DNA Damage and Repair PDF

This one usually flows over as opposed to finishing early. Okay. Happy Monday. How are we doing? Thank you. Yes, I'm so glad to hear that. So good. It's excellent. All right. All right. So today we're going to continue with DNA damage and repair. But I want to start off with our post questions fr...

This one usually flows over as opposed to finishing early. Okay. Happy Monday. How are we doing? Thank you. Yes, I'm so glad to hear that. So good. It's excellent. All right. All right. So today we're going to continue with DNA damage and repair. But I want to start off with our post questions from last time. So how do leading and lagging strands differ in DNA replication? What could we rename these two in terms of synthesis? Continuous and discontinuous, right? Because DNA is arranged anti- parallel, we can only add bases 5' to 3'. What's the reason for that? 3' hydroxyl. We can move smoothly toward the replication fork on one strand, continuously synthesizing bases, or we're stuck in the position where we have to run away from the direction of movement and keep getting pulled back in the direction of movement. And so if we sort of think about both of the polymerase sets on bungee cords, when you're just dragging along and able to move along, you're going to get pulled along with that helicase just fine. But if you are having to keep running away, you're going to keep getting snapped off the one strand and have to reset yourself. That's that lagging strand, very much fragmented synthesis. Make sense? So I already covered that one. What about this next question? What about this next question? What do we expect for early firing? Okay, let's rephrase. Okay, let's rephrase. What does every origin get all at the same time? Ork, the pre-replication complex, right? The most important piece of that being the origin- recognition complex, which is going to find the origin, and then the next piece it absolutely must call in is that helicase. So how does an early firing differ then if they all get those pieces at the same time, what makes it different? The pre- initiation complex, right? That second half of licensing. The other way in which this matters is where the origins are located. Not every cell in the organism is going to have the same origins. And this will be tied directly to what material that cell needs versus the other cells. Highly transcriptionally active regions fire early because we need them. We need them back as quickly as possible. We do not want that ork disrupting that area. And the best way to think about it is literally the origin of replication complex. The origin of replication is building stuff like a wall right at the start of a gene. Or if it's a weight firing region somewhere randomly in the middle. That is a wall. It is something that the machinery of transcription cannot get past. So if we leave that wall in place for the duration of replication, we've got a problem. So early firing regions have a higher rate of activity because we want that ork out of the way. Make sense? Okay. Oops, sorry. And then this is the every single origin gets recognized at the same time. What makes it different is that second part of licensing. Part two, pre- initiation complex. They all get the pre-replication complex at the same time. But we only call in the machinery of replication right before we fire. And so if we don't call it in, we fire later. What do I have to do to double-stranded material that I don't necessarily have to do to single-stranded? Got to break it open. Got to unzip it. Most of our means of replicating single-strand DNA is going to involve either the rolling circle or the hairpin loop. I've got to actually create regions that I can then replicate. I don't need to open single-stranded material. Even when it's self-twisting, right, even when it's doing hybridization within and of itself, it's not the same unzipping that we have to do for regular DNA. It's slightly different. How are these polymerases different? It's a really open-ended question, isn't it? They have fewer. They have less specificity. We have specific activities that we need. If you were to pinpoint one difference between the genetic material of a highly mutable organism versus the genetic material of us, what would it be? Tolerance for mutation, right? I refer to it as a highly mutable organism on purpose. Because their tolerance for base changes is much higher than our tolerance for base changes. So everything we have is set up to ensure integrity of our material. Everything we're doing today is all about fixing that which goes wrong and explaining how things go wrong in the first place. And then what are topos for? Torsional strain, making it possible for helicase to unwind. All right. So this is where we left off, yes? In terms of mutation, we have some things we need to think about. When you hear the word mutation, think change in DNA base. That is the connection you need to make. There is a mutation. The DNA has been altered. That is what mutation means. Technically, we should never say there is a mutated protein. We have an altered protein, but the mutation was literally a change in DNA. Do all changes in DNA have functional consequences? Those of you who are shaking your head no are correct. Tell me why. Because we don't have junk DNA, right? Go for it. That's a great answer. So what he was getting at is the downstream changes in gene code, right? We haven't gotten to reminding you about translation yet, but you have a triplet codon that confers an amino acid. But there is more than one triplet codon conferring the same amino acid. So that's the biggest one, right? Not all change changes the protein. Not all change actually changes the sequence of amino acids. But even before we get there, not all parts of your DNA change encoding regions. You have so many areas that are not encoding a product. If you change a regulatory element, you change the potential for amount of a product, not necessarily what the product looks like. Amount still matters, but the functional consequence is slightly different. What if I change a single base of a centromere? It's a highly repetitive, highly protected region. One base? Do you really think we can't handle one base change there? Let's play the devil's advocate on that. What if you did have a major problem from a single change at a centromere? One base throws off the whole ability to build a centromere. I love the facial story that you're making with this. It's like, right, there would be a terrible situation for maintaining the integrity of that whole chromosome, every cellular replication. So some areas can handle that. The nature of being repetitive gives you a little bit of leeway. So not every mutation is going to be held equal. Some are going to be way worse than others. Germline mutations are worse, air quotes, from the perspective of what? Next generation, right? Inheritance, down months, downstream in a family. All mutation has the potential for a consequence, depending on where it is. But germline can affect the very essence of what DNA is for, right? What about somatic mutations? Those are going to be correlated more with individual or internal problems, right? The exception to some of this is when you start to affect things like DNA repair mechanisms. When you start to impact critical processes, you start to increase the consequences for these changes. Spontaneous is what we refer to to all these naturally occurring mutations. When you have certain biological processes taking place, you make products, yes? Are all products good for you? Lots of excess. I just want to eat that pile of sugar over there. Who goes is good for me? Don't eat a pile of it. That's not a good plan, right? I don't know why I'm pointing to sugar like it's right there and you can see it. But excess of certain metabolites can be just as toxic as bringing things from the outside. Some of our errors lead to toxic buildup. One of the ones that we'll talk about for an inborn error of metabolism is going to be phenylalanine dehydrogenase because that is going to result in a toxic buildup of phenylalanine, which leads to a condition. I'm not listing it yet because you're going to learn it later. My point is that we want to pay attention to spontaneous as well as induced. Indogenous as well as exogenous mutation. We expect there to be some potential for DNA damage from our biological processes. What is a part of the cell that we talked about as being particularly prone to damage? Okay, we talked about a lot. Sorry, I didn't realize. I'm trying to lead you toward the powerhouse energy part of the cell. Mitochondria, right? What do mitochondria have? Circular genomes. So already we're in a position of we have a small genome that is capable of mutation and we've put them in an organelle that in and of itself has biological processes that can lead to DNA damage. Literally reactive oxygen species. Breaking DNA, changing bases, changing oxygenation. These are all spontaneous. So we have parts of us that are super critical that are also really sensitive to damage. If that was your DNA in your nucleus, you would be in a completely worse off place. We need to be able to fix the damage we cause. Environmental or exogenous damage, induced damage, is exactly what it sounds like. Something external to you that comes in and affects your DNA. We can have combination effects. One thing that breaks something and then another change that fixes it. Because you're human and really none of your genes are operating in a vacuum, you can have mutations that fix problems where you knock something down and so this other damage compensated by ramping up something else. This mechanism of change can actually lead cancer cells to get really good at evading drugs. But the system is in there to have redundancy to some things, especially the more important that thing is. But compensatory, we can have these sorts of suppressor or second mutations be in the gene, right, where one mutation shut it off and the other mutation fixed it, the intra, or it can be a different gene. You are not in isolated vacuum. Your cells are talking to each other. They are engaging each other. And even within a cell, there's molecules having a conversation, right? We remember signaling, probably not fondly. No? No? Okay. Cannot say this enough. Why is the big thing here? Why is replication the number one source of error? And it's because no replication machinery is perfect. Each level of genome has an increasingly better replication machinery. We have among one of the most efficient, and it's really good at fixing its mistakes, because we are the most sensitive to change. We don't have a system that can rapidly deal with major genome changes. So we need to make sure that everything we do is as good as it can be. Our polymerases are really, really accurate. But it's still the number one source of problem, because bases can take on different forms. Bases don't always look and act the way that the polymerase thinks it should. We have a system where everything we have is in a biochemically active place. So some of the things that we do to control that is separate things into different compartments, to not build things until we need it, to break things down quickly when we're finished with it. But overall, these four bases are really similar to each other. TC and U all look really similar. N and G look really similar. Take a look at this first one here. This right here is the keto form of thiamine. A simple shift referred to as the tautomeric shift, just moving a proton to right there, changes the way it binds, changes the number of hydrogen bonds it can have. This guanine, same thing, moving a proton. What is essentially a proton? A little positive charge, right? Moving its location. I go from being able to do a classic A-T combination of two hydrogen bonds to now preferentially bonding differently. Does oxygen like hydrogens, like protons? Yeah, oxygen likes protons. Hydroxyl groups are really, really important biochemically. So this is something that can happen relatively easily. A little bit of a battle over the plus. We have imino forms for cytosine and amine as well. Where we are losing that hydrogen. In this case, for a cytosine, there are two nitrogens battling. That feels like a pretty even battle to me. It's not quite, but still. When I do that, what do I look like now? Look here versus here. Look how structurally similar that new cytosine is to the original thiamine. They're not going to be identical, otherwise they would be the other thing, right? But a lot more structural similarity chemically. So when you have these different forms and your polymerase is doing the job of, oh, I see T, that has two hydrogens. I need to call in A. You can see how if G looks a lot like A, it's going to put G in wrong. Especially if either the original thiamine or the new G has an altered structure. Does it make sense? That's what we see. What do we expect? A goes with, C goes with, but changed them to their rare forms. And we can get preferential bonding with a different pairing. If you change them all to their rare forms, we go back. But if you keep them with only one of them being their rare form, think of this as a pool where I have, or maybe a ball pit is a better example. I think a ball pit is a little bit more specific. I have a ball pit with four different color *****. Blue and purple are supposed to go together. Orange and yellow are supposed to go together. Every time I grab orange, I should also get a yellow. Every time I see orange, it's going to make me grab a yellow. Blue and purple. But then I accidentally coat blue so that it looks a little orange. Don't know how I would do that. It was terrible color choices. But if the blue resembles orange, it's going to be grabbed when you have yellow accidentally. I'm going to occasionally, even if that blue, that new rare orange is only in there, one out of a thousand, there is still a chance I'm going to grab it. And so the simple reality that these rare forms exist mean that even when your polymerase is really good at grabbing the right base, the right nucleotide, it's going to sometimes mess it up. Because all it's going to see is, oh, hey, that's the right shape. That's the right number of bombs. But it's not going to be correct, is it? So sometimes it can catch itself. Sometimes it can go, nope, that's not right. And it can pull that out. That is that exonuclease activity we're talking about. It can move backwards, cut out the mistake it made, break that sugar backbone bond, give us back the hydroxyl group, and bring in the right base. But sometimes it's going to fail at that. And this actually helps to explain why it would fail. Because this looks like this. So if you look across, they match, right? This is what's supposed to happen. But when you have that rare form of cytosine, this is what can happen. This is what's supposed to happen. But when you have that rare form of guanine, this is what can happen. Does that make sense? And if your job is to be like, oh, that's a hexagon. Oh, that's a hexagon with a pentagon. You're not necessarily going to grab the right hexagon if they look so similar. Make sense? Okay. Most of the errors are going to be the obvious one, you would think. Is it more probable that I'm going to grab the same shape or a totally different shape? So transition mutations, where you swap C and T, A and G, are the more common. Most of those are what we expect. Transversions, where you should have grabbed C, but instead you grab A or G, are much more rare. But they also can happen. Want to hit this? Our actual error rate is very, very small, but it still exists. And now let's put that into scale, actually, for a second. How many cells do you have? Like you, personally. A lot feels like a right answer to that question. Every single one of your cells that has the potential to be replicatively active, presuming a 24-hour cycle for division, because I just like to make things easy, means across the span of your life, how many moments do you have the potential for error? And that's just the cell. Now we go to bases the polymerase is laying down per cell per replication. Now we certainly see why replication is the number one source of error, because across our cells, across our lifespan, we're doing it a lot. We are actually replicating more than we realize. But because all of them have the potential for error, we have to pay attention to what those errors could be. Transition errors are this simple. T becomes C, C becomes T, A becomes G, G becomes A. Transversions will swap the shape. Instead of purine to purine, I'm going purine to pyrimidine or pyrimidine to purine. I am literally changing the shape of what I'm incorporating. Question. I am spilling water on myself. Glad that wasn't on camera, but I still called myself out. I added a few elements to these questions that I hope will be helpful. And one of the elements I added was to give you what this could look like as an exam question in that kind of more open-ended form. How would incorporation of a different base affect replication? Start with that kind of open-ended. If I catch it as an error, what am I going to do? I'm going to try to repair it if I can. But ultimately, this is going to be a mutation. Putting the wrong base in is mutation. If I put in the wrong form, I also have to think about not just this moment of replication, but the next one. If I have an A, C base pairing, is that correct? Which, okay, what if I don't catch it? That doesn't necessarily matter in that moment unless that pair doesn't let us have a functional protein. But the next round of replication, how does replication work? What's the first step? Opening. Semi-conservative. The strand with A gets treated differently than the strand with C. And so now I have a chance for a daughter cell to have a completely different base pair if I don't fix it. So it not only matters for the current situation and what the impact for that cell would be, but also the next one. Keep the shape. It's a transition. Change the shape. You get a different outcome. That's a transversion. An **** form of guanine will preferentially match thymine. So this question gives us that hint at what I was just talking about. If I make this change before I have the chance to fix it or I fail to fix it, half my cells will be different. I will literally transition the entire base pair to a new base pair. And I'm no longer going to see that as error because they're just going to be different. I'm not going to see a mismatched pair. I've unzipped. I've laid down the right bases. This A now has a T with it. This C now has a G with it. Those are right, aren't they? But are they right for the system? Could be a beneficial mutation. But what's the probability of that? Okay. Common mutations. Many of our mutations are going to be thought of for the consequence they have on the protein. When we talk about sense, we are talking about making a protein that makes sense, that does its job. We are talking about the bases in order to create the right sequence of amino acids. So if we have a miss sense, we've messed up the sense a little bit. We have changed one of those amino acids. Silent mutations are what we were talking about before. Where the change doesn't affect function because it literally doesn't change the amino acid. It was supposed to be purine. Did we have a mutation? Yes. Did it matter for the final product? No. Miss sense changes it. In this example, we go from valley, where we are supposed to be, to leucine. That is a different amino acid, yes? The consequence of that is going to depend on what that amino acid did. What if it was a critical tyrosine holding a phosphate? What are phosphates for? They are important for application, they are important for literally enzymatic function. Didn't we phosphorylate a bunch of stuff in signaling? What if I just got rid of all their tyrosines, all their serines and threonines? Oh, my gosh, it's like I asked these questions back in block one for a reason. Sorry, sarcasm. Apologies. What happens if I make that change? I lose that function. So even some of our miss sense mutations can have insanely important impacts because they take away regulation. Losing a tyrosine, losing a serine, losing a threonine, losing a protein can change the way you bend. Some of them are less important because they are less reactive, glycines are really small. But changing a glycine becomes really important if you need things to pack together tightly. And so we will see losing some of our small things also have an impact. And this is going to be specific to the protein we are talking about. So miss sense mutations can have profound impacts. Nonsense. I stop making sense. I go from what I should have had to a fraction of it to nothing. Now it makes no sense. It's nonsense. These can also be thought of as truncation mutations. I have shortened the material I should have gotten. And how we do this is by introducing a premature stop. I change a base and it changes the code. So again, this is a functional consequence of mutation from the perspective of the protein. Frame shift is all about the encoding portion shifting its frame. Three bases matter. If I put in anything that is outside that number or remove anything that is outside that number, I can shift the entire molecule. I can make it go from reading, as in this example, met, lysine, glycine to lucinality. But please note you do have to actually insert outside the triplet codon. So if I have got 1, 2, 3, 1, 2, 3, 1, 2, 3, and I insert 1, 2, 3 in between those 1, 2, 3s, I insert three bases between triple codon 1 and triple codon 2, I am not shifting the frame. I am just adding what? 1 amino acid, right? That could have profound impacts, but it is not going to change the whole reading frame. If, however, I have got 1, 2, 3 here and I put 3 in the middle of that, that could shift the whole frame. So it is very much a positional consequence as well. But most of these frame shifts are going to be the addition or removal of bases in 1, 2, and 4 bases. Make sense? What would be the loss of three bases within frame? What would that do? I lose these three bases, and that is it. What would that do? I just lose that amino acid from the final product, right? So that is, again, not a frame shift. Make sense? Okay. The reason I bring that up before hitting this slide is because we already know about things called tandem duplications and deletions, right? At the chromosome level, what caused them? Non-allelic recombination, right? Non-allelic exchange. I can lose and gain material through the process of chromosomal rearrangement. For replication, it is all about slippage. Either the strand you are using as a template or the strand you are actually synthesizing can slip. When might that occur? Why might a strand get slippery and you start to get a little confused as a polymerase? We have all had to write long numbers, right? We have all had to do complicated math. How easy is it to write 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, and have the right number of 1's? It is not always easy, is it? How many times do you think you maybe write the wrong number? The same thing is true for this. We have two principles that actually apply to this. The same base over and over and over again. You are not always going to know that you needed to put 8 A's and you may only put 7 or you may put 9. But then we also have this tendency to hybridize. What does DNA like to be, especially our DNA? Double-stranded. If things are complementary, what does it try to do? Hydrogen bond, right? A's like, C's like. So if we see a pattern where I can create a loop, I may do that. The repetitive nature of your DNA lends itself towards slippage and mistakes. If the slippage is the newly synthesized strand, I am going to get extra bases. If the slippage is the template strand, I am going to have fewer bases. And that little bubble that is sticking out may be recognized and fixed before the next round. But if it is not, then it is the next round of replication I could expand that even further or I could reduce it even further. And we do in fact have human disorders where trinucleotide repeats, the number of a particular base matters. Those highly repetitive regions get shorter and shorter or longer and longer and it causes a phenotype. This is replication error. Topoisomerase, important. What if it falls off of the DNA? It depends on the topo. We get either a single-strand break or a double-strand break. Base deamination, that tautomeric shift of a proton is one way, but we can literally remove amino groups changing the base. This is actually a really important process for RNA. And so this is an important process going awry. We're moving a base where we still have the sugar, but we lost the base. It's like a gap in our ladder when we have half a lung. The backbone looks fine, but there's definitely a problem there. That can happen. And then of course, many of our biological processes require oxygen. Oxygen loves its electrons and it can do oxidative damage. Reactive oxygen species are a problem for our system that we have to pay attention to, and they can cause endogenous damage when they were produced from your system. And then we also have outside sources of damage, radiation being the one we're probably the most familiar with, right? UV light damage. Why do we wear sunblock? Because UV light directly damages DNA. It produces something called thymine dimers. Do you want your bases across two strands to bond to each other covalently? Do you want two bases in the same strand to bond to each other covalently? You want the sugar to, not the base. These are forms of damage. I can't unzip a covalent bond. If I am supposed to be matching this chair and very lightly associating, that's okay. I can open that. But if I suddenly hold onto this for dear life, like, no, it's not going anywhere, I'm not going to be able to get past that. That's a problem. Those are called interstrand cross-links. We have lots of things that cause damage. Teratogens are damage-causing agents that affect the fetus. Everything that you can imagine has the potential to damage your DNA. Some of the main ones that we want to talk about are toxins, but I can't possibly tell you every effect of every toxin. So we're going to focus instead on some more specific things, the aromatic amines that can lead to base substitution and frame shifts, environmental stress, but we're going to kind of focus on it this way. The main categories of damage are going to be having bases form covalent bonds they shouldn't, just inappropriately putting bases in, breaks, and bulky lesions. Things being attached to DNA that should not be attached to DNA. But this is what makes all of this okay. That bottom third is what makes all of these exposures, all of these processes that cause problems, still okay, because we can fix it. Our polymerase is really good at fixing things, but if they mess up and they don't catch it, we've got a bunch of alternatives too, because your entire system is designed to maintain its integrity. So we will come back to these next time. So we will come back to these next time. Close your eyes. Now let's get to that last part. Let's fix the damage we just made. We had lots of damage, right? Now let's fix it. All repair starts with finding the damage. What if I don't see the damage? Can I do anything about it? The first step in any type of repair is identify the damage. So if all types of damage are a little bit different, and we have multiple repair mechanisms, what does this mean? Where are we going to have the greatest degree of diversity in these different repair mechanisms? Okay, let me ask it this way. What does it mean to remove damage? If you've got, I'm trying to think, you've got a broken brick in a wall or a broken tile. What's the first thing you're going to do? See it? Oh, that's broken. Okay? What's the next part? Remove the tile probably, right? What helps you remove bases? What do you have to do to remove a base? I'm going to have to break the backbone, right? I'm going to have to have an exonuclease activity or an endonuclease activity of some kind. So that's kind of universal. What brings in new bases? Polymer bases. I'm going to use somewhat different polymer bases, but the basic action of reading a template and putting in new bases, that's pretty universal, right? What about sealing the backbone? What does that? Every single one of these is going to have that, right? So what's the only place left for there to be any sort of real difference? Recognition. All of the molecules that see damage are going to be very specific to your repair types. The general idea of pull the base out, you've got to, how you cut it, how big you cut it, that's going to be a little bit different, but recognition is going to be the thing that differs the most. The process of fixing is really straightforward. See it, remove it, replace it, seal it. This is our list. These are the repair mechanisms we're going to talk about. Direct repair is exactly what it sounds like. I'm going to directly fix a base without removing it. I'm going to change the DNA back. This obviously only applies to a base that has been altered in the strand. I can't fix something a polymerase puts in wrong. I can't make a C a T, but a C that has a problem with it. It's a thymine that's been covalently bound to another thymine. These are things I might be able to directly repair, except not us. We're not good at that. If you were looking for a shorthand of what to do with this, it's right here. It's your full list of every compare and contrast that I've got for this. It's one whole objective in one slide. What does it see is the second column. What are some additional features to distinguish it? Then what is the disorder that it's associated with? The last column is something we will spend more time with in block four. Each of these repair mechanisms repairs a different type of lesion because of the molecules of recognition. Direct repair, fix the base in place. Mess with the DNA as is. Don't cut anything out. Just make it go back to being okay. We can't really do that. Plants can. Plants can. Why might that be important? What is something a plant needs to be able to do that also has a detrimental impact on you? Photosynthesis. Why would I say photosynthesis, which is obviously a major part of our overall biosphere, be something that has a negative effect on you? Because what does the sun do to you? UV damage. Here we have an organism who needs the sun, but the sun can damage DNA. What if a plant couldn't deal with that? What would happen very quickly with too much sun? Probably what happens to you. We're going to turn bright red. I don't know. But it makes sense that some of these plants, bacteria, fungi can respond to UV damage differently than we do because it matters more to their system. We don't need direct repair when we can just cut that repair out. But it's important that we recognize that direct repair is possible because then it lets us actually say, okay, in what situations would we be capable of that? And why wouldn't we be capable of that? And in this case, we are actually going to use some of our transferase activity, some of our base-changing activity, to control and alter molecules downstream. And we'll see that again when we come back to RNA. This is our first one, base excision repair. What does it sound like? Just the title. We're going to cut out a base. Why would we cut out a base? It's a bad base. It's bad. Something happened to it. It was the wrong base, but it's bad. Yes. We see it. We say that shouldn't be there. Base excision and nucleotide excision repair are shockingly similar. Okay? So how you differentiate them is going to be based in what it is that it's recognizing. Base excision repair is not going to mess with anything that disrupts the helix. It can only deal with a single base. It's tiny repair. It's going to cut out a few bases sometimes or just a single base, but it cannot deal if the helix has been bent or twisted. If the DNA just doesn't look right on a bigger scale, it's like that's not my job because it can only deal with that tiny base. What is a nucleotide? Base, sugar, phosphate. Nucleotide excision repair is going to deal with large distorting errors. Base excision repair is going to focus on the base. There's no base. We're going to have a step where we cut the base. What do you see about the sugar backbone there? It's intact. So in order to replace a nucleotide now that we've removed a base, what do I got to do? I got to break the sugar. And we are out of time speaking of breaking the sugar. So we will pick up with base excision repair, compare it to nucleotide excision repair, and move forward for the next stuff. The next packet we'll probably be getting into. This is always the section that takes me a couple of days because it's a lot. But the next packet we'll move straight into after that so make sure you've downloaded the RNA stuff after that. We may take our time with this. We may have more. Questions or concerns? I feel like we kind of ended a little lower energy, and that's okay. This is a lot, but if you're starting from that chart, that's going to be everything that we really focus on. What you can do to this chart right here is add a couple of the specific pieces that we're going to talk about. For example, with this one, we're going to talk about glycosylase. So just sort of giving us some of those specifics. Does that make sense? Thank you so much. Happy Monday.