Genetics Lecture Notes PDF

Okay, it is one o'clock, it's time to get started. Happy Wednesday, remember we have secure review tonight, I believe. But one of the things that I want to start off this section with is the way that this kind of block works. Some sections I need to spend a little bit more time on, others less, so...

Okay, it is one o'clock, it's time to get started. Happy Wednesday, remember we have secure review tonight, I believe. But one of the things that I want to start off this section with is the way that this kind of block works. Some sections I need to spend a little bit more time on, others less, so I pretty much take all of the DNA content into one giant packet and just start working our way through it. Wherever we end up is where we stop, I will try to make sure I always stop on time, which I have been really bad at these last few times, but then we will continue on. but then we will continue on. If it's 10 minutes till, we're going to go on to the next section. Does that make sense? There are questions built in, it's sort of practice as we go, there's extra quizzes already available, all of that is stuff we'll work through over time. Makes sense? sense? Okay, so this is a, we're going to start off the bat with a slide you get to just put a giant X through. But I want to start from here because I want us to understand that everything we're doing is relatively recent. There are people still alive today who cannot give a definition of DNA because they literally didn't get exposed to it until adulthood. There are people that will have misconceptions about concepts in DNA, RNA, and protein because they literally didn't know those were things until they were much older. those were things until they were much older. So if you look at the concepts in basic genetics that we have, yeah, there's some time there, but in terms of actual DNA structures and actual processes, yes, there are still people alive today who didn't see that until they were 20 years old. Does that make sense? Does that make sense? And so especially in the realm of cancer genetic counseling, where much of that population would be at that age for highest risk of cancer, we have to pay attention to the level of information they are comfortable with. And so just remembering that genetics is a growing and changing thing and this only covers the background, these are all the more recent advancements. Recent being, you know, 1961 is on there. Not super recent, but still within lifespans. Genomics has been massively changing for the last 10 years. We went from sequencing a genome taking a decade to being able to do it in a single afternoon. A whole genome sequence for a human being in under four hours. That is a tremendous benefit to humanity. And we can now use things clinically that we never could before. So we have to have a fundamental understanding of how sequence contributes to biological function in order to understand how those new diagnostic tests actually benefit us. actually benefit us. So while you can somewhat ignore this slide, what would you say is one of the greatest advancements to genetics that you can think of? of? What do we think? I mean, that one thing that you know to be available that you think is a really good advancement for this field. Go for it. Absolutely. The discovery of polymerases that could handle high temperatures are thermophile bacteria giving us the ability to actually do sequencing revolutionized all of genetics. It's a great choice. CRISPR gene editing, right? When done ethically and when done appropriately has created incredible advancements in our ability to understand loss of function mutations. There are some amazing CRISPR activation plasmids that let us turn on genes and observe what overexpression of genes actually does. What our duplication mutations can actually do. It's too enough to be able to move on. Go for it. Right. Yeah, humans having model organisms where we can actually study genetics and genetic expression is so critical to applying that to human disorders. Love that. Okay. So that was just making sure we remember that this part has a purpose. Much of the mutation that we observe in human disease is going to be correlated with what we call single nucleotide polymorphisms. A single base change is going to cause a change in functionality. Since we understand that there is no junk DNA, we started this class from a perspective of cell. We're honed into the chromosome and heredity piece and now we're focused even more in on just DNA, RNA and protein. Sequences. Because those polymorphisms have consequences. The nine variation is not the same thing as pathogenic variation. And so we want to spend some time understanding how that variation is created and then what its consequences are. So our overall goal is going to be very much tied to understanding DNA, RNA and protein. And that starts with understanding what these things are. are. What is DNA? Define DNA for me. Deoxyribonucleic acid, genetic material, right? Deoxyribonucleic acid, genetic material, right? But what made it genetic, right? There's rules for what makes something genetic material. And here's where it becomes really important because we need to have working definitions of these words because they mean different things. The classic definition of a gene is a sequence of nucleotides that encodes for protein, right? Except there are lots of genes that never encode for proteins. So we have to adjust that definition to a sequence of nucleotides that encode for a product. Could be RNA, could be rRNA, could be tRNA, could be mRNA that eventually becomes a protein. The genome is all of the genetic material within an organism. Is all of the genetic material encoding a product? No. We have lots of regions that are important structurally but don't make products, right? So keeping in mind, we need to be able to do gene, genome, genotype. The actual sequences that make up your variation. Chromosome, chromatin, and chromatid, right? Longitudinal subunit versus arrangement of material versus the fully duplicated form. Transcriptome, proteome, and metabolome are going to be areas that we talk a bit about. The transcriptome is all of the transcription products. The proteome is all of the protein products. The metabolome is all of the metabolism that is taking place. So the functional downstream of the proteome. And, of course, phenotype, what we can physically see. More often than not, we are relying on phenotype to tell us what's going on. We are looking for a downstream functional consequence to identify what could have happened genetically. Sequencing is the diagnostics, but phenotype is what brings you in the door to reach a diagnosis. The central dogma of molecular genetics. This whole section is all about molecular genetics. Classic. DNA is transcribed into RNA, which is translated into protein. is translated into protein. Yes? DNA replicates. However, can you get functional chromosome level DNA without protein? DNA without protein? Nope. RNA also influences DNA. We need elements of RNA to actually confer replication, to actually confer transcription. Reverse transcription is a thing. Reverse transcription is a thing. Where do we rely on reverse transcription? Telomeres and telomerase, right? Telomeres and telomerase, right? RNA influences DNA. Proteins influence DNA as well. Literal higher order structures for DNA involve folding and an association with proteins. Transcription factors are necessary. Those are proteins. Without them, we can't express a gene. So when it comes to understanding human metabolism, human disease, just looking at the genome is not enough. We need to look at what genes are expressed where and when. We need to look at whether or not that gene created a functional protein. So transcriptomics, proteomics, all of the omics are necessary together. Metabolomics is a major area of current study, particularly as we become interested in the gut microenvironment. What is bacteria in your gut doing to your overall activity? There was a pretty limiting study on the role of synthetic sugars in terms of what impacted had on overall gut function and metabolism. That limiting study showed that if you are someone who consumes a large amount of diet coke, then you have a different metabolism potential than someone who doesn't, like someone who focuses on regular coke. Probably should phrase that differently. In terms of those differences, though, the study was very limiting and really only focused on one synthetic sugar, artificial sweetener, versus using sugar in general. And yet that focus, that shift in emphasis to how that affected metabolism was a huge piece. Because that gut microenvironment has a significant impact on metabolism. So in terms of why we look at all this, just because you have a sequence of DNA does not guarantee you function. Just because you have the presence of a gene does not guarantee that you have the product. And so we have to focus not just on the genome, but also what is transcribed, what the proteins are doing, and specifically how the proteins from one cell are impacting the whole organism. the proteins from one cell are impacting the whole organism. Are you producing an aberrant protein that is affecting the ability to carry oxygen? Are you producing an aberrant protein that is causing you to not be able to synthesize something like insulin? insulin? There is real human implications for not understanding all levels of molecular genetics. So specific concerns that we have. Pre-inplantation genetic diagnosis is a thing. In vitro fertilization is a thing. Being able to study and sequence and diagnose the potential for genetic anomaly is an important aspect to healthy prenatal care. That being said, we also have pharmacogenetics. If I am unlikely to properly metabolize a drug, should I be given that drug? And if there is a simple test that would tell me the likelihood of my ability to metabolize, shouldn't that be something that I have information for? We are not quite there yet, but we are getting there. We are getting to the point where we can very quickly and easily sequence individuals for these potential pathogenic variations. So that is my introduction to the why of all of this, understanding the connection to human disease. So now we get to just spend some time with DNA. And this entire section will move from just an introduction of structure to replication to error and repair. And each piece hopefully will build a whole picture. will build a whole picture. DNA is probably one of the most amazing molecules that there is. It can exist in multiple forms. It can take on multiple structures. And what we classically think of as DNA being this rigid, specific type of form, and this is what it looks like, turns out it has a little bit more flexibility. And it is really important that we understand the flexibility because studying DNA can be complicated by our lack of understanding. Why is DNA important? Well, because DNA is our genetic information. For years, we had this wrong. For years, we thought it was protein. We thought that the piece that mattered was protein. As long as you had the proteins, that was how what made you you was transmitted because DNA is too simple. There is no way that those four bases could encode for all of the variation we see. Right? Right? And then we had experiments that told us otherwise. We had the Hershey Chase experiments. We had the bacteriophage experiments. And we proved that DNA was the genetic material. And in proving that, we established what criteria there were to be genetic material. And we came up with four criteria. We, because I was totally involved. I was, I was, yeah, no, I'm not that old. I was, I was, yeah, no, I'm not that old. Anyway, it must contain information. What does it mean to contain information? A bunch of bases stacked together have no meaning, right? right? It's only if when reading those bases, we get something else that we contain information. So what's it saying is there needs to be genes. I need to be able to make something from this material. Can I do that with proteins? No, a protein has functioning of itself, but I can't make something new from that protein. I need to use that protein to make something. I know semantics, but trust me, it matters. Transmissibility. I have to be able to transmit it. It cannot just be something that only affects me. Heritable epigenetic signatures, the changes that happen to you in life matter because they're transmissible. They're heritable. Things you do that impact your DNA have the ability to impact future generations. That's important. Got to be able to copy it, right? Got to be able to make more than one of it. Would it be genetic material if it could never be used again? Not from the perspective of genetics equals heredity. So much of the previous block focused on that heredity piece. Some of this molecular genetics is just going to focus on the DNA, but we're still going to keep genetics equals heritable in mind. heritable in mind. A genetic change is important because it means it impacts the future. Now, here's where heritable gets a little fuzzy. A cell has daughter cells, yes? A cell has daughter cells, yes? That's heritable. But those daughter cells are not going to affect the next generation of organism unless they're what kind of daughter cells? Gammies. And thank you to those who mouthed it. I appreciate it. So we will talk about, especially in block four, the difference between somatic and germline mutations. We'll talk about it a little bit in a few lectures, too. And, of course, the last part of true genetic information, variation. What's the difference between a clone and a daughter? Variation. Variation. A clone is supposed to what? I love it when I softball these things. Come on. A clone is supposed to what? A clone is supposed to what? Be identical. It's supposed to be a perfect match. When you're doing clonal growth of a cell, you want all the cells to match. When B cells are triggered, we want all the cells to match. That B cell we triggered because, are we not there yet? Not there yet. Okay. Okay. Well, pick another cell. Because we want the product from those cells to all be the same, right? right? So humoral immunity was my tie-in here. I apologize if we weren't quite there yet, but I went first. Okay. So unlike a clone, a daughter cell has variation. Do I want perfect replicas of me? No, the world can't handle that. But I do have kids. And they have variation for me that gives them amazing strengths and different challenges. amazing strengths and different challenges. And that's part of what keeps these species as a whole. Coffee, right? Coffee, right? Just from an evolutionary perspective. I don't Okay. So because we know now that the genetic material has these criteria and that it is, in fact, genetic information, it is, in fact, genetic material, heritable, variation, all of those wonderful things, the structure becomes important. As someone who is trained in both genetics, biology, and biochemistry, I'm going to try to approach DNA structure a little bit with the biological significance impact. bit with the biological significance impact. And so in terms of structure, we all know ATCG, right? right? We know the sugar backbone. Deoxyribonucleic acid uses a ribo sugar that has an oxygen removed. This matters in terms of how these bases will stack. That second hydroxyl group matters. Removing a hydroxyl removes a component of reactivity. Hydroxyl groups are reactive. They interact with water. They interact with other things. So this gives us an increase in stability associated with this sugar. In most of our functional DNA, it's going to be double-stranded. There are single- stranded versions, but for humans, the predominant amount of time that our genetic information is organized, it is as double-stranded DNA. DNA. DNA has directionality. Those sugars have carbons, and those carbons are numbered according to a chemistry perspective. The three-prime carbon has a hydroxyl group. The five-prime carbon has a phosphate group, and it is the connection of that phosphate to the hydroxyl that confers the actual sequence. And so depending on whether you see the phosphate or the hydroxyl, and I realize that looks like the hydroxyl there, depends on which end you're looking at. If you see the phosphate, you're five-prime. If you see the hydroxyl, you're three-prime. Does that make sense? This matters because part of the reason the DNA is so stable is a combination of that changing sugar, the stacking of those sugars as a result, the aromatic base stacking. Van der Waals forces and double-stranded DNA particularly relies on hydrogen bonding. When we say stable, what do you think we mean? I'm sorry, say it again. Not easily broken down. When it comes to a sequence of bases, what are we talking about when we say not broken down? They're stuck together by their sugars, right? They're stuck together by their sugars, right? We're not breaking that bond easily. It's a covalent bond. It's a covalent bond. Between that hydroxyl and that phosphate is a covalent bond. Covalent bond, strong, weak, generally strong, right? It's going to take energy to break that. We're going to have to use enzymes or damaging agents to break that backbone. But then I said we exist as what? For our genetic material, it has to be double-stranded, right? it has to be double-stranded, right? Having those hydrogen bonds collectively are incredibly strong. And so unzipping that, opening that, or separating those two strands takes a higher melting temperature when we have more hydrogen bonds. So we're talking about literal stability, literal resistance to heat. I am harder to so DNA is an incredibly stable molecule. Incredibly. One of the best things about working with DNA is you can just leave it on the bench. I can have it in an aqueous environment. I can have it dry. I can ship it. It's fine. I can't stay like that for days. RNA, not so much. Protein, definitely not. And so the fact that it's so stable really does help reinforce that this makes sense as genetic material. Why do I want stability in genetic material? Do I want you to become hypermutable and have a bunch of changes exist in your lifetime? That sounds bad. Sounds like a direct route to cancer. And it is. Did not mean to sound that cheerful about that. DNA has to be stable to keep the system functioning. But we do want it to be capable of variation. And so we've built some of that into the system as well. DNA has to have the ability for change, but controlled regulated change. A bacteria needs a higher rate of change than you do. Because the bacteria's environment, its life cycle is shorter, more variable. its life cycle is shorter, more variable. So in order to respond to those changes in environment, it needs to have the ability to change its DNA on a much higher scale. And so we find that these variation, the ability to withstand variation, is very much dependent on need. We don't need a lot of variation. If it's cold, what do we do? Put on a sweater, right? Put on a sweater, right? Go inside. Our adaptability is functional adaptability, provided you have the means to do those things. We don't have to fundamentally change our body temperature to the same degree to survive a sudden change in cold. But a bacteria does, doesn't it? It has to be able to withstand rapid changes in temperature. And it can do that very quickly because for some, the replication is going to happen in every 30 minutes. Right? Vastly different. And so we find that this will be linked to replication error. And we'll talk about that later. The ability to change gives us the ability to do more. All right. Back to that zipped up double-strated DNA, the classics you're already familiar with, A to T, C to G, A and T make two hydrogen bonds, C and G make three. So tying this to stability, how would these two things impact stability? More GC equals more stable because it's going to raise that melting temperature slightly. This is the classic char graph based Rosalind Franklin proven Monson-Crick base pairing. They can't come up with this without char graph or Franklin. Just throwing that out there. They took the data of two other individuals to create this. Classic pairing is based on nitrogens, oxygens, and hydrogens. The hydrogen bond will exist between the two complementary bases. You'll see that nitrogens and oxygens form the basis of where those hydrogens are associating. Does that make sense? We have more than one way that these bases can arrange. The B form of DNA is the one that you have classically heard of. It's got major and minor grooves. It's the predominant form in which human DNA will be maintained. It is the majority of natural DNA. We are going to have two strands that are slightly offset from a helical center, giving you this twisting ladder-like appearance that I can never draw. that I can never draw. Is anybody really good at drawing this? drawing this? I can never seem to get it to twist evenly. But it's great that your DNA actually does twist evenly because those major and minor grooves matter for gene expression. Having the ability to have access points for machinery is critical. We can take this lovely, stable form of DNA where we have the capacity for binding versus areas where binding is a little bit blocked and transform it at changes in humidity or ionization to give us a different structure. We already know we have repetitive DNA, yes? Well, it turns out that some types of repetitive DNA can actually shift the way our DNA is arranged on a three- dimensional scale. And we have what is called A-form DNA. This will occur with a lack of nucleosomes. If we think back to what we had in terms of chromosome structure, we know that nucleosomes are part of that higher order. I can't get nucleosomes into this. I can't wrap this around nucleosomes. I am not going to have the same ability to organize this. And so this consequence of high humidity also results in exposed regions of DNA. So this is sometimes correlated with environmental changes in other organisms with potential for damage or inability to properly replicate. Does that make sense? There is also Z or Z-form, which has a different arrangement where we have different stretches. And this is essentially a 180 flip. And this is essentially a 180 flip. And it is going to give us a different form of the DNA, and it is going to be the result of high ionization. And so now we can sort of tie this back to our mitochondria as it stops to function properly. We have massive changes in ionization and osmolarity, right? Lots of material flowing in, lots of material flowing out. Mitochondria, does it have genomes? does it have genomes? And so we can see an impact on the genetic material of an already damaged mitochondria existing as a result of that change in ionization. Rapid cellular changes literally impacting DNA and the shape and structure of DNA. This can also impact chromatin structure and methylation states. So there is more than one type of DNA out there, even in the double-stranding. Then we have our single-stranded DNA. Why do we bother with that? Well, because single-stranded DNA viruses exist. Single-stranded DNA is still pretty stable. Not as stable as double-stranded. Bases want a hydrogen bond. So when they can't with another strand, they can with themselves. And so what you form is structures of folding across the sequence. High-wheel repetitive sequences give you hairpins, loops. We get hydrogen bonding together. But because we're single-stranded, we're actually considered hypermutable because mutation is a change in the sequence. We are affecting the bases. Mutation by definition changes bases of DNA, whether that's inappropriately shifting a particular guanine so that it takes on a different form, you know, removing or moving a hydroxyl group is what we do to create the enol form of guanine. There's more on that later. That type of change is a lot easier to do when that material is just a single strand. So single-stranded material is considered hypermutable. It is considered more sensitive to change. When it comes to single-stranded DNA viruses, you will see that they actually have a very high rate of mutation compared to double-stranded viruses, compared to other double-stranded DNA. It's an important aspect of that viral evolution. And then you get two RNA, goes up even higher for double-stranded, single-stranded RNA viruses because each of the molecular processes introduces a place for error. And the number one source of error, which you will get really tired of me saying, is replication. That is where the bulk of actual mutation comes from. Replication error. DNA is organized. We talked about this with chromosomes. At no time is your DNA ever in disarray. Even when you are chopping it up to kill that cell, it's organized. You are carefully using Nuclease activity to undergo apoptosis. Even when you're undergoing necrosis, the genetic material is organized. It's just not controlled well. Does that make sense? We have talked about Ploidy a lot. We have talked about Ploidy a lot. Ploidy is that chromosome complement. What are we? Deployed, right? Deployed, right? What are our gametes? Meaning they have one full chromosome complement set. C-value is a little bit different. C-value refers to the actual sequences of DNA, the actual genetic material. the actual genetic material. When we are dividing genetic material in meiosis, that first step separates homologous chromosomes, right? So at the end of meiosis one, we have two sets of material that has only one of each chromosome, chromosome one, chromosome one, right? right? But it really has two sister chromatids of that. that. So while we are technically at a point where we have only one chromosome complement, because those sister chromatids are stuck together and therefore by definition a chromosome, we have a C-value of two. Because we have twice the genetic material we need in that, but it's still just one complement. And so C- value is really about what genetic material is actually present. There is a reason, I didn't include that when we were doing meiosis, because I don't think it necessarily informs things. Because what matters is do you have the right gene dosage? the right gene dosage? And we were focused on chromosome as a gene dosage element. But when you talk about genetic material and you look at this from a scale of multiple organisms, you have to think about C-value a little bit because we have what is called the C-value paradox. Organismal complexity does not necessarily correlate to genetic material in that the amount of genetic material does not make you more complex. Here's the problem. Here's the problem. How do you define complexity? Isn't there an observational bias there? What I think is complex, right? What I think is complex, right? That looks pretty complex. complex. It might not be what you think is complex. But more importantly, what are you using to define complexity? But more importantly, what are you using to define complexity? In this case, well, fish. well, fish. What does a fish have? Stuff, right? Does it have bones? Does fish have bones? Does fish have bones? Has gills, right? Fins, stuff like that. Nobody is going to join me on my description of a random genetic fish. It only has 400 megabases of DNA. People have 3,000. Here's my favorite. Oh, this guy. That was pretty simple. No, he has bones. He has all kinds of stuff. It's actually quite complex. But my definition of simplicity might cause me to say, well, that's a simple organism. And yet, 90,000 megabases. That's the C-value paradox. Organismal complexity does not necessarily correlate with the amount of genetic material. But we do have to recognize observational bias of what complexity is. I think corn is pretty complex. It can do end-over-duplication. It can have complex triploidy during the formation of pollen, of its gametes. That's incredible. So it makes sense that it might need more genetic material to pull those things off. But if you just look at corn, it looks kind of basic, food- wise. Food-wise, it's not. It's kind of a side dish, right? It's an ingredient. So just remembering that C- value refers to the total DNA content of the haploid version and that the paradox's complexity is not really going to play a role in how much genetic information that you have, even if complexity is a bit of an odd phrasing. All right. So let's do our quick summary questions at the start of next time. So I'm going to skip ahead a bunch. Close your eyes. And we can move on to replication. So this is what will happen. We'll kind of – I don't want to do questions same day because that's what we decided to move them, but I'm keeping them in the same spots. Are we ready for replication? Number one source of error. We have some set objectives, including focusing on the actual processes. And here is why I am doing this. There are basics to replication. Just like DNA had rules for it to be the genetic material, we've got some basic concepts. I'm going to start off with the way that I will handle all processes for the rest of the block. Initiation, elongation, termination. You have to have a start. You have to maintain the process. process. And you have to stop the process. Replication should stop when? When the whole thing is copied, right? thing is copied, right? When it's all finished. Transcription stop is going to be different. Translation stop is going to be different. And we're going to find that many of the complexities for initiation are relayed throughout. Stopping is hard. Starting is hard. There is an aspect to all three of those stages. In order to replicate DNA, you have to access it. DNA replication is a semi-conservative process. I am taking a pre-existing double-strand. I am separating it. And I am replicating that material so that one old strand is connected to one new strand. There are benefits to doing that. Imagine a fully conservative process. What would that look like? First off, what would that mean to be fully conservative instead of semi-conservative? I'd have to have the old strands back together. I'd have to completely retain the original molecule and have an entirely new molecule. Given the methodology of replication, that would involve synthesis, unzipping, and putting back together the original strands. Let's pretend that that's not how polymerases work. Even if it was fully conservative, that would mean that as I reseal the old material, as I also try to seal new material, that feels like a lot of work. And it also would prevent me from being able to find the errors I put in there because I wouldn't have anything to go off of. The fact that an original strand exists helps us identify when our polymerase went bad. It's a really cute creep there. Words. It's a really critical part, that came out creep for some odd reason, really critical part of replication, of conferring consistent heritable information. consistent heritable information. Bi-directionality helps ensure that we do this quickly, that we can actually get the whole thing copied. Do you have a small genome or a big genome? I mean, compared to that snake, we have tiny, but it's pretty big, right? but it's pretty big, right? And yet replication occurs fast, slow. fast, slow. I've lost you somewhere along this lecture. Fast, right? You go through mitosis pretty quick. That's why if you want to stop it, you have to treat chemically to get things to stop so you can observe it, right? stop so you can observe it, right? That bi-directionality helps ensure speed. General steps. DNA double-stranded, right? What do we call that? Super stable, right? So the first step to replicating DNA is opening it. The next step is stabilizing that open DNA. I can't keep moving a helicase and having single-stranded material. Single-stranded DNA is hypermutable, yes? Do I want that as I'm trying to make my genetic material? So I've got to get that sealed or at least stabilized. From there, I've got to copy it. All synthesis relies on the three-prime hydroxyl. Without the three-prime hydroxyl, I cannot catalyze the covalent bond. That is why all synthesis takes place five-prime to three-prime. I am taking that three-prime hydroxyl and I am bringing in a five-prime phosphate, five to three. When I look down this strand, I see the five-prime end. But that means my complementary strand is the opposite. So polymerases move three-prime to five-prime so that they can always synthesize five to three. When it comes to this, this is going to give me a difference between lagging and leading strand, which we're going to get to. But DNA polymerase can't even get there unless the material is in the box. To replicate it, I need it to be single-stranded. I need to be open. But I can't land my polymerase unless it's double-stranded. land my polymerase unless it's double-stranded. What do I do? Well, it turns out it's not double-stranded DNA but a hybrid of DNA and RNA. I lay down a primer. I lay down a landing pad for polymerase to come and happen. I know a lot of this isn't new, but I'm still going to teach it with this gives us a little bit of a break from the new because we're about to get to some new. All right, who wants to draw? All right, who wants to draw? I do. Basic replication fork. What are we used to seeing? Right. Okay. Let's get away from this. Yes, I waited till you drew it. Go with this because this is a bi-directional fork. We are going to be having replication go this way and this way. this way and this way. What are the basics? What are those? Yep, that's the other case. What's going to be right here? What's going to be right here? What do I need to do to a material that I've opened up? I need to stabilize it. Those are single-strand binding proteins. This line actually matters because one strand is going to be one direction. Excuse me. One strand is going to be one direction. The other strand is going to be a different direction. That is going to influence leading and lagging strand synthesis. Heli-case movement determines our direction of replication. Heli-case is unzipping the material. When I have double-stranded material, nicely looped it gets tight, creates a knot. To relieve that, I need the ability to relieve pressure upstream. That is the activity of topoisomerase. So out here, way upstream, we have topos. And there are two of those, topo one and topo two, which we'll get into. I started to mention this part because we need to think of it like this within our replication fork. That which is going the same direction, the polymerase that is going the same way as Heli-case is going to be able to do that smoothly. They're going to be able to just hop on and go. But to get there, they need primers. And those primers, that little bit of RNA material, is laid by primase. In eukaryotic systems, that primase is going to be associated with Paul alpha, and we'll get into those different polymerases. But we're just drawing a basic replication fork here, and we've got a few more minutes to do that. If I am moving the same direction as my replication fork, I'm good. What is my direction of synthesis? Five to three. So I can move easily three to five. And so these two are going to be leading strand. These two are going to be lagging strand as they try to synthesize properly, but can't go the same direction as the fork. Because they can move three to five so that synthesis is always five to three. Once I have my sequence, which I'm now going to try to change color for. Come on. It's not going to let me. It wants to be red. Did you change the blue? Did I change it? Nope. It just wants to be red today. Super helpful for... Ah, there we go. So once I'm making my new strand, right, and I'm making my little bits of new strand all with the corresponding, all with the corresponding primers, I've got to get rid of those primers because DNA is not composed of a mixture of DNA and RNA. That's going to be a system, but I got to seal this backbone. What seals my backbone? DNA ligase. That's a basic bi-directional replication fork. fork. And we will pick up right there after the many questions to talk about the eukaryotic replication fork, or the prokaryotic. That's prokaryotic. That makes sense? That makes sense? That's a basic fork. That is my level of expectation for this, being able to do a basic replication fork. Yeah. I didn't specify. They're very, very similar. We're going to talk about which polymerases go together, like which ones have similar activity. I said Paul Alpha, which is eukaryotic. For prokaryotic, it's going to be Paul 1 and Paul 3. Makes sense? All right, questions or concerns? Thank you so much. I appreciate your time.