🎙️ Episode 44: Proofreading Precision — Unveiling the Cryo-EM Mechanism of Human DNA Polymerase ε Holoenzyme
🧬 In this episode of Base per Base, we delve into a groundbreaking study by Wang et al. (2025) in PNAS that illuminates the molecular choreography of the human leading-strand DNA polymerase ε (Polε) holoenzyme during mismatch proofreading. By combining in situ mismatch generation with high-resolution cryo-electron microscopy, the research captures authentic intermediates of the Polε–PCNA complex as it recognizes, backtracks, and excises replication errors, offering an unprecedented view of this essential fidelity mechanism.
🔍 Key highlights: The authors reveal that a primer containing a preformed mismatch is blocked from accessing the exonuclease site when PCNA is bound, underscoring the necessity of generating the mismatch within the polymerase active site; upon in situ synthesis of a mismatched nucleotide, three distinct proofreading states—mismatch-locking, polymerase backtracking, and mismatch-editing—are captured, showing an extended unwinding of six base pairs and out-of-register base pairing that guides the primer terminus into the exonuclease channel; detailed structural analysis uncovers how the PCNA sliding clamp imposes steric constraints that drive DNA translocation and how the unique P-domain of Polε contributes to duplex melting and primer repositioning; these findings challenge previous models derived from preformed mismatches and clamp-free systems by demonstrating a processive, clamp-retained proofreading pathway.
🧠Conclusion: This study inaugurates a new paradigm for understanding replicative fidelity by capturing the full proofreading cycle of a DNA polymerase–clamp holoenzyme in near-physiological conditions. The insights into PCNA’s role and P-domain dynamics promise to inform future research on replication errors and their links to cancer and genetic disease.
đź“– Reference:
Wang, F., He, Q., O’Donnell, M. E., & Li, H. (2025). The proofreading mechanism of the human leading-strand DNA polymerase ε holoenzyme. Proceedings of the National Academy of Sciences, 122(22), e2507232122. https://doi.org/10.1073/pnas.2507232122
đź“ś License: This episode is based on an open access article published under the Creative Commons Attribution 4.0 International license (CC BY 4.0). https://creativecommons.org/licenses/by/4.0/
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Episode Transcript
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(00:15):
Welcome to Base by Base. Today we're diving into
something absolutely fundamental, how our cells copy
their DNA with, well, almost unbelievable accuracy.
It has to be accurate. Errors or mutations can have
serious consequences. Right.
Things like cancer, inherited genetic diseases, the stakes are
incredibly high. Our focus is going to be on one
(00:37):
of the star players in this process, human DNA polymerase
epsilon. You'll often hear it called
Pulley. That's right, pulling is a real
workhorse enzyme. Its main job during DNA
replication is synthesizing whatwe call the leading strand.
But it's more than just a builder, it's got this fantastic
built in editing function too. Ah, so it checks its own work.
(00:58):
Exactly. It's a dual function enzyme.
It has the polymerase site whereit adds new DNA bases, the
ASTCS&G's and then it has another site, the three prime to
five prime exonucleus site. This is the editing or
proofreading site. And how far apart are these
sites? Does the DNA have to travel
much? There are about 40 angstroms
apart, but on a molecular scale is a fair distance within the
enzyme itself, maybe the width of a few DNA based pairs stacked
(01:21):
up. So if Polay accidentally puts in
the wrong base, a mismatch, it can sense this.
It then has to sort of pause synthesis and shuffle the end of
that newly made DNA strand over to the exonuclei site, the
editing Bay if you like to. Snip out the mistake.
Precisely. It cuts out the wrong base and
(01:42):
then synthesis can resume correctly.
This proofreading is absolutely critical for the high fidelity
of DNA replication. So it builds, makes a mistake,
hits undo basically, and then carries on.
Seems efficient. Very efficient and crucial.
But here's the interesting part,and really the core of what
we're discussing today. Even though we've known about
Poley and its proofreading for awhile, understanding precisely
(02:03):
how it carries up this error correction in its natural
context has been, well, a bit ofa black box.
It really has. Especially when it's working
with its essential partner, the PCNA sliding clamp.
Yes, the PCNA clamp is key. You can think of it like a ring
that slides onto the DNA double Helix.
Like a doughnut. Kind of, yeah.
Yeah. And it encircles the DNA and
tethers the pulmonaries, polanosthis case to the DNA strand.
(02:26):
Ah, so it stops the polymerase from just floating away after
copying a few bases. Exactly.
It confers processivity. That means Poly can synthesize
really long stretches of DNA without dissociating in the
cell. Poly always works with PCNA.
OK, so that's the first condition for seeing the real
process. Poland needs to be with PCNA.
What else? The second condition, and this
(02:47):
has been the real challenge, is that you need to see what
happens when the error, the mismatch, is created by the
enzyme itself during the ongoingsynthesis process.
And since you generated mismatchright as it happens.
Precisely, not just giving the enzyme DNA that already has a
mistake built into it. Why is that distinction so
important? Because the DNA has to follow
its natural path within the enzyme complex.
(03:09):
When a mismatch is made during synthesis, the DNA end is
already at the polymerase activesite for proofreading.
It needs to move from that site to the exonucleus site.
If you just provide DNA with a pre-existing mismatch from the
outside, it might not engage with the enzyme complex in the
same way. It might not be able to access
that path from the polymerase site to the exonucleus site
(03:29):
correctly, especially when the PCNA cramp is present.
I see, so previous studies mighthave struggled because they
either didn't have the PCNA clamp involved or they used
these pre made errors, or maybe both.
Often, yes, it was difficult to set up an experiment that met
both criteria simultaneously, the full pulling PCNA complex
and generating the error in situ.
(03:50):
This limited our understanding of the true physiological
mechanism. OK, so the stage is set.
The challenge capture this dynamic proofreading event in
action with all the key players present.
And that brings us squarely to the work we're focusing on
today. It's a study published in PNAS,
the Proceedings of the National Academy of Sciences in 2025 by
Doctor Feng Wang and colleagues.Yes, a really elegant piece of
(04:13):
work using cryoelectron microscopy or cryo EM.
Cryo EM allows scientists to getincredibly detailed, almost
atomic level pictures of biological molecules, right?
Exactly. They essentially flash freeze
the molecules in various States,and then use electron microscopy
and sophisticated image processing to reconstruct their
3D structures. So Doctor Wang and the team set
(04:35):
out to use cryo EM to visualize the human Poly enzyme bound to
its PCNA clamp interacting with DNA.
And crucially, they compared what happened when they use DNA
with a pre-existing mismatch versus allowing Pole to make the
mistake itself in situ. That comparison was key and the
results were quite striking. Let's start with the
pre-existing mismatch. What did they see when they gave
(04:57):
the polling PCNA complex the Holland enzyme DNA that already
had an error? They found something really
interesting. The complex essentially got
stuck. They turned it.
A blocked state blocked Tau. The DNA with the pre-existing
mismatch could bind to the hollow enzyme, but the mismatch
itself seemed unable to move into the correct position to be
proof read. It couldn't access the
exnuclease site. The interpretation is that the
(05:18):
PCNA clamp tightly encircling the DNA downstream of the
polymerase strongly constrains the DNA's flexibility and
movement. When the error is already
present, the DNA coming from theoutside just can't maneuver past
these constraints to get to the editing site.
Wow, so this suggests that many previous experiments using pre
made errors with clamped polymerases might have been
(05:40):
looking at an artifact, a blocked state that doesn't
represent actual proofreading. It strongly suggests that yes,
it implies that for POLA, when it's working with PCNA, the
error must arise during synthesis for the proofreading
pathway to be engaged correctly.So that makes the in situ
experiment where Polo made the mistake itself absolutely
critical. What did they find then?
(06:02):
That's where they capture the real action by setting up the
reaction. So POLI would incorporate A
mismatch. While bound to PCNA, they were
able to visualize intermediates in the proofreading process.
They identified 3 distinct structural states.
Three steps in the proofreading bands.
What were they? They called them the mismatch
locking state, the pull back tracking state, and the mismatch
(06:22):
editing state. OK, let's walk through those
mismatch locking state first. What happens there?
So this is right after Pulley incorporates the wrong
nucleotide. The enzyme sense is this part of
the polymerase. The fingers domain, which
usually closes around the correct incoming base, flips
open. This stops synthesis
immediately. Halts the production line.
(06:43):
Makes sense, yes? But here's a twist.
Structurally, they saw that the entire enzyme actually shifted
forward along the DNA by one base pair.
Forward, not backward. That seems weird.
Why forward? It does seem counterintuitive,
but the idea is that this forward translocation locks the
mismatch in place just downstream of the now empty
(07:03):
polymerase active site. It prevents the enzyme from
accidentally adding another baseonto the incorrect end.
OK, securing the error before dealing with it and PCNA still
attached. Oh yes, PCNA remains stated
bound throughout. It's the anchor.
Right, so state one mismatch made, synthesis stops, enzyme
nudges forward slightly, lockingthe error.
What's next? The pullback tracking state.
(07:25):
Pullback tracking, Yes. Now the system prepares to move
the error to the editing site. What they saw was the DNA
starting to unwind locally near the mismatch.
The very end of the primer strand is the strand being
synthesized, which now has the wrong base at a three foot tip
starts to peel away from the template strand.
It phrase like the end of a rope.
(07:47):
Exactly it Phrase and that ThreeFoot N begins its journey
towards the Exonucleosite's entry channel.
How does the enzyme help this happen?
The observed specific movements in the pulley structure.
The thumb domain, another part of the plum race, shifts away
from a region called the P domain.
The P domain is actually unique to pulley among replicative
polymerases. OK, so the thumb moves away from
(08:08):
this special P domain. Which seems to create the
necessary space allowing the DNAto unwind and the primer end to
start moving. Importantly, even with these
internal rearrangements, Pulley maintains its strong three-point
connection to the PCNA clamp. So the clamp isn't just holding
it on the DNA, it's also kind ofguiding these internal movements
during proofreading. That appears to be the case.
(08:29):
It provides a stable platform and likely helps orchestrate
the. Fascinating.
OK so mismatch locked then DNA phrase and the error starts
moving. What's the final state they
captured? The mismatch editing state this
is the destination. The error arrives at the delete
button. Precisely the three bit end of
the primer with the incorrect base has now translocated about
(08:52):
15 angstroms from where it was initially locked, and it's now
sitting perfectly within the active site of the exonuclease
domain, ready to be cleaved off.15 angstroms.
That's that significant internalmovement we talked about
earlier. Yes, and again, even in this
editing configuration, the main Poland core is still stably
associated with PCNA. Incredible.
Capturing these three states is a huge step forward, but you
(09:14):
mentioned surprises in the structural details, especially
in that editing state. Yes, some really unexpected
findings when they look closely the DNA structure within the
editing state, specifically comparing it to models based on
other polymerases often studied without the clamp.
What was the biggest surprise? The extent of DNA unwinding.
Previous studies on related polymerases suggested maybe
(09:35):
three base pairs of the DNA duplex unwind during
proofreading transfer. Just enough to expose the error,
presumably. Right.
But in the polling PCNA Hollomanzyme caught in the act
of proofreading and in situ mismatch, Doctor Wang and
colleagues observed a much more dramatic unwinding.
How much more? Six base pairs double the
previously expected amount. A whole segment of the newly
(09:57):
synthesized DNA duplex is meltedapart.
Six base pairs. That's a significant stretch of
DNA to unzip just for proofreading.
Was that the only surprise? No, perhaps even more surprising
was what happened to those unwound strands.
They didn't just hang loose. What did they do?
They kind of stretched up the single template strand and the
single primer strand folded backon themselves, and actually form
(10:20):
new out of register base pairs with each other downstream from
the externucleoside. Wait, the unwound strands paired
up again, but incorrectly mismatched.
Yes, their structure showed the scrunched region contained I
think 4 mismatched base pairs and kind of randomly 2 pairs
that happened to be correct GC pairs just by chance in their
(10:41):
sequence. Out of register pairing?
Scrunching. That sounds messy.
Why would the enzyme allow or even promote that?
What's the point? The researchers proposed a
really clever functional role for this.
By forming this scrunched out ofregister structure, the inner
part of the primer strand and the bases just behind the actual
error at the three feet end getstucked away and protected.
(11:03):
So it physically ensures that only the very last base, the
incorrect one, is exposed and accessible in the Exonuclease
active site. That's the hypothesis.
It provides exquisite specificity, making sure the
enzyme only cuts off the single terminal mismatch and doesn't
accidentally chew back further into the correctly synthesized
DNA. That's incredibly elegant,
actually, turning potential messiness into precision.
(11:26):
Do they have any idea why Poly does this particular scrunching?
They noted that Poly has a relatively short beta hairpin
loop structure within its exonuclease domain compared to
some other polymerases. They suggest the shorter loop
might not be able to keep the two unwound DNA strands
effectively separated over that six base period distance,
potentially allowing or even encouraging them to snap back
(11:47):
together in this scrunched out of register way.
So the enzymes own structure might facilitate this unusual
DNA confirmation for a functional reason.
Amazing. It really highlights how protein
structure and function are intertwined with the dynamic
behavior of the DNA itself. And how are these major DNA
rearrangements, the six BP unwinding, the translocation,
the scrunching connected back tothe enzymes interaction with
(12:10):
PCNA? The study suggests that the
stable multi point interaction between POLI and PCNA is
critical. It likely drives and guides the
necessary movements of the POLI domains, like that P domain
tilting and the thumb domain shifting we mentioned earlier.
These domain movements, orchestrated by the PCNA
interaction are what facilitate the extensive DNA unwinding and
(12:30):
the precise translocation of theprimer end.
The clamp doesn't just provide processivity, it actively shapes
the proofreading pathway. So the clamp imposes constraints
that lead to this very specific,maybe even unique mechanism
involving massive unwinding and refolding.
That seems to be the picture emerging from this study.
It's a fundamentally different view compared to what you might
(12:51):
see or predict for Pulley operating without its essential
partner, PCNA. OK, let's try to summarize the
core findings then. This study by Doctor Wang and
colleagues using cryo EM gives us the first real glimpse of
human pulley proofreading as it happens physiologically bound to
PCNA, correcting an error it just made.
Right, and it reveals A mechanism constrained by PCNA.
(13:11):
A mechanism involving much more DNA unwinding than we thought.
Six base pairs. And this unexpected but
functional scrunching and out ofregister pairing of the unwound
DNA strands to ensure editing precision.
It really changes the textbook picture, doesn't it?
It certainly refines it significantly and highlights the
importance of studying these enzymes in their complete
(13:33):
functional context. So what are the broader
implications here? You mentioned this likely
represents the true physiological mechanism.
Yes, that's a major implication.Capturing these states with the
whole syme and the in situ mismatch strongly suggests this
is how holy proofreads in our cells, which, as we discussed,
forces a re evaluation of conclusions drawn from earlier
(13:54):
studies that lacked the clamp orused preformed errors.
Those might not reflect the biological reality for this
enzyme. And what about that P domain you
mentioned? It's unique to Pulley.
We knew it was involved in processivity and maybe helping
load PCNA. Right.
But this study has a third crucial role.
It appears to be actively involved in manipulating the DNA
during proofreading, helping to melt it reposition.
(14:15):
Does that mean Pulley's proofreading strategy might be
fundamentally different from other polymerases like pole
delta, which replicates the lagging strand and also works
with PCNA? It's a very strong possibility
the unique P domain's role combined with the extensive six
base pair unwinding and the scrunching behavior.
It does suggest Pulley might have evolved a distinct
(14:36):
proofreading choreography, perhaps optimized for its
meeting strand duties. We'll need similar in situ
studies on pull delta with PCNA to really know for sure how they
compare. Let's talk about the clinical
side. Pulling mutations are notorious
in Cancer Research, right? Especially mutations in the
proofreading exonuclease domain.Absolutely.
Mutations that cripple the pulling exonuclease lead to a
(14:58):
mutator phenotype. The enzyme loses its editing
ability and the mutation rate skyrockets, driving cancer
development, particularly in colorectal and endometrial
cancers. This is well established.
So this study obviously reinforces the importance of
that exonucleus function. It does, but it also raises
another possibility, given how crucial the stable interaction
(15:19):
with PCNA appears to be for the entire proofreading process, the
unwinding, the translocation, the scrunching.
It suggests that mutations that don't directly hit the
exonuclease active site, but instead disrupt the key contact
points between Pulley and PCNA could also impair proofreading
fidelity. So weakening the pulley PCNA
connection might indirectly leadto more replication errors
(15:42):
because the proofreading mechanism itself gets derailed.
Potentially, yes. It broadens the scope of
mutations within Pulley or even potentially in PCNA, that could
impact genomic stability and contribute to cancer.
It's an important area for future investigation.
That's a really significant clinical implication.
They're going back to that fascinating DNA scrunching and
(16:02):
out of register pairing. The paper linked this to
replication slippage. Yes, they speculated about a
potential connection. Replication slippage is a known
source of mutations, particularly insertions or
deletions, that often happens inrepetitive DNA sequences like
long strings of the same base, sometimes called homonucleotide
runs. How might the scrunching be
involved? The idea is this, after the
(16:24):
exonucleus removes the mismatch,the enzyme needs to reposition
the now corrected primer end back into the polymerase active
site to resume synthesis. If the DNA is still in that
scrunched out of register state from the proofreading step,
especially in a repetitive region where incorrect pairings
might be temporarily stable, it might be difficult for the
(16:45):
enzyme to perfectly realign the primer and template strands
before adding the next base. So a slight misalignment during
this re engagement step could lead to adding an extra base or
skipping 1. Exactly.
It could manifest as an insertion or deletion error, the
hallmark of replication slippage.
So ironically, a feature designed for proofreading
precision might, under certain circumstances or in certain
(17:08):
sequence context, contribute to a different type of error.
If real aim, it fails. Wow, the complexity is just
astounding. One process potentially
influencing another. It really shows how dynamic DNA
replication is. And finally, it's worth
emphasizing the power of the experimental approach itself.
Generating the mismatch in situ within the fully assembled pole
clamp complex. Yes, this methodology, now
(17:30):
validated by Doctor Wang and colleagues for Poli, provides a
powerful tool. It can and likely will be
applied to study proofreading byother polymerases that function
with sliding clamps like the aforementioned pole delta, or
even polymerases from different organisms.
Giving us a clearer picture across the board.
Hopefully yes. Understanding the nuances of how
(17:51):
each enzyme ensures fidelity is crucial.
Absolutely. OK, so to wrap up, here's a
final thought for you, our listeners to consider.
We've learned about these uniqueaspects of the Pulley PCNA
proofreading mechanism, the extensive six base pair
unwinding, the active role of the P domain in handling the
DNA, this potential link betweenscrunching and replication
(18:11):
slippage. Given this incredibly detailed,
almost mechanistic understanding, how might this
knowledge open up new avenues? Could we potentially develop
novel therapeutic strategies, perhaps for cancers driven by
pollen mutations, by targeting not just the exonuclease
activity itself, but maybe theseunique structural transitions or
interactions revealed in the study?
Something to think about A. Very pertinent question for
(18:33):
future research. This episode is based on an Open
Access article under the Creative Commons Attribution 4.0
International License, CCBY 4.0.You'll find the DOI and a link
to the license in the episode description.
Welcome to Base by Base. Today we're diving into
something absolutely fundamental, how our cells copy
their DNA with, well, almost unbelievable accuracy.
It has to be accurate. Errors or mutations can have
serious consequences. Right.
Things like cancer, inherited genetic diseases, the stakes are
incredibly high. Our focus is going to be on one
(00:37):
of the star players in this process, human DNA polymerase
epsilon. You'll often hear it called
Pulley. That's right, pulling is a real
workhorse enzyme. Its main job during DNA
replication is synthesizing whatwe call the leading strand.
But it's more than just a builder, it's got this fantastic
built in editing function too. Ah, so it checks its own work.
(00:58):
Exactly. It's a dual function enzyme.
It has the polymerase site whereit adds new DNA bases, the
ASTCS&G's and then it has another site, the three prime to
five prime exonucleus site. This is the editing or
proofreading site. And how far apart are these
sites? Does the DNA have to travel
much? There are about 40 angstroms
apart, but on a molecular scale is a fair distance within the
enzyme itself, maybe the width of a few DNA based pairs stacked
(01:21):
up. So if Polay accidentally puts in
the wrong base, a mismatch, it can sense this.
It then has to sort of pause synthesis and shuffle the end of
that newly made DNA strand over to the exonuclei site, the
editing Bay if you like to. Snip out the mistake.
Precisely. It cuts out the wrong base and
(01:42):
then synthesis can resume correctly.
This proofreading is absolutely critical for the high fidelity
of DNA replication. So it builds, makes a mistake,
hits undo basically, and then carries on.
Seems efficient. Very efficient and crucial.
But here's the interesting part,and really the core of what
we're discussing today. Even though we've known about
Poley and its proofreading for awhile, understanding precisely
(02:03):
how it carries up this error correction in its natural
context has been, well, a bit ofa black box.
It really has. Especially when it's working
with its essential partner, the PCNA sliding clamp.
Yes, the PCNA clamp is key. You can think of it like a ring
that slides onto the DNA double Helix.
Like a doughnut. Kind of, yeah.
Yeah. And it encircles the DNA and
tethers the pulmonaries, polanosthis case to the DNA strand.
(02:26):
Ah, so it stops the polymerase from just floating away after
copying a few bases. Exactly.
It confers processivity. That means Poly can synthesize
really long stretches of DNA without dissociating in the
cell. Poly always works with PCNA.
OK, so that's the first condition for seeing the real
process. Poland needs to be with PCNA.
What else? The second condition, and this
(02:47):
has been the real challenge, is that you need to see what
happens when the error, the mismatch, is created by the
enzyme itself during the ongoingsynthesis process.
And since you generated mismatchright as it happens.
Precisely, not just giving the enzyme DNA that already has a
mistake built into it. Why is that distinction so
important? Because the DNA has to follow
its natural path within the enzyme complex.
(03:09):
When a mismatch is made during synthesis, the DNA end is
already at the polymerase activesite for proofreading.
It needs to move from that site to the exonucleus site.
If you just provide DNA with a pre-existing mismatch from the
outside, it might not engage with the enzyme complex in the
same way. It might not be able to access
that path from the polymerase site to the exonucleus site
(03:29):
correctly, especially when the PCNA cramp is present.
I see, so previous studies mighthave struggled because they
either didn't have the PCNA clamp involved or they used
these pre made errors, or maybe both.
Often, yes, it was difficult to set up an experiment that met
both criteria simultaneously, the full pulling PCNA complex
and generating the error in situ.
(03:50):
This limited our understanding of the true physiological
mechanism. OK, so the stage is set.
The challenge capture this dynamic proofreading event in
action with all the key players present.
And that brings us squarely to the work we're focusing on
today. It's a study published in PNAS,
the Proceedings of the National Academy of Sciences in 2025 by
Doctor Feng Wang and colleagues.Yes, a really elegant piece of
(04:13):
work using cryoelectron microscopy or cryo EM.
Cryo EM allows scientists to getincredibly detailed, almost
atomic level pictures of biological molecules, right?
Exactly. They essentially flash freeze
the molecules in various States,and then use electron microscopy
and sophisticated image processing to reconstruct their
3D structures. So Doctor Wang and the team set
(04:35):
out to use cryo EM to visualize the human Poly enzyme bound to
its PCNA clamp interacting with DNA.
And crucially, they compared what happened when they use DNA
with a pre-existing mismatch versus allowing Pole to make the
mistake itself in situ. That comparison was key and the
results were quite striking. Let's start with the
pre-existing mismatch. What did they see when they gave
(04:57):
the polling PCNA complex the Holland enzyme DNA that already
had an error? They found something really
interesting. The complex essentially got
stuck. They turned it.
A blocked state blocked Tau. The DNA with the pre-existing
mismatch could bind to the hollow enzyme, but the mismatch
itself seemed unable to move into the correct position to be
proof read. It couldn't access the
exnuclease site. The interpretation is that the
(05:18):
PCNA clamp tightly encircling the DNA downstream of the
polymerase strongly constrains the DNA's flexibility and
movement. When the error is already
present, the DNA coming from theoutside just can't maneuver past
these constraints to get to the editing site.
Wow, so this suggests that many previous experiments using pre
made errors with clamped polymerases might have been
(05:40):
looking at an artifact, a blocked state that doesn't
represent actual proofreading. It strongly suggests that yes,
it implies that for POLA, when it's working with PCNA, the
error must arise during synthesis for the proofreading
pathway to be engaged correctly.So that makes the in situ
experiment where Polo made the mistake itself absolutely
critical. What did they find then?
(06:02):
That's where they capture the real action by setting up the
reaction. So POLI would incorporate A
mismatch. While bound to PCNA, they were
able to visualize intermediates in the proofreading process.
They identified 3 distinct structural states.
Three steps in the proofreading bands.
What were they? They called them the mismatch
locking state, the pull back tracking state, and the mismatch
(06:22):
editing state. OK, let's walk through those
mismatch locking state first. What happens there?
So this is right after Pulley incorporates the wrong
nucleotide. The enzyme sense is this part of
the polymerase. The fingers domain, which
usually closes around the correct incoming base, flips
open. This stops synthesis
immediately. Halts the production line.
(06:43):
Makes sense, yes? But here's a twist.
Structurally, they saw that the entire enzyme actually shifted
forward along the DNA by one base pair.
Forward, not backward. That seems weird.
Why forward? It does seem counterintuitive,
but the idea is that this forward translocation locks the
mismatch in place just downstream of the now empty
(07:03):
polymerase active site. It prevents the enzyme from
accidentally adding another baseonto the incorrect end.
OK, securing the error before dealing with it and PCNA still
attached. Oh yes, PCNA remains stated
bound throughout. It's the anchor.
Right, so state one mismatch made, synthesis stops, enzyme
nudges forward slightly, lockingthe error.
What's next? The pullback tracking state.
(07:25):
Pullback tracking, Yes. Now the system prepares to move
the error to the editing site. What they saw was the DNA
starting to unwind locally near the mismatch.
The very end of the primer strand is the strand being
synthesized, which now has the wrong base at a three foot tip
starts to peel away from the template strand.
It phrase like the end of a rope.
(07:47):
Exactly it Phrase and that ThreeFoot N begins its journey
towards the Exonucleosite's entry channel.
How does the enzyme help this happen?
The observed specific movements in the pulley structure.
The thumb domain, another part of the plum race, shifts away
from a region called the P domain.
The P domain is actually unique to pulley among replicative
polymerases. OK, so the thumb moves away from
(08:08):
this special P domain. Which seems to create the
necessary space allowing the DNAto unwind and the primer end to
start moving. Importantly, even with these
internal rearrangements, Pulley maintains its strong three-point
connection to the PCNA clamp. So the clamp isn't just holding
it on the DNA, it's also kind ofguiding these internal movements
during proofreading. That appears to be the case.
(08:29):
It provides a stable platform and likely helps orchestrate
the. Fascinating.
OK so mismatch locked then DNA phrase and the error starts
moving. What's the final state they
captured? The mismatch editing state this
is the destination. The error arrives at the delete
button. Precisely the three bit end of
the primer with the incorrect base has now translocated about
(08:52):
15 angstroms from where it was initially locked, and it's now
sitting perfectly within the active site of the exonuclease
domain, ready to be cleaved off.15 angstroms.
That's that significant internalmovement we talked about
earlier. Yes, and again, even in this
editing configuration, the main Poland core is still stably
associated with PCNA. Incredible.
Capturing these three states is a huge step forward, but you
(09:14):
mentioned surprises in the structural details, especially
in that editing state. Yes, some really unexpected
findings when they look closely the DNA structure within the
editing state, specifically comparing it to models based on
other polymerases often studied without the clamp.
What was the biggest surprise? The extent of DNA unwinding.
Previous studies on related polymerases suggested maybe
(09:35):
three base pairs of the DNA duplex unwind during
proofreading transfer. Just enough to expose the error,
presumably. Right.
But in the polling PCNA Hollomanzyme caught in the act
of proofreading and in situ mismatch, Doctor Wang and
colleagues observed a much more dramatic unwinding.
How much more? Six base pairs double the
previously expected amount. A whole segment of the newly
(09:57):
synthesized DNA duplex is meltedapart.
Six base pairs. That's a significant stretch of
DNA to unzip just for proofreading.
Was that the only surprise? No, perhaps even more surprising
was what happened to those unwound strands.
They didn't just hang loose. What did they do?
They kind of stretched up the single template strand and the
single primer strand folded backon themselves, and actually form
(10:20):
new out of register base pairs with each other downstream from
the externucleoside. Wait, the unwound strands paired
up again, but incorrectly mismatched.
Yes, their structure showed the scrunched region contained I
think 4 mismatched base pairs and kind of randomly 2 pairs
that happened to be correct GC pairs just by chance in their
(10:41):
sequence. Out of register pairing?
Scrunching. That sounds messy.
Why would the enzyme allow or even promote that?
What's the point? The researchers proposed a
really clever functional role for this.
By forming this scrunched out ofregister structure, the inner
part of the primer strand and the bases just behind the actual
error at the three feet end getstucked away and protected.
(11:03):
So it physically ensures that only the very last base, the
incorrect one, is exposed and accessible in the Exonuclease
active site. That's the hypothesis.
It provides exquisite specificity, making sure the
enzyme only cuts off the single terminal mismatch and doesn't
accidentally chew back further into the correctly synthesized
DNA. That's incredibly elegant,
actually, turning potential messiness into precision.
(11:26):
Do they have any idea why Poly does this particular scrunching?
They noted that Poly has a relatively short beta hairpin
loop structure within its exonuclease domain compared to
some other polymerases. They suggest the shorter loop
might not be able to keep the two unwound DNA strands
effectively separated over that six base period distance,
potentially allowing or even encouraging them to snap back
(11:47):
together in this scrunched out of register way.
So the enzymes own structure might facilitate this unusual
DNA confirmation for a functional reason.
Amazing. It really highlights how protein
structure and function are intertwined with the dynamic
behavior of the DNA itself. And how are these major DNA
rearrangements, the six BP unwinding, the translocation,
the scrunching connected back tothe enzymes interaction with
(12:10):
PCNA? The study suggests that the
stable multi point interaction between POLI and PCNA is
critical. It likely drives and guides the
necessary movements of the POLI domains, like that P domain
tilting and the thumb domain shifting we mentioned earlier.
These domain movements, orchestrated by the PCNA
interaction are what facilitate the extensive DNA unwinding and
(12:30):
the precise translocation of theprimer end.
The clamp doesn't just provide processivity, it actively shapes
the proofreading pathway. So the clamp imposes constraints
that lead to this very specific,maybe even unique mechanism
involving massive unwinding and refolding.
That seems to be the picture emerging from this study.
It's a fundamentally different view compared to what you might
(12:51):
see or predict for Pulley operating without its essential
partner, PCNA. OK, let's try to summarize the
core findings then. This study by Doctor Wang and
colleagues using cryo EM gives us the first real glimpse of
human pulley proofreading as it happens physiologically bound to
PCNA, correcting an error it just made.
Right, and it reveals A mechanism constrained by PCNA.
(13:11):
A mechanism involving much more DNA unwinding than we thought.
Six base pairs. And this unexpected but
functional scrunching and out ofregister pairing of the unwound
DNA strands to ensure editing precision.
It really changes the textbook picture, doesn't it?
It certainly refines it significantly and highlights the
importance of studying these enzymes in their complete
(13:33):
functional context. So what are the broader
implications here? You mentioned this likely
represents the true physiological mechanism.
Yes, that's a major implication.Capturing these states with the
whole syme and the in situ mismatch strongly suggests this
is how holy proofreads in our cells, which, as we discussed,
forces a re evaluation of conclusions drawn from earlier
(13:54):
studies that lacked the clamp orused preformed errors.
Those might not reflect the biological reality for this
enzyme. And what about that P domain you
mentioned? It's unique to Pulley.
We knew it was involved in processivity and maybe helping
load PCNA. Right.
But this study has a third crucial role.
It appears to be actively involved in manipulating the DNA
during proofreading, helping to melt it reposition.
(14:15):
Does that mean Pulley's proofreading strategy might be
fundamentally different from other polymerases like pole
delta, which replicates the lagging strand and also works
with PCNA? It's a very strong possibility
the unique P domain's role combined with the extensive six
base pair unwinding and the scrunching behavior.
It does suggest Pulley might have evolved a distinct
(14:36):
proofreading choreography, perhaps optimized for its
meeting strand duties. We'll need similar in situ
studies on pull delta with PCNA to really know for sure how they
compare. Let's talk about the clinical
side. Pulling mutations are notorious
in Cancer Research, right? Especially mutations in the
proofreading exonuclease domain.Absolutely.
Mutations that cripple the pulling exonuclease lead to a
(14:58):
mutator phenotype. The enzyme loses its editing
ability and the mutation rate skyrockets, driving cancer
development, particularly in colorectal and endometrial
cancers. This is well established.
So this study obviously reinforces the importance of
that exonucleus function. It does, but it also raises
another possibility, given how crucial the stable interaction
(15:19):
with PCNA appears to be for the entire proofreading process, the
unwinding, the translocation, the scrunching.
It suggests that mutations that don't directly hit the
exonuclease active site, but instead disrupt the key contact
points between Pulley and PCNA could also impair proofreading
fidelity. So weakening the pulley PCNA
connection might indirectly leadto more replication errors
(15:42):
because the proofreading mechanism itself gets derailed.
Potentially, yes. It broadens the scope of
mutations within Pulley or even potentially in PCNA, that could
impact genomic stability and contribute to cancer.
It's an important area for future investigation.
That's a really significant clinical implication.
They're going back to that fascinating DNA scrunching and
(16:02):
out of register pairing. The paper linked this to
replication slippage. Yes, they speculated about a
potential connection. Replication slippage is a known
source of mutations, particularly insertions or
deletions, that often happens inrepetitive DNA sequences like
long strings of the same base, sometimes called homonucleotide
runs. How might the scrunching be
involved? The idea is this, after the
(16:24):
exonucleus removes the mismatch,the enzyme needs to reposition
the now corrected primer end back into the polymerase active
site to resume synthesis. If the DNA is still in that
scrunched out of register state from the proofreading step,
especially in a repetitive region where incorrect pairings
might be temporarily stable, it might be difficult for the
(16:45):
enzyme to perfectly realign the primer and template strands
before adding the next base. So a slight misalignment during
this re engagement step could lead to adding an extra base or
skipping 1. Exactly.
It could manifest as an insertion or deletion error, the
hallmark of replication slippage.
So ironically, a feature designed for proofreading
precision might, under certain circumstances or in certain
(17:08):
sequence context, contribute to a different type of error.
If real aim, it fails. Wow, the complexity is just
astounding. One process potentially
influencing another. It really shows how dynamic DNA
replication is. And finally, it's worth
emphasizing the power of the experimental approach itself.
Generating the mismatch in situ within the fully assembled pole
clamp complex. Yes, this methodology, now
(17:30):
validated by Doctor Wang and colleagues for Poli, provides a
powerful tool. It can and likely will be
applied to study proofreading byother polymerases that function
with sliding clamps like the aforementioned pole delta, or
even polymerases from different organisms.
Giving us a clearer picture across the board.
Hopefully yes. Understanding the nuances of how
(17:51):
each enzyme ensures fidelity is crucial.
Absolutely. OK, so to wrap up, here's a
final thought for you, our listeners to consider.
We've learned about these uniqueaspects of the Pulley PCNA
proofreading mechanism, the extensive six base pair
unwinding, the active role of the P domain in handling the
DNA, this potential link betweenscrunching and replication
(18:11):
slippage. Given this incredibly detailed,
almost mechanistic understanding, how might this
knowledge open up new avenues? Could we potentially develop
novel therapeutic strategies, perhaps for cancers driven by
pollen mutations, by targeting not just the exonuclease
activity itself, but maybe theseunique structural transitions or
interactions revealed in the study?
Something to think about A. Very pertinent question for
(18:33):
future research. This episode is based on an Open
Access article under the Creative Commons Attribution 4.0
International License, CCBY 4.0.You'll find the DOI and a link
to the license in the episode description.
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