problems for very long molecules. Just think of it. You cannot easily unwind this very long DNA molecules. It would be a little bit like trying to pull apart a rope that is coiled. So basically, in addition to this, there are other issues that have to be considered and it is obvious that the initial proposal of how DNA is copied required decades of experimentation so that all the nuances of the process could really be understood and so that all the associated machinery could be characterized, investigated, analyzed and this is the kind of problem that will be