Episode Transcript
[00:00:00] Speaker A: Foreign.
[00:00:14] Speaker B: Welcome to Base by Bass, the papercast that brings genomics to you wherever you are. Thanks for listening and don't forget to follow and rate us in your podcast app.
[00:00:22] Speaker C: It's fantastic to be back. We have a real biochemical detective story on our hands today.
[00:00:28] Speaker B: We really do. And to kick things off, I want to step away from the lab bench for a moment. I want you to imagine you are running a.
A high end, very specialized auto repair shop.
[00:00:40] Speaker C: Okay?
[00:00:41] Speaker B: You have a strict division of labor in this garage. You have one mechanic, he only touches engines. You have another, she only touches tires.
[00:00:49] Speaker C: Got it. Efficient.
The classic assembly line model. Everyone stays in their lane.
[00:00:54] Speaker B: Exactly. But one day you decide to run a sort of chaotic experiment. You fire the engine guy, and just to be safe, you fire the tire guy, too. You remove the specialist entirely.
[00:01:04] Speaker C: I assume the whole shop just grinds to a halt, cars start piling up.
[00:01:07] Speaker B: That's exactly what you would expect. But strangely, no. The cars keep rolling out of the garage, fully repaired, the engines are purring, tires are balanced. Business as usual, that is.
[00:01:18] Speaker C: Well, it's unsettling. It implies someone else is doing the work when you aren't looking.
[00:01:23] Speaker B: Right? If the mechanics are gone, who is turning the wrench?
For decades, this was the exact paradox facing scientists who study DNA mismatch repair.
[00:01:32] Speaker C: We thought we had the clue list.
[00:01:33] Speaker B: We thought we knew exactly which enzymes were the mechanics responsible for, you know, cutting out errors in our DNA.
[00:01:39] Speaker C: And when those enzymes were removed in experiments, the system, it just didn't crash.
[00:01:44] Speaker B: It didn't crash. It barely even slowed down.
So today we're doing a deep dive into the hidden hands of DNA repair. We are going to find out how the character we thought was just the.
[00:01:54] Speaker C: Manager, everyone just shouting orders, might actually.
[00:01:57] Speaker B: Be the one rolling up its sleeves and doing the dirty work itself.
[00:01:59] Speaker C: This is a major, major shift in how we understand genomic stability.
We are looking at the article titled DNA mismatch repair mediated by MLH1PMS1 endonucles catalyzed misbehre excision.
[00:02:13] Speaker B: A bit of a mouthful, but we'll break it down base by base.
[00:02:15] Speaker C: We will. And before we get into the weeds, we should recognize the team behind this. This is a massive effort. The research is led by Tatiana Palacio and Felipe A. Khalil, along with Richard D. Collodner and their colleagues.
[00:02:28] Speaker B: And this is all coming out of ucsd, the University of California San Diego.
[00:02:32] Speaker C: School of Medicine, a real powerhouse in this field. They are really pushing the boundaries of what we know about, you know, DNA maintenance.
[00:02:39] Speaker B: So let's start with the basics. We mentioned DNA mismatch repair or mmr. I usually describe this to friends as the spell checker for our genetic code. Is that.
Am I oversimplifying?
[00:02:49] Speaker C: No, that is the perfect analogy. Think about it. When your DNA replicates, it's copying billions of letters. A with T, C with G. But the copier, the polymerase, it's fast and it makes mistakes.
[00:03:01] Speaker B: Like typing a text message too quickly.
[00:03:03] Speaker C: Exactly. Maybe it puts a C where an A should be. That's the mismatch. Right. And if that tipo stays there, it becomes a permanent mutation the next time the cell divides.
And mutations, well, they can drive cancer, genetic diseases. So MMR is the system that scans the fresh DNA, spots that typo, and fixes it before the ink is dry.
[00:03:23] Speaker B: Okay, so that's the goal. Fix the typo. Now, walk me through the old worldview. Before this new paper, how did we think the spell checker worked?
[00:03:31] Speaker C: The textbook model, the one I learned, the one still in many textbooks, is a relay race. Okay, first you have the recognition team. These are proteins called MS.2 and MH6. They are the proofreaders. They slide along the DNA and stop when they feel a bump, a distortion caused by the mismatch.
[00:03:47] Speaker B: So proofreaders find the error, they flag it. Then what?
[00:03:50] Speaker C: Then they recruit the scissors. In the classical view, these are specialized enzymes called exonucleases.
The big celebrity here is one called EXO1. EXO1. EXO1 dot its job is to bind to the DNA and just chop out a whole section of the strand with the error. It unzips it, basically.
[00:04:07] Speaker B: And once that bad chunk is gone.
[00:04:08] Speaker C: Polymerase comes in and fills the gap with the right letters. Find cut paste.
[00:04:13] Speaker B: So find cut paste and x01 does the cutting.
[00:04:18] Speaker C: That was the dogma. X01 is a primary cutter. There's a backup called Rad27, but x01 is the star.
[00:04:23] Speaker B: But this is where the story gets weird, where that auto shop analogy comes back. You said the system didn't crash when they removed these tools. How strong is that evidence?
[00:04:31] Speaker C: Oh, it's overwhelmingly strong.
And baffling. Years ago, researchers deleted XO1 in yeast cells. They expected the mutation rate to just.
[00:04:41] Speaker B: Skyrocket because the repair system should be broken.
[00:04:43] Speaker C: It should be, but it didn't. They saw less than a 1% defect compared to a totally broken system.
[00:04:50] Speaker B: Wait, wait. 1%? So 99% of the repairs were still happening without the main pair scissors.
[00:04:55] Speaker C: Correct. And even when they got rid of both, X01. And the backup, Red 27. Most of the repair was still happening.
[00:05:01] Speaker B: That's wild. That's like removing the engine from a car and it still does. Zero to 60 in four seconds.
[00:05:06] Speaker C: Exactly. The field knew there was this. This dark matter in DNA repair. They called it the X01 and RAD27 independent pathway. We knew that it happened, but we had no idea how.
[00:05:16] Speaker B: Who was cutting the DNA?
[00:05:18] Speaker C: Who was cutting the DNA?
[00:05:19] Speaker B: And that brings us to this paper. They went hunting for that ghost mechanic. How do you even begin to look for something like that? You can't just stare at cells under a microscope.
[00:05:28] Speaker C: No, Living cells are way too messy. Thousands of proteins all buzzing around. If you want to find the hidden mechanic, you have to build the garage from scratch.
[00:05:36] Speaker B: Reconstitution.
[00:05:37] Speaker C: Yes. They used a reconstituted system. They purified the individual proteins, MHO, MLH1, all the parts, and mixed them in a test tube.
[00:05:46] Speaker B: So they built a biological engine on a workbench?
[00:05:49] Speaker C: Pretty much. They took circular loops of DNA and engineered them to have a specific typo, like a CC misbehav. And they also put a little nick in the DNA nearby to simulate a replicating strand.
[00:06:01] Speaker B: So they have the broken DNA, they have the repair crew, but they left out X01 and RAD27.
[00:06:06] Speaker C: Deliberately. They withheld the known scissors. They wanted to see if the remaining proteins could fix the error on their own. And they had their eyes on one specific suspect, a complex called MLH1PMS1.
[00:06:17] Speaker B: Now, I've heard of MLH1, PMS1 in the old model. Isn't this just the matchmaker, the middleman?
[00:06:23] Speaker C: That was the assumption, yeah.
We thought it just coordinated things. It would come in, maybe it make a tiny nick to mark the spot, and then call X01 to do the heavy lifting.
[00:06:33] Speaker B: But the researchers suspected the middleman might be hiding a weapon.
[00:06:37] Speaker C: They suspected it might be doing the cutting itself. But proving that is incredibly difficult. You have to see exactly what's happening to the DNA molecule.
[00:06:45] Speaker B: You need to see the cuts, the.
[00:06:47] Speaker C: Gaps, the actual physical changes. And this is where the paper gets really creative. I mean, they use standard methods, sure, but then they use something called the Apobec 3 a trick.
[00:06:57] Speaker B: Oh, this is clever. This is a highlight for listeners who love the biochemical details.
[00:07:01] Speaker C: It's so clever.
[00:07:02] Speaker B: Break it down for us. What is Apobic 3A?
[00:07:05] Speaker C: It sounds like a droid from Star Wars. Right? Apobic 3A is an enzyme that attacks DNA. But, and here's the key, it only attacks single stranded DNA.
[00:07:15] Speaker B: Okay. Why is that useful here?
[00:07:17] Speaker C: Well, DNA is usually a double helix, zip together. But when an enzyme repairs a mistake by excision, it has to chew away one strand, creating a gap. Exactly. That gap leaves the other strand exposed. It's single stranded because its partner has been eaten.
[00:07:32] Speaker B: And that's when APOBEC strikes.
[00:07:34] Speaker C: Yes. They threw APOBEC into the mix. So if MLH1PMS1 was creating gaps, Apobec would attack that exposed strand and chemically change the letters. It turns cytosines into uracils.
[00:07:46] Speaker B: It's like dusting for fingerprints.
[00:07:48] Speaker C: It is exactly like that. Or footprints in wet cement. By sequencing the DNA later, they could look for those changes. Wherever they found them, they knew. Aha. The DNA was single stranded here. The enzyme chewed this part up.
[00:08:00] Speaker B: That is unbelievably precise. Usually you just see repaired or not repaired.
[00:08:04] Speaker C: This tells you how it visualizes the invisible and maps the repair down to the single letter.
[00:08:09] Speaker B: So they have the test tube engine, a fingerprint powder. What did they find? Was the manager, MLH1PMS1 actually fixing the car?
[00:08:16] Speaker C: The manager is a master mechanic.
The study proved unequivocally that MLH1, PMS1 can drive the repair entirely on its own. It does not need X01. It does not need RAD27.
[00:08:27] Speaker B: So it's not just pointing fingers and coordinating. It's holding a knife.
[00:08:30] Speaker C: I like to call it the wood chipper.
[00:08:32] Speaker B: The wood chipper. That sounds aggressive. For a biological process, it is aggressive.
[00:08:37] Speaker C: Compared to X01, X01 is like a zipper. It starts at a cut and just unzips in a continuous line. It's very orderly.
[00:08:43] Speaker B: One long segment.
[00:08:44] Speaker C: One long segment. MLH1PMS1. It's messy. It makes multiple cuts. It chops.
The electron microscopy, where they took literal pictures of the DNA, showed these gaps being formed by distinct chopping actions. It essentially shreds the DNA around the air.
[00:09:00] Speaker B: So it turns the bad DNA into confetti.
[00:09:02] Speaker C: Effectively. And here's a crucial detail that explains why this was missed for so long. This wood chipper mode. It has a specific fuel source.
[00:09:10] Speaker B: A fuel source? What do you mean?
[00:09:12] Speaker C: Well, enzymes often need metal ions to work.
EXO1, the classic enzyme. One loves magnesium. Most lab buckers are made with magnesium.
[00:09:20] Speaker B: Okay, so that's the standard.
[00:09:21] Speaker C: But MLH1, PMS1, to go into this beast mode, it prefers manganese or zinc.
[00:09:26] Speaker B: Manganese, yes.
[00:09:28] Speaker C: So if you're only looking at these reactions in magnesium, which is standard, you see a little bit of activity, but you might Miss just how powerful it really is. With manganese present, it becomes a robust excision machine.
[00:09:39] Speaker B: That explains why it might have been hidden in other experiments. We weren't giving it the right fuel.
[00:09:44] Speaker C: Precisely. It was there all along. But we were feeding it the wrong diet.
[00:09:48] Speaker B: I want to go back to the woodchipper visual. When they looked at the gaps, were they just random? Does it just shred the whole strand? Because that sounds dangerous.
[00:09:58] Speaker C: No. And that's the beauty of it. It's controlled chaos.
The mapping revealed two specific types of gaps.
[00:10:05] Speaker B: What are they?
[00:10:06] Speaker C: First, you have what they call polar gaps. These are long. Hundreds of letters long.
They start at that initial nick in the DNA, and they extend toward the error.
[00:10:16] Speaker B: Okay, so it starts at the cut and eats its way down. Directional. That makes sense.
[00:10:20] Speaker C: But then they found dispersed gaps. And these were fascinating. Short, scattered gaps right around the error itself. Sometimes not even connected to the main cut.
[00:10:29] Speaker B: That sounds risky. If you're chopping around the error, aren't you worried about chopping the wrong strand? The template.
[00:10:35] Speaker C: That is the number one rule of DNA repair. Do not touch the template. If you alter the master copy, you aren't fixing an error. You're cementing it.
[00:10:43] Speaker B: You're creating a mutation.
[00:10:45] Speaker C: So how does the woodchipper know which strand to shred? It has strict strand specificity. It only targets the strand that is already nicked. The new strand. The study showed it the gaps were exclusively on that broken strand. The template was pristine.
[00:10:59] Speaker B: That is just such a sophisticated system. It's aggressive, but it's precise. It knows exactly which rail of the track is broken.
[00:11:06] Speaker C: And it's versatile.
Once MLH1, PMS1 has shredded the bad section, a polymerase has to come fill it in. They found that multiple types, Polepsilon or Pol Delta, can do the job.
[00:11:17] Speaker B: It's not picky about the cleanup crew.
[00:11:19] Speaker C: Exactly. As long as the AD sequence is gone, the cell has tools to patch the hole.
[00:11:23] Speaker B: So we've established that the manager, MLH1, PMS1, is actually this secret agent who can do the whole job solo. But I have to ask the why question.
[00:11:31] Speaker C: It's the evolutionary question.
[00:11:33] Speaker B: Yeah. If we have X01 and we have RAD27 and they work, why does the cell need this third way of doing things? Isn't that overkill?
[00:11:41] Speaker C: In biology, there is no such thing as overkill when it comes to DNA integrity. Redundancy is survival.
[00:11:46] Speaker B: It's the backup for the backup.
[00:11:48] Speaker C: Think about the stakes. If this system fails, I mean, really fails, the cell accumulates mutations that Leads to cell death or worse, cancer.
Evolution does not take chances with this.
[00:12:01] Speaker B: So it's like wearing a belt and suspenders and then also duct taping your pants to your waist.
[00:12:07] Speaker C: That is a vivid image. But yes.
This third pathway explains why those mice we talked about didn't get massive cancer rates. You knock out the main plan, X01, the cell has a backup. RAD27, you knock that out. MLH1PMS1 steps in with the wood chipper.
[00:12:21] Speaker B: It creates a safety net that is almost impossible to fall through.
[00:12:24] Speaker C: And it solves a massive mystery in the field. Scientists always wondered, why is MLH1PMS1 required for all mismatch repair?
[00:12:31] Speaker B: Right. Because even in the old model, if you deleted MLH1PMS1, the whole system just broke.
[00:12:36] Speaker C: Exactly. If it was just a helper for X01, you'd think X01 could manage without it. But it can't. This paper suggests MLH1PMS1 is the central hub. It might be doing some level of cutting and processing in every repair event.
[00:12:48] Speaker B: Not just as a backup.
[00:12:49] Speaker C: Not just as a backup. It's likely the core processor of the whole operation.
[00:12:53] Speaker B: So the authors propose a new model. Right. The three pathway model.
[00:12:56] Speaker C: They do. It's a hierarchy. Pathway one, exO1 dependent, the classic zipper. Pathway two, RAD27 dependent, bit different. And then pathway three, this MLH1PMS1 mediated excision, the wood chipper.
[00:13:09] Speaker B: And all three work together to keep our code clean.
[00:13:11] Speaker C: It seems so. And we should mention there are still things we can't see.
The electron microscopy, I mean, it's amazing. But it had a resolution limit. They couldn't see gaps smaller than about 30 nucleotides.
[00:13:24] Speaker B: So there might be even tinier microsurgery happening that we're missing.
[00:13:28] Speaker C: Almost certainly. MLH1PMS1 might be making tiny nicks that didn't even show up on camera. But what we can see is robust proof that this protein is a powerhouse.
[00:13:38] Speaker B: It really changes the narrative. We used to look at MLH1PMS1 and say, oh, good job coordinating. Thanks for the help. Now we realize it's the one one holding the chainsaw.
[00:13:45] Speaker C: A very precise manganese activated chainsaw.
[00:13:48] Speaker B: I want to zoom out a bit. We've gone deep into the biochemistry, the enzymes, the manganese. For the listener at the gym right now, why does this matter? What's the big takeaway?
[00:13:57] Speaker C: The core insight is that our view of biological machinery is often way too simple. We like to put proteins in boxes. This one cuts, this one signals. This paper proves that nature is far more versatile. MLH1PMS1 is a robust standalone machine that guarantees that even if the specialists fail, the job gets done.
[00:14:16] Speaker B: It speaks to the resilience of life. We are built to survive our own errors.
[00:14:21] Speaker C: We are. And it creates a really fascinating prompt for us to consider, you know, when it comes to medicine. So we'll think about cancer treatment. Cancer cells are often defined by their instability. Their DNA repair is often broken or rewired. That's why they mutate so fast.
[00:14:35] Speaker B: Right.
[00:14:36] Speaker C: So if we know that healthy cells have three distinct layers of redundancy, but a cancer cell might be relying heavily on just one of them because the other two are broken.
[00:14:44] Speaker B: Ah, we could target that specific pathway.
[00:14:47] Speaker C: Exactly. This is the concept of synthetic lethality. If we understand the backup mechanics, we can find ways to close the garage on cancer cells permanently. If we know a tumor is surviving solely because of the MLH1PMS1 pathway, and we create a drug to block that.
[00:15:01] Speaker B: Wood chipper, the cancer cell can't repair itself and it dies.
[00:15:05] Speaker C: While the Helki cells, which still have their other pathways, might be totally fine, it opens up new doors for precision medicine using the cancer's own weaknesses against it.
[00:15:15] Speaker B: That's a powerful thought. It takes this from abstract biochemistry to something that could actually save lives.
[00:15:21] Speaker C: Absolutely. The more we know about the parts list, the better we can tune the engine.
[00:15:25] Speaker B: Well, that is where we will have to leave it for this deep dive. A dense paper, but a really, really important one. A huge thank you to the team at UCSD for shedding light on these hidden hands.
[00:15:37] Speaker C: It was a pleasure unpacking this mystery with you.
[00:15:39] Speaker B: This episode was based on an Open Access article under the CC BY 4.0 license. You can find a direct link to the paper and the license in our episode description. If you enjoyed this, follow or subscribe in your podcast app and leave a five star rating. If you'd like to support our work, use the donation link in the description now. Stay with us for an original track created especially for this episode and inspired by the article you've just heard about. Thanks for listening and join us next time as we explore more science. Bass by bass.
[00:16:22] Speaker A: Neon lanes in a double helix City headlights flicker where the copy slips One wrong spark in the rolling rhythm A tiny clash in the traffic of flips I feel the stutter in the spinning line Hear the hush before the alarms ignite A quiet era Trump trying to hide in the perfect rush of the night but the sentries circle Patient and precise hands on the pulse eyes on the fracture they don't shout they lock in close they hold the moment longer Cut it clean Let it go Let it fall away Leave a break out where the shadow used to stay Ring clicks shut and the signal turns to true we me the lane and we carry it through no skies loud Just a light in the groove Capturing a long oh got it clean Let it go Let it fall away in the sky Breathing God.
A clamp of midnight A circle of promise Slide through the nick like a magnet in a rain Then the blade you never saw coming Open space without breaking the frame not one long chase not one straight tunnel Sometimes many small windows appear Little missing pieces of doubt so the truth can steer and the gap becomes a pathway not alone A measured falls where the future can turn no drama, just a craft A spy that doesn't burn Cut it clean and let it go Let it fall away Leave a bright gap where the shadow used to stay Ring click shut and the signal turn true we mend the lane and we carry it through no stars loud Just the light in the groove Momentum capture and along the hole Let it clean, let it go Let it fall away in the skyline breathing go.
All.
Hear the freeway hum under open air Salt wind rising through a lattice of light Every mistaken step we don't keep Is a quieter world tonight Fill the space with a steady hand Seal the seam like a vow you can trust so the next thousand miles of living.
Don't turn to run.
Cut it clean Let it go Let it fall away Leave a bright gap where the shadow used to stay we click shut then the signal turns true we mend the lane that we carry now lift it high Let the harmonies move the mones some Capturing along the hole Got it clean it go Let it fall away in the skyline breathing Go.
