Optical tweezers and fluorescence imaging show human Topoisomerase IIIα–RMI1–RMI2 (TRR) processively relaxes highly negatively supercoiled DNA faster than PICH loops.
0:00Welcome to Base by Base, the paper cast that brings genomics to you wherever you are. Thanks for listening, and don't forget to follow and rate us in your podcast app. Today involves a journey into, um, the absolute mechanical limits of life.
0:13We're zooming in on a process that is violent, incredibly fast, and operates on a razor's edge between success and catastrophe. like the sound of that. Yeah. And to set the scene, I want you and everyone listening to visualize the most high stakes moment in the life of a cell, the very end of the line, mitosis.
0:33The great divide. Right. You know, we usually see those textbook diagrams where chromosomes line up all politely in the middle, and then they just sort of drift apart to opposite sides, like they're doing a synchronized swim.
0:43But the reality is much more physical, isn't it? Oh much more. It's a biological tug of war. You have these massive forces pulling the genetic material to opposite poles. It is extremely physical. And while it might look clean from a distance or, you know, under a standard microscope, the separation is rarely perfect.
0:59Biology is messy. That is the understatement of the year. So what happens in that messiness? Well, as the 2 new daughter cells are trying to pull away from each other, they often remain tethered. There are these invisible threads connecting them.
1:13Invisible threads. We call them ultrafine and a phase bridges or UFBs. So essentially, the DNA hasn't fully untangled. Correct. Imagine you have 2 balls of yarn and you pull them apart violently. Sometimes a few strands just get knotted in the middle.
1:30These UFBs are those knots. They're threads of DNA that didn't get the memo to separate. And the stakes here. I mean, they aren't small. If the cell keeps pulling and that thread snaps. You're looking at genomic chaos, shattered chromosomes, this is how you get translocations, genomic instability, potential cancer, or just cell death.
1:49Or just immediate cell death. It's a complete disaster scenario. So the cell needs a bomb squad. It needs something to cut or untie that knot before the tension breaks the thread, but here's where the mystery has been for a long time.
2:00Mitosis is a sprint. It happens fast. Extremely fast. We're talking about a window of just seconds. So the question is, does the cell actually have a machine fast enough to untie a microscopic nod in that split 2nd before the bridge breaks?
2:16The math? For a long time, it just didn't seem to add up. That is the core tension. We knew who the crew was, but we didn't know if they had the speed to actually pull off the rescue. Which brings us to the source of today's deep dive.
2:28We're looking at a really impressive piece of work that finally sheds light on this high-speed rescue mission. We certainly are. And we really need to take a moment to celebrate the large collaborative team behind this.
2:39We're talking about Diane Spackman, Andreas S. Britcher, Anna H. Bizard, Andy Hickson, Irwin J.G. Peterman, E.J.S. J.L. White, and Graham A. King. That is a serious lineup. And looking at the affiliations.
2:52This is a heavy hitter collaboration. You've got Ria University, Amsterdam, the University of Copenhagen, and University College, London, all represented here. A truly international effort to solve a well, a microscopic problem.
3:05We're diving into their paper, titled Mechanistic Basis for Relaxation of DNA Super Coils, by Human to Poison Race, Alpha RMI1RMI2, which was published in PNAS on January 23, 2026. It's a dense title, but it tells you exactly who the stars of the show are.
3:21It does, but to Poison Race the 3rd, RMI1RMI2 is a bit of a mouthful for a casual conversation. It is, yeah. The paper abbreviates the complex as TRR. I think we should probably stick with that. TRR it is.
3:35So let's strip this down. Who are the characters in this drama? We have the knot, and we have the untanglers. Okay, so first, you have to understand the family of enzymes known as to poissom races. I like to think of them as the architects of DNA topology.
3:48Architect. Well, DNA is a double helix. It's coiled. If you try to pull it apart or access it for any reason. It just gets tangled and twisted to Poissom races, solve this by temporarily cutting the DNA to relieve that tension.
4:00Cutting the DNA sounds counterintuitive if you're trying to save it from snapping. It does, but it's a controlled demolition. Specifically, we're looking at type one a to poison races. They cut just one single strand of the DNA.
4:13Just one. Pass the other strand through that gap and then seal it right back up. Okay, so it's like that magic trick where solid rings pass through each other, they cut pass, seal, no harm done. Precisely.
4:24Now, TRR is a specific team of these magicians. The heavy lifter, the one holding the scissors, so to speak, is human toys Amarace the third, but it doesn't work alone. It's combined with 2 regulatory proteins called RMI1 and RMI2.
4:40Hence, TRRR. So TRR is the hero. But every hero needs a conflict. In this case, it's the not on the bridge. But that knot isn't just there by accident, is it? There's another character involved. There is, enter the partner, or maybe the instigator, depending on how you look at it.
4:55A protein called P-I-C-H, P-I-C-H. And what's PSCH doing on this invisible bridge? P-A-C-H is a translocates a motor protein. It patrols the bridge. Its job is essentially to bind to the DNA and stretch it out, trying to help resolve the connection, but in doing so, PICH creates these loops of DNA.
5:14And this is where the physics gets a little tricky. These loops are negatively super coiled. break that down for us. Okay, imagine you have a piece of rope that's twisted really tight. If you grab the middle and pull it apart to make a loop, all that twist has to go somewhere.
5:26PICH essentially underwinds the DNA in these loops. It twists it against the spiral. So if normal DNA is like a coiled phone cord. PICH is twisting it the wrong way to open it up. Exactly. These are negatively super coiled loops.
5:40And this is the scientific gap we're addressing. Right. The working model for years was that PICH creates these loops to expose the DNA, and then our hero, TRR, is supposed to come in and relax them. Relax the meaning, remove the twists.
5:55Yes, and this is crucial. If TRRR relaxes the negative coils, the basic mechanics of the DNA cause the remaining section to become positively super coiled. Okay, and why do we want positive super coils?
6:07Because a completely different enzyme, topotata alpha specifically needs those positive super coils to recognize where to make the final putt that separates the chromosomes for good. Ah, so it's an assembly line.
6:18PICH twists it open, TRR relaxes that twist, which winds up the rest of it just enough for Topo 2 to come in and say snip. That's the elegant choreography. But the doubt, the thing keeping researchers up at night was the speed.
6:31Right. We're in anaphase. The cell is splitting now. Exactly. PICH loops are fleeting. If TRR is too slow, the loop collapses before it's relaxed, the bridge isn't resolved, and you get that genomic chaos we talked about.
6:45The question was, is TRRR physically capable of moving fast enough to beat the clock? To answer that, you can't just look at a cell under a standard microscope. It's too small, too fast. You need to get hands on with the molecules.
6:58And this is where the paper's methodology becomes just beautiful engineering. They use something called dual trap optical tweezers. Optical tweezers is one of those terms that always sounds like it belongs in science fiction.
7:09It really does, but the concept is actually pretty straightforward. Imagine a microscopic rack. They take a single strand of DNA. They attach one end to a tiny bead held by a laser beam, and the other end to another bead held by another laser beam.
7:23So they have the DNA suspended between 2 tractor beams. Essentially, yes. And they used a technique called ODS optical DNA super coiling. By rotating one of the lasers, they can actually spin the bead.
7:36So they can wind up the DNA on demand, like winding a watch. Exactly. They can crank it to create those specific negative super coils, mimicking exactly what PICH does in the cell. But they didn't just pull and twist it.
7:49They needed to see the enzyme working. Right. So they combine the tweezers with fluorescence microscopy. They took the TRR complex and labeled it with MM Sherry, a red glowing tag. So they can physically see the protein landing on the DNA while they're twisting it with lasers.
8:05That's incredible. And to make it even more sophisticated, they did all this in a microfluidic flow cell. The authors described the setup almost like a car wash. A car wash. so? Well, imagine you have these extremely thin lanes of fluid flowing side by side.
8:20Because of the physics at that scale. It's called laminar flow. The liquids don't mix. They just flow parallel to each other. Okay, got it. So they can hold the DNA in a buffer lane, twist it up with the lasers, and then literally move the lasers to dip the DNA into the lane containing the protein.
8:37Like they just dip it? They dip it in, let the enzyme bite, and then move it back out to watch what happens. That is wild. So how do you measure relaxation? If I'm watching this movie, what am I looking for?
8:48You're looking at the length, the extension of the DNA strand. Because coiled DNA is shorter. Exactly. Think of that phone cord again. If it's all twisted up on itself, it's bunched up and short. As the enzyme relaxes those coils, the cord straightens out and gets longer.
9:03So by measuring how fast the DNA lengthens, they can calculate exactly how fast the enzyme is working. Yes. They calculate something called the linking number, or dollars, they can literally count how many twists the enzyme is removing per 2nd in real time.
9:18That is just incredible resolution. Okay. So they've built this microscopic torture rack. They've got the glowing enzymes, and they're cranking the handle. What did they find? Is TRR the speed demon we needed to be?
9:29It turns out TRR is an absolute powerhouse. Give me the stats. How fast are we talking? A single TRR complex? Just one can relax negative super coils at a rate of roughly $35 per second. 35 twists per second.
9:45It's a blur. Now, to be fair, if you compare it to his bacterial cousin, E. coli topo I, it's actually about 10 times slower. Bacteria are just speed demons in general because they have to replicate so fast, but for a human enzyme doing this specific, delicate job, it's very impressive.
10:03But speed isn't just about how fast you spin. It's about efficiency, right? Because they do one twist and then just fall off. And that was the other major finding. The data showed that TRR is highly percessive.
10:13Meaning it holds on. It holds on tight. It doesn't just snip, fix one twist and let go. That would be incredibly inefficient because it would have to find the spot and rebind every single time. Instead, it acts in bursts.
10:25Or a machine gun. Once it grabs the DNA, it performs 1000s of strand passages, 1000s of relaxation events without falling off. It just runs down the line. That explains the speed. It creates a stable gate and just pumps the DNA through it.
10:38Correct. And interestingly, they found this rate follows the Arhenius law. Arhenius law. You're bringing me back to high school chemistry. Refresh my memory. In this context, it just means the rate depends on the force.
10:52The tension on the DNA, the more you pull on the DNA with the tweezers, the harder it is for the enzyme to do its job. But the data showed that even under significant tension, TRR still manages these massive bursts.
11:03Okay, so it's fast and it works in long bursts. But DNA isn't a uniform string. Does TRRR care where it binds? It cares a lot. The fluorescence imaging, let them see exactly where those little red dots, the TRR complexes were landing, and they found a really strong preference for AT rich sequences.
11:22Add any thine mean regions. Why those specifically? Well, think about the bonds holding the DNA helix together. GC pairs, guanine cytocene. They have 3 hydrogen bonds. They're tight, difficult to pull apart.
11:32But AT pairs only have 2 hydrogen bonds. They're weaker. So they're the weak links in the chain. Exactly. When you negatively supercoil DNA, when you underwind it, like PICH does, it wants to relieve that stress by popping open, it melts.
11:47These melted spots or bubbles of single stranded DNA happen most easily at those 8 T rich sections. And TRR needs single stranded DNA to work. Because it has to cut one strand. Precisely. So TRR is a hunter.
12:02It hunts for these flimsy AT rich bubbles. It latches on there and creates a stable gate in that single strand to pass the other one through. So far, this is painting a perfect picture. It's fast. It's efficient.
12:13It hunts for the right spots. But then, I read the section about the sticky surprise, and this seemed to throw a wrench in the gears. This was the finding that really made the researchers pause. It's the plot twist of the paper.
12:25Tell us about the stickiness. So usually you expect an enzyme to do its job and leave. It finds the knot, unties it and moves on to the next problem. Right Job done. Clock out. But in these experiments, they found that even after the DNA was fully relaxed, meaning the job was completely finished, the linking number was back to normal.
12:42The TRR complex did not let go. It just sat there. It sat there. In the data, they show TRR staying bound to the DNA for over 30 minutes after the relaxation was finished. 30 minutes. Half an hour. In the cellular world, that's not just a long time.
12:58That's an eternity. Mitosis takes minutes, total. 30 minutes is like staying at a party 3 days after it ended. It is remarkably persistent, and it wasn't just sitting on double stranded DNA. It actually seemed to be stabilizing those bubbles.
13:12It was actively holding the DNA open. Wait, if it holds the DNA open, isn't that keeping the DNA damaged or at least vulnerable? That is the big question. Why would an enzyme that's supposed to fix DNA refuse to leave the scene of the crime?
13:26Here's where it gets really interesting for me. We had to reconcile this stickiness with the job it's supposed to do. So, let's go to the discussion. How do we connect these lab findings back to that tug of war on the anaphase bridge.
13:39Let's look at the numbers again. It really is a math problem. Remember PICH. The motor making the loops. Right. Previous research established that PICH extrudes a loop of DNA that only lasts for about one.
13:503 seconds. One.3 seconds. That is the window of opportunity. That's the deadline. Correct. If the loop collapses before the super coils are relaxed, you're in trouble. But now we know TRR relaxes at $35 per second.
14:02So in one. seconds, it can relax. What, about 45 twists? Roughly. And the average PICH loop only contains about 6 super coils. Oh. So TRRR isn't just fast enough. It's overkill. It's massive overkill. This confirms mechanically that TRR is more than capable of resolving those loops before PICH lets them go.
14:23The speed matches the biological requirement perfectly. It validates the entire model of how we think UFBs are resolved. So the hero arrives on time. But the hero doesn't leave. Let's go back to that conundrum.
14:36If TRR stays stuck for 30 minutes, but the cell divides in 5 minutes. Isn't that a problem? It would be, yes. If TRRR stayed stuck to the DNA indefinitely in a living cell. It could actually cause genomic instability itself.
14:49It would be a literal roadblock for other machinery trying to read or copy the DNA. So what's happening? Is the experiment wrong? Not wrong, but isolated. Remember, this is a single molecule experiment in a clean buffer, just the DNA and the enzyme.
15:03Ah, the vacuum of space approach, no other variables. In a real cell, it's a crowded dance floor. The expert speculate that the stickiness we see in the lab is likely regulated by something else in the cell.
15:14A bouncer. Exactly. A molecular bouncer. There are a few theories. One is facilitated dissociation. And basically, other proteins might come along and physically push it off. Or think about the mechanics again.
15:29We said TRR relaxes negative super coils, which helps generate positive super coiling elsewhere. Right. It's possible that the buildup of positive supercoiling acts as a kind of mechanical ejector seat, forcing the enzyme to detach, or maybe it's partner protein BLM changes the shape of the complex and makes it let go.
15:47So the stickiness shows us that TRR has a really high affinity for these bubbles. It really wants to be there, but the cell must have checks and balances to make sure it doesn't overstay its welcome. Precisely.
15:58And that's what makes the study so important. It isolates the intrinsic behavior of the enzyme. It tells us, this is what TRR wants to do. Now we can go looking for the factors that tell it to stop. And beyond just TRRR.
16:09This whole method seems like a game changer. Oh, absolutely. The combination of ODS, the twisting, and the fluorescence is incredibly powerful. They mentioned in the paper that this can now be used to decode other poison rises.
16:22We can watch how drugs interact with them, how mutations affect them. It opens a whole new window into DNA topology. It's like upgrading from a still photo to a high-def movie of the enzymes at work. That is a very fair analogy.
16:35And when you're dealing with things as fleeting and dynamic as Anaphase Bridges, You really need that movie. So let's wrap this up. What is the big take-home message for everyone listening? The takeaway is that TRR is a highly processive, specialized machine.
16:49It targets the weak points in our DNA, those AT rich bubbles, and relaxes them with incredible speed. It is perfectly tuned to keep up with the rapid fire mechanics of cell division, ensuring those invisible threads don't snap and cause chaos.
17:02It's the bomb squad that diffuses the explosive with .one seconds left on the clock every single time your cells divide. And it does it by holding on tight and not letting go until presumably something else tells it.
17:14The job is truly done. Which leads us to our provocative thought for the day. We know TRR is sticky. We know it refuses to lead the DNA on its own in the lab. So if you're a cell biologist listening to this.
17:26Who is the bouncer? That is the $10000 question. What is the molecular signal that breaks that incredibly strong bond once the DNA is safe? Is it a push, a twist, or some kind of chemical modification?
17:39If you figure it out, let us know. We'd love to do a deep dive on that paper, too. This episode was based on an open access article under the CCBY 4.0 license. You can find a direct link to the paper and the license in our episode description.
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