Single-molecule imaging reveals target-search mechanisms during DNA mismatch repair.
ABSTRACT The ability of proteins to locate specific targets among a vast excess of nonspecific DNA is a fundamental theme in biology. Basic principles governing these search mechanisms remain poorly understood, and no study has provided direct visualization of single proteins searching for and engaging target sites. Here we use the postreplicative mismatch repair proteins MutSα and MutLα as model systems for understanding diffusion-based target searches. Using single-molecule microscopy, we directly visualize MutSα as it searches for DNA lesions, MutLα as it searches for lesion-bound MutSα, and the MutSα/MutLα complex as it scans the flanking DNA. We also show that MutLα undergoes intersite transfer between juxtaposed DNA segments while searching for lesion-bound MutSα, but this activity is suppressed upon association with MutSα, ensuring that MutS/MutL remains associated with the damage-bearing strand while scanning the flanking DNA. Our findings highlight a hierarchy of lesion- and ATP-dependent transitions involving both MutSα and MutLα, and help establish how different modes of diffusion can be used during recognition and repair of damaged DNA.
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ABSTRACT: Vesicular stomatitis virus (VSV) is the prototype for negative sense non segmented (NNS) RNA viruses which include potent human and animal pathogens such as Rabies, Ebola and measles. The polymerases of NNS RNA viruses only initiate transcription at or near the 3' end of their genome template. We measured the dissociation constant of VSV polymerases from their whole genome template to be 20 pM. Given this low dissociation constant, initiation and sustainability of transcription becomes nontrivial. To explore possible mechanisms, we simulated the first hour of transcription using Monte Carlo methods and show that a one-time initial dissociation of all polymerases during entry is not sufficient to sustain transcription. We further show that efficient transcription requires a sliding mechanism for non-transcribing polymerases and can be realized with different polymerase-polymerase interactions and distinct template topologies. In conclusion, we highlight a model in which collisions between transcribing and sliding non-transcribing polymerases result in release of the non-transcribing polymerases allowing for redistribution of polymerases between separate templates during transcription and suggest specific experiments to further test these mechanisms.PLoS Computational Biology 12/2014; 10(12):e1004004. · 4.87 Impact Factor
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ABSTRACT: The DNA mismatch repair (MMR) system plays a crucial role in the prevention of replication errors and in the correction of some oxidative damages of DNA bases. In the present work the most abundant oxidized pyrimidine lesion, 5,6-dihydro-5,6-dihydroxythymidine (thymidine glycol, Tg) was tested for being recognized and processed by the E. coli MMR system, namely complex of MutS, MutL and MutH proteins. In a partially reconstituted MMR system with MutS-MutL-MutH proteins, G/Tg and A/Tg containing plasmids failed to provoke the incision of DNA. Tg residue in the 30-mer DNA duplex destabilized double helix due to stacking disruption with neighboring bases. However, such local structural changes are not important for E. coli MMR system to recognize this lesion. A lack of repair of Tg containing DNA could be due to a failure of MutS (a first acting protein of MMR system) to interact with modified DNA in a proper way. It was shown that Tg in DNA does not affect on ATPase activity of MutS. On the other hand, MutS binding affinities to DNA containing Tg in G/Tg and A/Tg pairs are lower than to DNA with a G/T mismatch and similar to canonical DNA. Peculiarities of MutS interaction with DNA was monitored by Förster resonance energy transfer (FRET) and fluorescence anisotropy. Binding of MutS to Tg containing DNAs did not result in the formation of characteristic DNA kink. Nevertheless, MutS homodimer orientation on Tg-DNA is similar to that in the case of G/T-DNA. In contrast to G/T-DNA, neither G/Tg- nor A/Tg-DNA was able to stimulate ADP release from MutS better than canonical DNA. Thus, Tg residue in DNA is unlikely to be recognized or processed by the E. coli MMR system. Probably, the MutS transformation to active "sliding clamp" conformation on Tg-DNA is problematic.PLoS ONE 08/2014; 9(8):e104963. · 3.53 Impact Factor
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ABSTRACT: Unlike other members of the methyl-cytosine binding domain (MBD) family, MBD4 serves as a potent DNA glycosylase in DNA mismatch repair specifically targeting (m)CpG/TpG mismatches arising from spontaneous deamination of methyl-cytosine. The protein contains an N-terminal MBD (MBD4MBD) and a C-terminal glycosylase domain (MBD4GD) separated by a long linker. This arrangement suggests that the MBD4MBD either directly augments enzymatic catalysis by the MBD4GD or targets the protein to regions enriched for (m)CpG/TpG mismatches. Here we present structural and dynamic studies of MBD4MBD bound to dsDNA. We show that MBD4MBD binds with a modest preference for(m)CpG as compared to mismatch, unmethylated and hydroxymethylated DNA. We find that while MBD4MBD exhibits slow exchange between molecules of DNA (intermolecular exchange), the domain exhibits fast exchange between two sites in the same molecule of dsDNA (intramolecular exchange). Introducing a single-strand defect between binding sites does not greatly reduce the intramolecular exchange rate, consistent with a local hopping mechanism for moving along the DNA. These results support a model in which the MBD4MBD4 targets the intact protein to (m)CpG islands and promotes scanning by rapidly exchanging between successive (m)CpG sites which facilitates repair of nearby (m)CpG/TpG mismatches by the glycosylase domain.Nucleic Acids Research 09/2014; · 8.81 Impact Factor
Single-molecule imaging reveals target-search
mechanisms during DNA mismatch repair
Jason Gormana,1, Feng Wangb,2, Sy Reddingc,2, Aaron J. Plysd,3, Teresa Fazioe, Shalom Winde, Eric E. Alanid,
and Eric C. Greeneb,f,4
Departments ofaBiological Sciences,bBiochemistry and Molecular Biophysics, andcChemistry, andfHoward Hughes Medical Institute, Columbia University,
New York, NY, 10032;dDepartment of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853; andeDepartment of Applied Physics and Applied
Mathematics, Center for Electron Transport in Molecular Nanostructures, NanoMedicine Center for Mechanical Biology, Columbia University, New York,
Edited* by Kiyoshi Mizuuchi, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD, and approved September 4, 2012 (received for
review July 5, 2012)
nonspecific DNA is a fundamental theme in biology. Basic principles
governing these search mechanisms remain poorly understood, and
for and engaging target sites. Here we use the postreplicative
mismatch repair proteins MutSα and MutLα as model systems for
understanding diffusion-based target searches. Using single-mole-
cule microscopy, we directly visualize MutSα as it searches for DNA
MutLα complex as it scans the flanking DNA. We also show that
MutLα undergoes intersite transfer between juxtaposed DNA seg-
ments while searching for lesion-bound MutSα, but this activity is
suppressed upon association with MutSα, ensuring that MutS/MutL
remains associated with the damage-bearing strand while scanning
the flanking DNA. Our findings highlight a hierarchy of lesion- and
ATP-dependent transitions involving both MutSα and MutLα, and
help establish how different modes of diffusion can be used during
recognition and repair of damaged DNA.
increases the fidelity of DNA replication up to 1,000-fold, and
MMR defects in humans cause hereditary nonpolyposis colorectal
MutLα are conserved eukaryotic protein complexes necessary for
MMR. MutSα is responsible for recognition of mismatches and
small insertion/deletion loops (1–3), whereas MutLα harbors an
endonuclease activity necessary for cleavage of the lesion-bearing
DNA strand (4, 5).
The challenges faced during MMR can be illustrated by con-
sidering that Saccharomyces cerevisiae should incur only approxi-
mately two mismatches per cell cycle (6). MutSα must find these
rare lesions, MutLα must search for lesion-bound MutSα, and the
lesion-bound MutSα/MutLα complex must search the flanking
(1–3). Models describing how DNA-binding proteins search for
specific targets include 3D diffusion (i.e., jumping), 1D hopping,
1D sliding, and intersegmental transfer; the latter three are cate-
gorized as facilitated diffusion because they allow target associa-
tion rates exceeding limits imposed by 3D diffusion (7–10). New
single-molecule and NMR techniques have led to resurgent in-
terest in understanding how proteins locate targets (11–13), and
using single-molecule imaging we previously demonstrated that
MutSα and MutLα can undergo facilitated diffusion on un-
damaged DNA through 1Dsliding and 1D hopping, respectively
(14, 15). However, no single-molecule study has directly revealed
proteins searching for and subsequently engaging a target site
through 1D diffusion (i.e., 1D sliding or 1D hopping) (7), and the
inability to visualize target capture also prevents investigation of
questions regarding downstream MMR events.
Here we used nanofabricated DNA curtains and total internal
reflection fluorescence microscopy (TIRFM) to watch MutSα and
synthesis before they lead to genomic instability (1–3). MMR
asked how these proteins conduct their respective target searches
throughout the early stages of MMR. We show that MutSα can be
targeted to mismatched bases through either 1D sliding or 3D dif-
fusion, that MutLα locates mismatch-bound MutSα through 1D
and MutSα/MutLα are released upon binding ATP and scan the
flanking DNA for strand-discrimination signals by 1D diffusion.
While searching for lesions, the movement of MutSα is consistent
with a model wherein the protein rotates to maintain constant
register with the helical contour of the DNA (14). However, once
released from a mismatch, MutSα is altered so that mismatches no
longer are recognized as targets, and the protein slides much more
rapidly, suggesting its motion no longer is coupled to rotation
MutSα/MutLα complex undergoes an ATP-dependent functional
These data provide a detailed view of how diffusion can contribute
to the early stages of MMR.
Visualization of Mismatch Recognition by MutSα on DNA Curtains.
We have used DNAcurtainspreviously toinvestigate thebehavior
of MutSα and MutLα on undamaged DNA (14, 15). Here we
sought to determine how MutSα and MutLα behave on substrates
with defined mismatches. For these experiments, we engineered
a λ-DNA (47,467 bp) harboring three tandem G/T mismatches
separated from one another by 38 bp (SI Appendix, Fig. S1; three
single-tethered DNA curtains, the DNA was anchored to a lipid
bilayer on the surface of a microfluidic sample chamber, and hy-
drodynamic force was used to push the DNA into nanofabricated
barriers (Fig. 1A) (16). The DNA was aligned along the barriers,
enabling visualization of hundreds of molecules by TIRFM (Fig. 1
B and C and Movie S1). At 150 mM NaCl and 1 mM ADP MutSα
showed preferential binding to the mismatches, as evidenced by
Author contributions: J.G., F.W., S.R., and E.C.G. designed research; J.G., F.W., and S.R.
performed research; J.G., F.W., S.R., A.J.P., T.F., and S.W. contributed new reagents/ana-
lytic tools; J.G., F.W., S.R., and E.E.A. analyzed data; and J.G., F.W., S.R., E.E.A., and E.C.G.
wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
See Commentary on page 18243.
1Present address: Vaccine Research Center, National Institutes of Health, Bethesda, MD,
2F.W. and S.R. contributed equally to this work.
3Present address: Department of Biological Sciences, University of Cyprus, 2109 Nicosia,
4To whom correspondence should be addressed. E-mail: email@example.com.
See Author Summary on page 18251 (volume 109, number 45).
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| Published online September 24, 2012 www.pnas.org/cgi/doi/10.1073/pnas.1211364109
the “lines” of QD-MutSα that spanned the DNA curtains at the
mismatches (Fig. 1B and Movie S1) and as also was evident from
histograms of the MutSα binding distributions (Fig. 1D). MutSα
disappeared when flow was interrupted and reappeared when flow
was resumed, verifying that the proteins were bound to the DNA
and were not stuck to the surface of the sample chamber (Fig. 1 B
and C and Movie S1). MutSα exhibited a half-life of 9.6 ± 1.5 min
60; SI Appendix, Fig. S2).
MutSα Is Targeted to Mismatches Through a Combination of 1D Sliding
and 3D Diffusion. Next, to determine how MutSα located the
mismatches, we used double-tethered DNA curtains where the
DNA was aligned and anchored by both ends, allowing the mole-
cules to be viewed in the absence of buffer flow (Fig. 2A) (17).
MutSα wasinjectedinto the samplechamber,flowwas terminated,
and the proteins were observed in real time as they searched the
DNA. At physiological ionic strength, MutSα located the mis-
matches either through 1D sliding (42.5% of observed events; n =
distances up to 3.7 µm (∼14.6 kbp), or through apparent 3D dif-
five consecutive frames; any submicroscopic 1D sliding events be-
low this resolution were scored as apparent 3D diffusion. There-
fore, the 42.5% of events attributed to 1D sliding represents the
minimal fraction that can be described by this mechanism (SI
MutSα Scans DNA Flanking the Mismatch by 1D Diffusion. The
mechanism by which MMR proteins search for strand-discrimi-
nation signals remains controversial (1–3, 18). Three proposed
models are (i) translocation, in which MutSα uses the free energy
released by ATP hydrolysis to move along DNA (19, 20); (ii) the
molecular-switch model, in which ATP binding triggers a con-
formational change enabling MutSα to scan DNA by 1D diffusion
G/T mismatches (MM)
flow on flow off flow on
040 3020 10
nanofabricated barriers. (B) Images of a three-tiered DNA curtain with flow on (Left), during a transient pause in flow (Center), and after flow has been
resumed (Right). Flow is from top to bottom; DNA is green, and proteins are magenta. The location of the three tandem G/T mismatches (MM) is indicated. (C)
Kymogram generated from a single DNA molecule subjected to transient pauses in buffer flow (light blue arrowheads) followed by quickly resuming flow
(green arrowheads). (D) Distribution of MutSα bound to mismatch-containing DNA. Error bars in this and subsequent figures represent the SD from
N bootstrap samples (44).
Mismatch recognition by MutSα. (A) Schematic of single-tethered DNA curtains. DNA substrates are anchored to the bilayer and aligned along
Gorman et al. PNAS
| Published online September 24, 2012
(21–23); and (iii) static transactivation, in which ATP-binding
allows stationary MutSα to search for distal strand-discrimination
signals through DNA looping (Fig. 3A) (24–26). Each model
makes unique predictions as to how MutSα should behave in the
DNA curtain assay: Translocation predicts that MutSα should
undergo ATP hydrolysis-dependent unidirectional motion; the
molecular-switch model predicts that MutSα should exhibit ATP-
binding–dependent 1D diffusion; and static transactivation pre-
dicts that MutSα should remain at the mismatch while awaiting
looping-mediated interactions with flanking DNA.
curtains to investigate what happened when mismatch-bound
MutSα was chased with ATP. When mismatch-bound MutSα was
chased with ATP at physiological ionic strength, most proteins
(85%; n = 60/71) were released from the mismatches after a brief
delay (t1/2= 14.6 s; n = 60), consistent with the 8.0 ± 2.7 s half-life
reported for ATP-triggered release from G/T mismatches in bio-
stationaryand did notrespondto ATP. Ofthosethat were released
DNA with no evident sliding, whereas the remaining 85% (n = 51/
60) were released from the mismatch and scanned the flanking
DNA through 1D diffusion (Fig. 3B, SI Appendix, Fig. S4, and
Movie S2). Analysis of the mean squared displacement revealed
25) after ATP-triggered mismatch release. Experiments conducted
at 50 mM NaCl revealed significantly less ATP-dependent release
upon ATP injection, and the remaining proteins either diffused
(18%) or directly dissociated (4%) from the lesions (n = 78; SI
Appendix, Fig. S5), indicating that ATP-triggered release and 1D
diffusion were favored at physiological ionic strength. Our results
also revealed changes in the lifetime of the complexes, as has been
reported for Taq MutS (27). As demonstrated above, MutSα can
that at 150 mM NaCl the lifetime of Mutsα while scanning DNA
3D collision MM
1D diffusion MM
lipid bilayer, are aligned along the nanofabricated barriers, and then are anchored at their downstream ends through a digoxigenin–antibody linkage. (B)
Example of MutSα undergoing 1D diffusion until encountering the lesion. MutSα is magenta, the DNA is not labeled, and gaps in the trajectories reflect QD
blinking. The lower panels highlight the first few seconds of the trajectory. (C) Example of MutSα capturing the mismatch through a direct 3D diffusion.
Experiments in B and C were conducted with double-tethered curtains, and flow was terminated after MutSα entered the sample chamber.
Mechanisms of mismatch targeting by MutSα. (A) Schematic of the double-tethered DNA curtains. DNA substrates are anchored by one end to the
| www.pnas.org/cgi/doi/10.1073/pnas.1211364109Gorman et al.
the MutSα diffusion trajectories after lesion release yielded a lower
bound for the lifetime, t1/2≥ 198 ± 23.4 s (Fig. 3 and SI Appendix,
Fig. S4). MutSα also diffused along the DNA when chased with
ATPγS (62% diffused, 23% dissociated, and 15% remained sta-
tionary; n = 26), indicating that nucleotide binding was sufficientto
trigger mismatch release (Fig. 3C and SI Appendix, Fig. S4). These
findings support the molecular-switch model in which MutSα scans
the flanking DNA by 1D diffusion (21).
MutSα Must Remember Whether It Has Encountered a Mismatch. The
problem: Once MutSα is released from a mismatch and starts
scanning the flanking DNA by 1D diffusion, it must not reengage
the mismatch; otherwise it could become nonproductively trapped
This problem can be illustrated by considering that when MutSα
takes a single diffusive step away from the mismatch, it has a 50%
probability of re-encountering the mismatch on the very next step,
and the average number of times MutSα would re-encounter the
mismatch is equal to N−1, where N is the distance in 1-bp diffusion
steps between the mismatch and the nearest strand discrimination
signal (SI Appendix, Fig. S6). These considerations suggest that
MutSα must be functionally distinct after ATP-triggered release
from a mismatch to avoid redundant lesion recognition.
To evaluate this hypothesis, we assessed the efficiency of lesion
the mismatches. Of the MutSα molecules that recognized the
lesions through a 1D search, none diffused past the lesions (n = 0/
17) (Fig. 2B and SI Appendix, Fig. S3), indicating that initial target
Kymogram and tracking showing the response of mismatch-bound MutSα upon injection of 1 mM ATP. Experiments were conducted with double-tethered
curtains, MutSα was prebound to the mismatch, ATP was injected at 0.1 mL min−1, and flow was terminated after ATP entered the sample chamber. The DNA
was not labeled. “Flow on” indicates when ATP injection was initiated, and “ATP arrival” indicates when ATP entered the sample chamber. The difference
between these time points corresponds to the dead volume of the microfluidics. (C) Response of mismatch-bound MutSα upon injection of 1 mM ATPγS. (D)
Example of spontaneous, ATP-independent release of MutSα, followed by ATP-dependent release.
ATP binding provokes 1D diffusion of mismatch-bound MutSα. (A) Models showing how MutSα might search for strand-discrimination signals (SS). (B)
Gorman et al. PNAS
| Published online September 24, 2012
recognition must be efficient. Moreover, when MutS spontane-
ously escaped from the mismatches (i.e., ATP-independent re-
lease), the proteins typically diffused a short distance along the
DNA and then quickly rebound to the lesions (n = 101 escapes, of
which 97 resulted in rebinding to the lesions without bypass) (Fig.
3D, SI Appendix, Fig. S7, and Movie S3). Considered together,
these data show that before the addition of ATP, MutSα stopped
moving upon encountering the lesions during 1D searches in 97%
of all observed cases (n = 114/118), with only 3% of the observed
encounters leading to diffusion past the lesions. In contrast, after
ATP- (or ATPγS)-triggered mismatch release, we observed a total
of 325 independent, microscopically observed bypass events (n =
51 proteins, corresponding to an average of approximately six
bypasses per protein), none of which led to detectable rebinding;
these values represent the lower bounds for the number of po-
tential bypass events, because the proteins often continued dif-
fusing on the DNA beyond the duration of our observations.
Notably, each microscopically observed bypass reflects ∼1,000
submicroscopic encounters with the lesions; these encounters are
undetectable asindependentevents givencurrent resolution limits
(SI Appendix). These results indicate MutSα no longer recognizes
mismatches as viable targets after ATP-triggered release.
MutSα Diffuses More Rapidly After Mismatch Recognition. The mean
D1Dof MutSα before lesion recognition was 0.009 ± 0.011 µm2
s−1(at 150 mM NaCl; n = 25) (14), but there was a 6.3-fold
increase (Student t test, P = 1.5 × 10−9) in this value to 0.057 ±
0.064 µm2s−1(n = 25) after ATP-mediated release from the
mismatches. Before lesion recognition, the diffusion coefficient of
MutSα is consistent with 1D sliding wherein lateral motion of the
proteinis coupledtoobligatory rotationasittracks the helicalpitch
of the DNA (14). However, after lesion recognition, the mean dif-
fusion coefficient of MutSα exceeded the theoretical threshold for
rotation-coupled 1D diffusion (Drot,theor= 0.024 µm2s−1) (14) and
was physically incompatible with motion involving an obligatory
rotational component (12, 28–30). Structures of MutS and MutSα
reveal the proteins are in intimate contact with DNA along an in-
terface that completely encircles the duplex (24, 31–33). This con-
figuration could accommodate 1D sliding or could allow MutSα to
make very small hops on the DNA as a closed ring, provided there
we cannot yet distinguish between these two possibilities experi-
mentally. However, we can conclude that the rapid movement of
MutSα after mismatch release is most consistent with 1D diffusion
(hopping or sliding) in the absence of an obligatory rotational
molecule measurements of Taq MutS bound to mismatch-con-
to rotation-uncoupled diffusion upon lesion recognition and ATP-
binding may be a common feature of the MutS family of proteins.
Colocalization of MutLα with Mismatch-Bound MutSα We next asked
whether QD-tagged MutLα colocalized with mismatch-bound
MutSα on the single-tethered DNA curtains (Fig. 4). We have
shown previously that MutLα binds DNA, but rather than
remaining stationary, most MutLα (≥95%) diffuses rapidly along
the DNA by a 1D hopping mechanism (Movie S4) (15). We
detected no colocalization of MutLα and MutSα on DNA that
lacked mismatches (n ≥ 2,000; see below), and MutLα alone did
not bind the G/T mismatches in the absence of MutSα but instead
diffused past the lesions without stopping (Fig. 4E and Movie S5).
However, when MutSα was bound to the mismatch, MutLα stop-
ped diffusing at lesion-bound MutSα (Fig. 4 A and B). In the ab-
sence of ATP, both proteins remained at the lesions (Movies S6
andS7),withMutLα exhibiting a half-life of7.8± 0.4min(n= 65)
when colocalized with mismatch-bound MutSα (SI Appendix, Fig.
S2). Mismatch colocalization of MutLα was observed with both
QD-tagged MutSα and untagged MutSα (Fig. 4 C and D). We
conclude that MutLα was targeted specifically to mismatch-
MutLα Is Targeted to Lesion-Bound MutSα by 1D Hopping and 3D
Diffusion. We next watched MutLα as it searched for mismatch-
bound MutSα on double-tethered DNA curtains. MutLα could lo-
cate mismatch-bound MutSα by a 1D-hopping mechanism (55% of
observed events; n = 33/60) or by apparent 3D diffusion (45% of
observed events; n = 27/60) (Fig. 5A and SI Appendix, Fig. S8);
the percentage of events attributed to 1D diffusion represents the
0 4030 2010
binding to mismatch-bound MutSα on single-tethered DNA curtains. MutSα
was bound to the mismatch, followed by injection of MutLα into the sample
chamber. MutLα and MutSα were labeled with different colored QDs. (Top)
MutSα. (Middle) MutLα. (Bottom) Overlay with MutSα (magenta) and MutLα
(green). The DNA was not labeled. Imperfect correspondence between all
individual QD green/magenta pairs reflects the presence of “dark” proteins.
(B) Kymogram generated from a single DNA molecule showing that MutLα
remains stationary and colocalized with mismatch-bound MutSα over time;
the green (MutLα) and magenta (MutSα) signals appear white in the overlay.
Blue and green arrowheads indicate transient pauses in buffer flow, and the
coincident disappearance of the QD signals verifies that neither protein was
stuck to the sample chamber surface. (C) Distribution of QD-tagged MutLα in
the presence of QD-tagged MutSα. (D) Distribution of QD-tagged MutLα
with unlabeled MutSα. Insets in C and D show kymograms illustrating that
MutSα/MutLα remains at the mismatch. (E) Distribution of QD-tagged MutLα
on a single-tethered curtain in the absence of MutSα. MutLα diffuses on DNA
continually in the absence of MutSα (Inset), so the distribution histogram in
E represents the instantaneous distribution of mobile MutLα molecules,
whereas the distribution peaks observed in C and D represent proteins that
are stably bound to the lesions and are not moving along the DNA.
Colocalization of MutLα with mismatch-bound MutSα. (A) MutLα
| www.pnas.org/cgi/doi/10.1073/pnas.1211364109 Gorman et al.