ARTICLE
Received 27 Aug 2015 | Accepted 4 Jan 2016 | Published 3 Feb 2016
DOI: 10.1038/ncomms10607 OPEN
Dynamic DNA binding licenses a repair factor to bypass roadblocks in search of DNA lesions
Maxwell W. Brown1, Yoori Kim1, Gregory M. Williams2, John D. Huck2, Jennifer A. Surtees2 & Ilya J. Finkelstein1,3
DNA-binding proteins search for specic targets via facilitated diffusion along a crowded genome. However, little is known about how crowded DNA modulates facilitated diffusion and target recognition. Here we use DNA curtains and single-molecule uorescence imaging to investigate how Msh2Msh3, a eukaryotic mismatch repair complex, navigates on crowded DNA. Msh2Msh3 hops over nucleosomes and other protein roadblocks, but maintains sufcient contact with DNA to recognize a single lesion. In contrast, Msh2Msh6 slides without hopping and is largely blocked by protein roadblocks. Remarkably, the Msh3-specic mispair-binding domain (MBD) licences a chimeric Msh2Msh6(3MBD) to bypass nucleosomes. Our studies contrast how Msh2Msh3 and Msh2Msh6 navigate on a crowded genome and suggest how Msh2Msh3 locates DNA lesions outside of replication-coupled repair. These results also provide insights into how DNA repair factors search for DNA lesions in the context of chromatin.
1 Department of Molecular Biosciences, Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas 78712, USA.
2 Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, New York 14214, USA.
3 Center for Systems and Synthetic Biology, The University of Texas at Austin, Austin, Texas 78712, USA. Correspondence and requests for materials should be addressed to I.J.F. (email: mailto:[email protected]
Web End [email protected] ).
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DNA-binding proteins must rapidly locate specic sites amidst a vast pool of non-specic DNA. To accelerate the search process, these proteins reduce the total search
space by employing a combination of three-dimensional (3D) diffusion through the nucleus and facilitated one-dimensional (1D) diffusion along the DNA1. During 1D diffusion, proteins can either slide along the helical pitch of the DNA backbone, or can transiently dissociate and associate with the DNA via a series of microscopic hops. Both sliding and hopping have been observed in vitro via single-molecule and ensemble biochemistry approaches, and have also been inferred via single-molecule imaging in live cells26. Indeed, 1D-facilitated diffusion is a common feature of nearly all proteins that scan both DNA13 and RNA7,8 for specic sequences, structures or lesions.
In the eukaryotic nucleus, these proteins must also navigate on chromatin crowded with nucleosomes and other DNA-binding proteins. While the role of nucleosomes and other roadblocks in modulating facilitated diffusion has been considered computationally9,10, there is scant direct evidence that diffusing proteins can bypass nucleosomes and other DNA-bound roadblocks while still recognizing specic DNA sequences or structures. To experimentally address this question, we investigated facilitated diffusion by yeast Msh2Msh3 and Msh2Msh6, two heterodimeric MutS homologue (Msh) complexes that participate in the rst step of eukaryotic mismatch repair (MMR)11,12. Both Msh complexes form sliding clamps on DNA and scan the genome for a partially overlapping but distinct spectrum of DNA mismatches and other extrahelical lesions1315. Once a lesion is found, the Msh complex binds and recruits downstream protein factors to initiate repair. In vitro studies have established that Msh2Msh6 can scan naked DNA for lesions via 1D facilitated diffusion along the DNA track1416. However, both yeast and human Msh2Msh6 diffusion is blocked by nucleosomes in vitro17,18. This led to a model in which Msh2Msh6 mainly scans newly synthesized DNA at the replication fork, which is transiently nucleosome-free1922.
How Msh2Msh3 scans a crowded DNA remains unexplored and the in vivo interactions between Msh2Msh3 and the replication fork are less clear. Msh2Msh3 is also implicated in other genome maintenance pathways that occur outside of replication-coupled MMR, suggesting that it must scan DNA in the context of nucleosomes21,2326. Thus Msh2Msh3 may employ a unique strategy for navigating protein-bound DNA. Here we use single-molecule uorescence microscopy to reveal that Msh2Msh3 scans DNA via a facilitated diffusion mechanism comprised of both 1D sliding and microscopic hopping. Msh2Msh3s DNA interactions are sufciently dynamic to allow the bypass of nucleosomes and other protein obstacles, while still allowing the complex to recognize a single DNA lesion. In contrast, Msh2Msh6 does not hop on DNA and is largely blocked by nucleosomes. Remarkably, a chimeric version of Msh2Msh6 that encodes the Msh3 mispair-binding domain (MBD) imparts roadblock bypass activity to Msh2 Msh6. Thus the Msh3 MBD is sufcient to license Msh complex hopping. Our studies contrast how Msh2Msh3 and Msh2Msh6 navigate a crowded genome and suggest how Msh2Msh3 functions outside of replication-coupled repair. More broadly, we provide a model for how dynamic uctuations within DNA-encircling protein domains may facilitate bypass of other protein roadblocks during 1D-facilitated diffusion.
ResultsVisualizing Msh2Msh3 sliding on DNA curtains. We investigated how Msh2Msh3 slides on DNA by directly monitoring the proteins movement via total internal reection uorescence
microscopy of uorescently labelled Msh2Msh3. Yeast Msh2Msh3 with a hemagglutinin (HA) epitope tag on the Msh2 subunit was overexpressed and puried from yeast cells (Supplementary Fig. 1). To uorescently label Msh2Msh3, we conjugated the protein with anti-HA antibody-coupled quantum dots (QDs). Gel shift and ATPase assays indicated that the QD-tagged Msh2Msh3 retained biochemical activities similar to wild-type protein and remained responsive to specic DNA templates (Supplementary Fig. 1). These data indicate that the QD does not compromise communication between the DNA-binding and ATPase domains of Msh2Msh3. This epitope-labelling strategy has also been used successfully with yeast Msh2Msh6 (refs 17,27).
We used a high-throughput DNA curtain assay for assembling precisely positioned arrays of DNA molecules on the surface of a microuidic owcell (Fig. 1a)17,28,29. In this double-tethered DNA curtains assay, a microscope slide was passivated with a uid lipid bilayer. l-phage DNA (48,502 bp long) was deposited on the surface of the slide and tethered between lithographically
a Linear Cr
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Figure 1 | Visualizing protein diffusion on aligned arrays of DNA molecules. (a) An illustration of the DNA curtains assay (Supplementary Information). A quartz microscope slide is fabricated with an alternating pattern of linear chromium (Cr) diffusion barriers and oval pedestals (B30 nm tall; 13 mm separation). The pedestals are coated with anti-digoxigenin antibodies. The owcell surface is passivated with a uid lipid bilayer (B5 nm tall), and DNA (from l-phage, 48,502 bp) is afxed to the bilayer via a biotin-streptavidin linkage. Buffer ow is used to organize
DNA molecules at the linear diffusion barriers and the free DNA end is immobilized at the Cr pedestals via a digoxigeninantibody interaction. DNA molecules that are tethered at both ends remain extended when buffer ow is turned off. (b) A double-tethered DNA curtain. DNA is stained with YOYO-1, a uorescent intercalating dye (green; top). Quantum dot (QD)-conjugated Msh2Msh3 binds specically to the DNA molecules (magenta; bottom). We did not observe any QD signal when Msh2Msh3 was omitted from the incubation, or when Msh2Msh3 was incubated with an unconjugated QD. YOYO-1 was omitted from subsequent experiments because it can cause laser-induced DNA damage. Scale bar: 10 mm.
(c) Kymograph of a single diffusing Msh2Msh3 protein. QDs blinking (white arrows) indicates that these traces arise from single uorescent particles.
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fabricated chromium (Cr) diffusion barriers. One end of the DNA molecule was biotinylated and afxed to a uid lipid bilayer via a biotinstreptavidin linkage. The second DNA end was labelled with digoxigenin (DIG) and captured at an anti-DIG antibody-coated Cr pedestal positioned 13 mm away from the linear diffusion barrier28. Double-tethered DNA remains in an extended state, allowing us to image Msh2Msh3 in the absence of any hydrodynamic force29. Following DNA curtain assembly, we injected uorescently labelled Msh2Msh3 into the owcells, and observed protein co-localization with the extended DNA molecules (Fig. 1b). DNA-bound QDs were only detected when Msh2Msh3 and anti-HA-conjugated QDs were pre-incubated before injection into the owcell. Pre-incubating Msh2Msh3 with unconjugated QDs did not result in any DNA-bound QDs30. In our typical imaging buffer conditions (40 mM Tris-HCl pH 8, 25150 mM NaCl, 2 mM MgCl2, 2 mM DTT, 0.2 mg ml 1 BSA), we observed intermittent uorescent emission (blinking) from the diffraction-limited uorescent particles. Blinking is an intrinsic property of single QDs and is partially suppressed by including 12 mM DTT in the imaging buffer31. As two QDs are unlikely to blink simultaneously, these blinking events indicate that our uorescent trajectories are from individual uorescent QD-protein particles (Fig. 1c). We conclude that Msh2Msh3 is singly labelled via its HA epitope tag and that the HA-tagged Msh2Msh3 specically binds DNA.
Msh2Msh3 scans DNA via hopping and 1D diffusion. Fluorescently labelled Msh2Msh3 readily associated with the double-tethered DNA curtains and 490% (n 584) of the
DNA-bound molecules exhibited sliding behaviour in the absence of buffer ow (Fig. 1c). These observations are consistent with Msh2Msh3s high afnity for homoduplex DNA in both gel-shift and surface plasmon resonance based assays3235. The time-dependent uorescent signals were t to a two-dimensional Gaussian36 function and the resulting trajectories were used to analyze the movement of Msh2Msh3 along the DNA molecule (Fig. 2a). Msh2Msh3 trajectories had a net displacement of zero base pairs, as would be expected for molecules that are undergoing thermally driven diffusion (Supplementary Fig. 2). To characterize how Msh2Msh3 scans the DNA, we computed the mean-squared displacements (MSD, examples in Fig. 2b) and diffusion coefcients (Fig. 2c) for each sliding molecule. The range of observed diffusion coefcients is consistent with a scanning mode where Msh2Msh3 partially tracks the helical twist of the DNA duplex (see below)3,27,3740.
Msh2 and Msh3 each harbour non-equivalent Walker-type ATP hydrolysis sites, and ADP to ATP exchange is a key feature of mismatch release by all Msh proteins4146,35. To probe the impact of nucleotides on Msh2Msh3 interactions with DNA, we varied the nucleotides that were included in the ow buffer and measured their effect on diffusion (Fig. 2c). Msh2Msh3 diffusion coefcients were nucleotide dependent, increasing approximately twofold from 1 mM ADP (mean 0.0250.021 mm2 s 1;
n 72, range indicates s.d.) to 1 mM AMPPNP
(mean 0.0530.058 mm2 s 1; n 50) or ATP in the absence
of Mg 2 (mean 0.0380.039 mm2 s 1; n 56). In contrast,
the diffusion coefcients of yeast Msh2Msh6 were nucleotide independent27. A complete summary of these results is provided in Supplementary Table 1. Biochemical studies with both human and yeast proteins have suggested that the Msh2 and Msh3 subunits differ in their nucleotide binding and hydrolysis activities when the Msh2Msh3 complex is bound to homoduplex DNA35,42,47. Here we show that ADP and ATP also alter the interactions of the yeast Msh2Msh3 complex with homoduplex DNA, presumably through conformational changes
that are communicated from the ATPase domains to the DNA-binding domain. The lowest diffusion coefcients were with ADP, suggesting that the ADP-bound state interacts most strongly with the DNA and is thus poised for lesion recognition.
Proteins can scan non-specic DNA via 1D sliding, hopping and/or intersegmental transfer. During 1D sliding, the protein retains continuous contact with the DNA, while hopping is characterized by a series of correlated microscopic detachment and reattachment events. Intersegmental transfer can occur when a protein transfers between two DNA sites by directly binding both sides of a DNA loop. Intersegmental transfer is unlikely in our experiments because the DNA molecules are kept in an extended state, precluding looping. Furthermore, intersegmental transfer over distances larger than B1 kb would appear as punctate trajectories with rapid protein re-localization between two distal DNA sites. Observation of over 300 diffusing Msh2Msh3 molecules did not reveal any such discontinuous trajectories on extended DNA molecules.
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Figure 2 | Msh2Msh3 scans DNA via one-dimensional (1D) sliding.(a) Representative traces of diffusing Msh2Msh3 molecules with 1 mM of the indicated nucleotide and 50 mM NaCl in the imaging buffer (black: ADP; blue: ATP; orange: ATPMg 2; green: AMPPNP; pink: no nucleotide). (b) The trajectories in a were used to calculate mean squared displacements (MSD) and the MSDs for each molecule were used to obtain an apparent 1D diffusion coefcient (black: ADP; blue: ATP; orange: ATP-Mg2; green: AMP-PNP; pink: no nucleotide). Solid lines indicate linear ts through the MSD points. (c) Diffusion coefcients for at least 50 molecules in each nucleotide state (with 50 mM NaCl). Red diamonds indicate the mean of the distribution. *P value o0.05 and ***P value o0.001. There is a statistically signicant twofold increase in the mean diffusion condents with non-hydrolyzable nucleotides (P values:2.5 10 2, 1.4 10 4, and 1.2 10 2 for ATPMg2, AMPPNP, and no
nucleotide, respectively). Dashed line: theoretical limit for sliding with rotation along the DNA backbone. Supplementary Table 1 summarizes the means, s.d., and additional P values for each nucleotide condition.(d) Msh2Msh3 diffusion coefcients increase with higher ionic strength. Error bars represent the s.e.m. A linear t to the loglog plot has a slope of 1.30.2, suggesting B1.5 chargecharge interactions between
Msh2Msh3 and DNA are disrupted at increasing ionic strengths. Dashed line: theoretical limit for sliding with rotation along the DNA backbone. Each data point represents the mean of at least 47 diffusing particles, and all results are summarized in Supplementary Table 2.
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a
To differentiate between sliding and hopping, we measured Msh2Msh3 diffusion coefcients at increasing ionic strengths (Fig. 2d and Supplementary Table 2). A higher ionic strength increases electrostatic screening between a protein and DNA. This reduces the fraction of time that a protein is in contact with the DNA and results in increased diffusion coefcients at higher ionic strengths. This approach has recently been used to differentiate between sliding and hopping for a variety of DNA-binding proteins40,48,49. Msh2Msh3 diffusion coefcient increased fourfold, from 0.0310.027 mm2 s 1 at an ionic strength of 51 mM (n 47) to 0.120.14 mm2 s 1 (n 49;
P value: 7.5 10 7) at an ionic strength of 176 mM (Fig. 2d
and Supplementary Table 2). For a protein that mainly interacts with DNA via electrostatic interactions, the diffusion coefcient reports on KD, the microscopic dissociation constant37,50. The number of chargecharge interactions between the protein and DNA can be estimated from the slope of the log(D1D) versus
log(I), where I is the total ionic strength. The slope of the t to the data in Fig. 2d was 1.30.2 (root-mean-square error), which corresponds to 1.50.2 (root-mean-square error) screened charges with an ion condensation parameter of 0.88 for double-stranded DNA50. In contrast, Msh2Msh6 diffusion was not salt-dependent27 and MutS diffusion was weakly salt dependent(0.230.01 screened charges)45. We conclude that Msh2Msh3 hops while diffusing, and that the hopping is facilitated by weak electrostatic contacts between Msh2Msh3 and DNA.
The MutS DNA-binding clamp can undergo large conformational rearrangements in the absence of DNA44,51 and when bound to homoduplex DNA51. Thus, we reasoned that Msh2Msh3 may hop on DNA by transiently opening and closing its DNA-binding clamp, which encircles the DNA duplex in co-crystal structures of Muts homologs with DNA43,44,52,53. Transient opening of the DNA clamp could allow Msh2 and/or Msh3 to briey detach from the DNA, while rapid re-closing would prevent the protein from dissociating into solution (Fig. 3a, top panel). This model suggests three testable hypotheses: (i) diffusing Msh2Msh3 will dissociate from both internal sites (clamp opening) as well as free DNA ends (sliding off the DNA);(ii) Msh2Msh3 dwell times will be sensitive to the addition of competitor DNA; and (iii) Msh2Msh3 may hop between two closely positioned DNA molecules. First, we measured the dissociation positions and dwell times of Msh2Msh3 on single-tethered DNA curtains (Fig. 3a). In this assay, one of the DNA ends is attached to the lipid bilayer surface, while the second end remains free in solution29. Continuous buffer ow is used to keep the DNA extended and also biases protein diffusion towards the free DNA end. The availability of a free DNA end allowed us to measure both Msh2Msh3 dissociation from internal sites and sliding off from the free DNA ends (Fig. 3a). We observed that 80% (n 40/50; 50 mM NaCl, 1 mM ADP) of
diffusing Msh2Msh3 molecules dissociated from internal DNA sites, with nearly half of those molecules (n 21/40) sliding for at
least B1 kb before dissociation (Fig. 3a, middle panel). The remaining 20% (n 10/50) of the molecules slid off the free
DNA end. We also measured the dwell times of Msh2Msh3 on single-tethered DNA curtains. In the absence of competitor DNA, the Msh2Msh3 half-lifes.e. was 761.0 s (n 50; Fig. 3b).
The Msh2Msh3 half-life was reduced threefold (250.4 s, n 50) after addition of homoduplex competitor DNA (4 mM
39-mer double-stranded oligo; Fig. 3c). In contrast, the half-life of Msh2Msh6 was not dependent on the addition of competitor DNA (Supplementary Fig. 3 and ref. 27).
We also observed that Msh2Msh3 could transfer between two closely positioned DNA molecules (Fig. 3d). For these experiments, we assembled high-density double-tethered DNA curtains and analysed regions of the owcell where two extended DNA
molecules were laterally separated by B1 mm, the closest spacing between our Cr pedestals. We observed that Msh2Msh3 complexes could transfer between two such adjacent DNA molecules (Fig. 3d). Here Msh2Msh3 scans the left DNA molecule, followed by transfer and diffusion on a neighbouring DNA. We observed such transfer events for 46% (n 23/50) of
diffusing Msh2Msh3 molecules (with 1 mM ADP, 100 mM NaCl in the imaging buffer). These observations do not stem from binding by two different Msh2Msh3 complexes because all free enzymes have been ushed out of the owcell. We also ruled out the possibility that these observations are due to several Msh2Msh3 proteins per QD by conjugating fewer than one antibody per QD54. Msh2Msh3 can transfer between the two DNA strands via either intersegmental transfer or by hopping.
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Figure 3 | Msh2Msh3 transiently dissociates from DNA during 1D sliding. (a) Cartoon illustration (top) and a kymograph (bottom) of Msh2Msh3 dissociating from a single-tethered DNA molecule. In the absence of competitor DNA (mock injection), Msh2Msh3 slides along the DNA and dissociates from both internal sites (white arrow) and from free DNA ends (yellow arrow). Msh2Msh3 dissociates from DNA curtains more rapidly after competitor DNA is injected in the owcell (dashed line). Quantication of the Msh2Msh3 lifetimes (b) without or (c) with competitor DNA. Lifetimes are t to a single exponential decay and the half-livess.e. are reported in the panels. (d) Msh2Msh3 (magenta) can transfer between adjacent DNA molecules. Initially, Msh2Msh3 diffuses on the left DNA molecule. After 3 s, the complex transfers to an adjacent DNA. After the diffusion data was acquired, the DNA molecules were stained with YOYO-1 (green). Scale bar, 2 mm (e) A trace of the complete trajectory (white) is superimposed on the locations of the two DNA molecules. The starting and ending points are indicated by yellow andred triangles, respectively.
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The DNA molecules were uorescently labelled after the Msh2Msh3 diffusion traces were acquired, so we cannot unambiguously distinguish between these two mechanisms. Regardless, our results demonstrate that Msh2Msh3 scans the genome by a combination of hopping and 1D sliding, and that these facilitated diffusion modes are consistent with transient opening of the DNA-binding clamp.
Diffusing Msh2Msh3 can bypass protein obstacles on DNA. We next tested whether dynamic opening of the Msh2Msh3 DNA clamp may also facilitate bypass of other DNA-bound proteins that would be encountered during the lesion search process. First, we investigated whether two diffusing Msh2Msh3 complexes can bypass each other as they slide on the same DNA molecule. For these experiments, two Msh2Msh3 fractions were each conjugated with spectrally distinct QDsthe rst emitted in the green channel (605 nm peak uorescence emission) and the second in the magenta channel (705 nm emission). The differentially labelled proteins were mixed in a 1:1 ratio and injected into a owcell with pre-assembled DNA curtains (Fig. 4a). As expected, both species readily diffused on DNA. We observed bypass events when two differentially labelled molecules collided (Fig. 4a), suggesting that one Msh2Msh3 can bypass a second diffusing molecule on the same DNA strand. Next,
we determined whether Msh2Msh3 also bypasses other protein roadblocks. We used EcoRI(E111Q), a hydrolytically defective restriction enzyme that is frequently used as a model protein roadblock55. Fluorescent EcoRI(E111Q) retains sub-nanomolar binding afnity to the ve EcoRI sites in our DNA substrate30 and has previously been shown to block diffusing MutS16. Remarkably, Msh2Msh3 readily bypassed uorescent EcoRI(E111Q), indicating that it can diffuse past both moving and stationary protein roadblocks (Fig. 4b).
Nucleosomes are the most frequent DNA obstacles that Msh2Msh3 encounters in vivo. To explore how Msh2Msh3 navigates on a nucleosome-coated DNA track, we puried recombinant histone octamers with an N-terminal triple-FLAG epitope tag on the H2A subunit (Supplementary Fig. 4a). Epitope-labelled and wild-type histone octamers were indistinguishable in gel-based reconstitution assays (Supplementary Fig. 4b). We deposited an average of 4.51.7 nucleosomes on the DNA substrate via a salt-dialysis protocol (Supplementary Fig. 4 and Methods). Next, we determined whether Msh2Msh3 bypassed nucleosomes that are not conjugated with QDs (unlabelled nucleosomes). In these assays, nucleosome-coated DNA was rst assembled into DNA curtains after which uorescent Msh2Msh3 was ushed into the owcell. After 10 min of monitoring Msh2Msh3 diffusion, the experiments were stopped and nucleosomes were labelled in situ with uorescently labelled antibodies. Msh2Msh3 readily bypassed unlabelled nucleosomes as it diffused on DNA (Fig. 4c, top). We ruled out the possibility that these observations are due to several Msh2Msh3 proteins per QD by observing hopping with HA antibodies labelled with the much smaller Alexa488 uorescent probe and also when Msh2Msh3 was incubated with a large excess of QDs (1:5 protein:QD ratio; Supplementary Fig. 4e). To quantify the nucleosome bypass frequency, we scored all Msh2Msh3 diffusion trajectories that entered a 750 bp collision zone centred on a nucleosome. The collision zone was dened as three times the s.d. of our precision in localizing a uorescent nucleosome. A collision was scored as a bypass event when Msh2Msh3 diffused from one side of a nucleosome to the other through the collision zone. We observed nucleosome bypass events in 46% of all collisions (53/115 collisions; 58 trajectories). Msh2Msh3 also bypassed QD-labelled nucleosomes (605 nm emission, B10 nm radius39), creating a much larger barrier to 1D diffusion (Fig. 4c, middle). Remarkably, the bypass probability was 43% (12/28 collisions; 17 trajectories), nearly identical to unlabelled nucleosome obstacles. Next, we monitored collisions between Msh2Msh3 and dense nucleosome arrays. For these experiments, DNA curtains were reconstituted at fourfold higher octamer to DNA ratios, such that we could no longer resolve individual nucleosomes. After reconstitution at these increased ratios, we estimated 20 or more nucleosomes per DNA molecule. Surprisingly, 58% (n 30/52) of Msh2Msh3 molecules
continued to show diffusive motion on these nucleosome-coated DNA substrates (Fig. 4c, bottom). As nucleotide binding modulated the apparent diffusion coefcient on naked DNA (Fig. 2c), we determined the effect of nucleotide on Msh2Msh3s ability to bypass nucleosomes (Supplementary Fig. 5). Msh2Msh3 hopped over nucleosomes in the presence of all nucleotides, but we observed fewer nucleosome bypass events when AMPPNP (28%, n 14/50) or ATPMgCl2 (33%,
n 16/50) was included in the imaging buffer (Supplementary
Fig. 5). These results further support the model that nucleotide binding triggers conformational changes that are transmitted to the DNA clamp domain, ultimately altering Msh2Msh3 facilitated diffusion on both naked and crowded DNA35,42,56. In contrast, Msh2Msh6 is rarely able to pass unlabelled nucleosomes, and is completely blocked by QD-conjugated
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Figure 4 | Diffusing Msh2Msh3 bypasses protein roadblocks.(a) Cartoon illustration (top) and kymograph (bottom) of green and magenta Msh2Msh3 complexes bypassing each other on the same DNA molecule. The bypass events are indicated with white arrowheads. (b) Kymograph of Msh2Msh3 (magenta) bypassing EcoRI(E111Q) (green). (c) Kymographs of Msh2Msh3 bypassing unlabelled nucleosomes (top, green) and QD-labeled nucleosomes (middle, green). Msh2Msh3 also diffuses on dense nucleosome arrays (bottom, green). These arrays appear completely green due to the large quantity of post-labelled nucleosomes. Msh2Msh3 (magenta) appears as white when co-localized with nucleosomes.
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nucleosomes (Supplementary Fig. 6). As with Msh2Msh3, we conrmed that Alexa488-aHA labelled Msh2Msh6 was also blocked by nucleosomes, indicating that QDs do not contribute to the observed differences in facilitated diffusion between the two Msh complexes (Supplementary Fig. 6d). These results highlight that Msh2Msh3, unlike Msh2Msh6, has the potential to hop over nucleosomes and other protein obstacles as it scans the genome for DNA lesions.
The Msh3 MBD enables roadblock bypass. Our results highlighted dramatic differences between the scanning modes of Msh2Msh3 and Msh2Msh6 on homoduplex and nucleosome-coated DNA. As the Msh2 subunit is present in both heterodimers, we reasoned that the MBDs of the Msh6 and Msh3 subunits may regulate these differences between the two complexes. To test this hypothesis, we characterized a chimeric Msh2Msh6 in which the Msh6 MBD is swapped for the Msh3 MBD20. This Msh2Msh6(3MBD) chimera (Fig. 5a) partially rescues Msh3-null phenotypes in vivo and exhibits increased specicity for Msh2Msh3-like lesions in vitro20. We introduced an HA epitope tag into the Msh2 subunit of Msh2Msh6(3MBD), and assayed the activity of the chimeric protein via ATPase and electrophoretic mobility shift assays (EMSA) (Supplementary Fig. 7). The ATPase activity of the chimeric complex was responsive to DNA both with and without QDs (Supplementary Fig. 7b). In accordance with previous studies, Msh2 Msh6(3MBD) had an increased afnity for a 8 insertion/
deletion loop (Supplementary Fig. 7c)20. For single-molecule assays, Msh2Msh6(3MBD) was labelled with QDs, as described for Msh2Msh3 and Msh2Msh6. Fluorescent Msh2Msh6(3MBD) readily bound DNA curtains with 76%
(n 269) of the molecules showing diffusive trajectories on DNA
(Supplementary Fig. 7).
Remarkably, replacing the Msh6 MBD substantially altered the dynamic behaviour of the Msh complex on DNA, more closely resembling Msh2Msh3 than Msh2Msh6. Msh2Msh6(3MBD) diffusion coefcients increased monotonically with increasing ionic strength (Fig. 5b and Supplementary Table 3), indicating that, like Msh2Msh3, this construct diffuses via a combination of sliding and hopping. Conversely, Msh2Msh6 did not exhibit this behaviour (Fig. 5c and ref. 27). Furthermore, Msh2Msh6(3MBD) readily transferred between adjacent DNA molecules (44%, n 22/50), and also bypassed nucleosome
roadblocks (Fig. 5d). Remarkably, both of these activities occurred with nearly the same frequencies for Msh26(3MBD) and Msh2Msh3 (Fig. 5e). In contrast, only 8% (n 4/50) of
Msh2Msh6 molecules transferred between adjacent DNA strands. These results are consistent with structural and functional studies that have shown distinct DNA-binding modes for Msh2Msh3 and Msh2Msh6 (refs 43,57). Our single-molecule data indicate that these structural differences are translated into distinct dynamic behaviours on homoduplex DNA substrates that have important implications for the respective search mechanisms. We conclude that the Msh3 MBD is sufcient to alter the dynamics within the Msh2Msh6 DNA-binding clamp, which stimulates a combination of facilitated diffusion and roadblock bypass activities of the Msh2Msh6(3MBD) chimera.
Msh2Msh3 bypasses nucleosomes during lesion recognition. Msh2Msh3 recognizes and facilitates processing of 30 single-stranded non-homologous tail DNA structures during single-strand annealing (SSA), which can occur throughout the cell
a hMsh2 hMsh6
MBD
b
c
5
Msh2Msh6
Msh2Msh3
Msh2Msh6 (3MBD)
100
***
***
***
1 mM ADP
Diffusion coefficient
(m2 s1 )
Relative diffusion
coefficient
3
101
1
Msh3
Msh6
102
1
1,047
51 76 101 126 176 Ionic strength (mM)
51 176 Ionic strength (mM)
126
MBD (155285)
MBD
76
101
1
1,242
Msh6(3MBD)
e
1304 4221,242
Msh2Msh6
Msh2Msh3
Msh2Msh6 (3MBD)
9%
45%
44%
0%
43%
40%
50%
Percent bypass
d
Unlabelled nucleosomes
30%
Bypass
5 m
5 s
10%
QD-labelled nucleosomes
Bypass
5 m
5 s
Unlabelled nucleosomes
Labelled nucleosomes
Figure 5 | Characterizing the sliding of a chimeric Msh2Msh6(3MBD). (a) Top: hMSH2MSH6 structure (PDB: 2O8B). Msh2, Msh6 and DNA are shown in blue, grey and green, respectively. The Msh6MBD is coloured in orange and makes multiple contacts with the DNA. Below: domain map of yeast Msh2Msh6(3MBD) chimera. (b) Msh2Msh6(3MBD) diffusion coefcients as a function of total ionic strength (nZ50 for each condition). Red diamonds indicate the mean diffusion coefcients. Asterisks indicate ***P value o0.001. Diffusion coefcients increase at higher ionic strengths (see Supplementary
Table 3 for P values). (c) Summary of the relative diffusion coefcients for Msh2Msh6 (grey, from ref. 27), Msh2Msh3 (orange, this study) and Msh2Msh6(3MBD) (blue, this study). Diffusion coefcients for each protein are normalized to their respective values at the lowest ionic strength. Error bars are the s.e.m. (d) Kymographs of Msh2Msh6(3MBD) (magenta) bypassing unlabelled (top, green) and QD-labelled (bottom, green) nucleosomes. (e) Quantication of the nucleosome bypass frequencies for each of the three heterodimers (unlabelled nucleosomes: n 100 for
Msh2Msh6, Msh2Msh3, and Msh2Msh6(3MBD); pre-labelled nucleosomes: n 31, 28 and 25 for Msh2Msh6, Msh2Msh3 and
Msh2Msh6(3MBD)). The data for Msh2Msh6 are acquired in this study and agree with a previous study17.
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cycle and is not coupled to DNA replication21. Msh2Msh3 may thus need to bypass nucleosomes as it scans the genome to recognize these single-strand DNA (ssDNA) aps. Therefore, we explored Msh2Msh3s ability to recognize specic lesions on a nucleosome-coated DNA track. We introduced an 18-nucleotide 30-ssDNA ap 20 kb downstream of the biotinylated DNA end (Supplementary Fig. 8a and Supplementary Table 4). Over 95% of the DNA molecules incorporated the 30-ssDNA ap (Supplementary Fig. 8c and Information), and site-specic incorporation was conrmed by both gel assays and single-molecule uorescence imaging (Supplementary Fig. 8). We next incubated Msh2Msh3 with the lesion-containing DNA and measured the locations of DNA-bound Msh2Msh3 complexes (Fig. 6a). The DNA-binding histogram showed a strong
enrichment at the lesion, indicating that the complex specically binds the 30-ssDNA ap (Fig. 6a). We also observed an increased afnity for 12 nucleotide 50-ssDNA ends over homoduplex regions, with 9% of the molecules localizing to the vicinity of these free DNA ends in our single-tethered DNA curtain assay (inset, Fig. 6a). These results agree with previous studies that have reported increased afnity of Msh proteins for ss/double-stranded DNA junctions34.
Msh2Msh3 has the potential to recognize lesions by one of two non-exclusive mechanisms: (i) 1D scanning (Fig. 6b) and(ii) 3D collisions (Fig. 6c). To observe lesion recognition, we assembled the lesion-containing DNA substrate into a double-tethered DNA curtain and imaged the lesion-recognition reaction in real-time (with 1 mM ADP, in the absence of buffer ow). We saw evidence of both lesion recognition mechanisms, with 27% of the molecules (n 4/15) directly binding the lesion via a
3D collision mechanism (within our B300 bp resolution). The remaining 73% of molecules diffused for at least 1 kb along the DNA before stopping at the lesion site. Next, we deposited nucleosomes on the lesion-containing DNA (Fig. 6b). Remarkably, diffusing Msh2Msh3 could readily bypass a nucleosome en route to binding the 30-ssDNA ap (Fig. 6b).
We limited our analysis to nucleosomes that were at least 1 kb away from the DNA lesion, ensuring that we could resolve both the hopping and lesion recognition events by the same Msh complex. These results show that Msh2Msh3 can hop over nucleosomes while maintaining its ability to recognize a single DNA lesion.
DiscussionMsh proteins form sliding clamps on DNA to recognize mismatches and other DNA structures that arise during DNA replication and homologous recombination (HR). We show that Msh2Msh3 scans DNA via a combination of sliding and hopping, a fundamentally different mechanism than previously reported for prokaryotic MutS and eukaryotic Msh2Msh6 (refs 27,45,51,58). Hopping is facilitated by rapid opening and closing of the Msh2Msh3 DNA-binding clamp, as revealed by three lines of evidence: (i) the apparent 1D diffusion coefcient increased Bfourfold when the total ionic strength is varied from 51 to 176 mM (Fig. 2d); (ii) Msh2Msh3 dwell times on DNA are dependent on the addition of competitor DNA (Fig. 3b,c); and (iii) Msh2Msh3 can dynamically transfer between two neighbouring DNA molecules (Fig. 3d). In addition to increasing
a
Normalized
frequency
0.10
0.05
0
N = 503Xc= 202.7 kb
0
10 20 30 40
Position (kb)
b
t
3 m
3 s
Flap
Position
(kb)
20
28
Bypass
Nuc.
Nuc.
24
0
10 20 30 40
Time (s)
c
t
Figure 6 | Msh2Msh3 recognizes DNA lesions via both 1D slidingand 3D collisions. (a) Distribution of Msh2Msh3 molecules on lesion-containing DNA. The red line is a Gaussian t to the data (n 503).
The center of the peak corresponds to the expected location of the DNA ap (20 kb from the top DNA barrier). The inset shows seven representative DNA molecules with ap-bound Msh2Msh3. (b) Cartoon illustration (top) and kymograph (middle) of Msh2Msh3 (magenta) hopping over a nucleosome (post labelled; green) and stopping at a DNA lesion (30-ssDNA ap; red octagon). The corresponding single-particle trajectory is shown below. The Msh2Msh3 trajectory is in magenta, the nucleosome position is represented with a solid green line, and the ap position is indicated as a dashed red line (also see Supplementary Fig. 8). (c) Cartoon (top), kymograph (middle), and single-particle trajectory of Msh2Msh3 (magenta) recognizing a 30-ssDNA ap via 3D collision (bottom). (d) A model for how Msh2Msh3 (left) and Msh2Msh6 (right)
scan DNA to nd a lesion. Msh2Msh3 diffuses via a combination of 1D sliding (1) and hopping (2). Msh2Msh3 dynamics facilitate transient release from the DNA track and hopping over nucleosomes (3) but still support lesion recognition (red octagon) (4). In contrast, Msh2Msh6 does not hop on DNA and is blocked by a nucleosome roadblock.
3 m
3 s
Nuc.
Position
(kb)
20 Flap
16
24
3D collision
0
5 10 15
20
Time (s)
d 1
3
X
2
4
X
Msh2Msh3 Msh2Msh6
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the apparent diffusion coefcient, hopping permits Msh2Msh3 to bypass diverse protein obstacles (Fig. 4), and to recognize a lesion on a nucleosome-coated DNA (Fig. 6b). In contrast, sliding of Escherichia coli MutS on DNA is blocked by EcoRI(E111Q)16 and sliding of human and yeast Msh2Msh6 is inhibited by nucleosomes17,18 (also see Supplementary Fig. 6).
How do Msh2Msh3 and Msh2Msh6 differ in their ability to bypass protein obstacles? We propose a model where the Msh2Msh3 DNA-binding clamp transiently opens and closes as the protein slides on DNA (Fig. 6d). Msh2Msh3 scans DNA via a combination of 1D sliding and hopping (steps 1 and 2 in Fig. 6d). Transient opening and re-closing of the DNA-binding clamp allows the protein to hop by briey dissociating and re-engaging the DNA track (step 3 in Fig. 6d). Hopping facilitates long-range movement between two segments of DNA, as well as bypass of protein obstacles. Remarkably, Msh2Msh3 is able to re-establish contact with the DNA such that it can recognize an extrahelical lesion after it hops over a nucleosome (step 4 in Fig. 6d). In contrast, we propose that Msh2Msh6 and bacterial MutS have less dynamic DNA-binding clamps, which precludes hopping and obstacle bypass (Fig. 6d, right panel).
We reasoned that the different dynamic behaviours of Msh2Msh3 and Msh2Msh6 stem from the distinct types of DNA lesions that are recognized by each complex. Msh2Msh6 recognizes mismatches and small insertion/deletion loops14,15. These lesions may lead to relatively moderate distortions of overall DNA duplex structure5961. In contrast, Msh2Msh3 chiey recognizes some mismatches, large insertion/deletion loops and ssDNA aps14,15. Such lesions are likely to lead to larger DNA distortions and are reected in the differences between the Msh3 and Msh6 MBDs. The Msh3 MBD lacks a highly conserved phenylalanine-X-glutamate motif that directly interacts with DNA in bacterial MutS and Msh6 family proteins43,44. Instead, the Msh3 MBD encodes a conserved tyrosinelysine pair that may result in looser contact with homoduplex DNA34,43,44,52. Similarly, the position of Msh2 is distinct in the context of the Msh3 MBD versus that of Msh6, making direct contacts with the lesion52. We tested our hypothesis by characterizing a chimeric Msh2Msh6 that encodes the 130-residue MBD from Msh3. Remarkably, this chimeric construct gained the ability to hop on naked DNA and to bypass nucleosomes (Fig. 5).
Post-replicative MMR largely occurs during DNA replication, where B250 bp of newly replicated DNA is nucleosome-free for a short time19. Msh2Msh6 is present at the replication fork and can thus scan this nucleosome-free region before the DNA is fully chromatinized21. Indeed, human Msh2Msh6 is recruited to chromatin early in S-phase via the Msh6-encoded PWWP domain62. After DNA replication, iterative cycles of Msh2Msh6 loading delays nucleosome deposition and may displace existing nucleosomes6366, extending the time window for MMR on nucleosome-free DNA. Both Msh2Msh6 and Msh2Msh3 also function in HR, where these proteins block recombination between divergent DNA sequences (homeologous recombination)21,26. In homeologous recombination, Msh2Msh6 recognizes mismatches within D-loops and clears RAD51 laments, which are likely to be nucleosome-free26,67. In contrast, Msh2Msh3 must locate ssDNA aps that occur during HR and single-strand annealing. Both repair processes occur throughout the cell cycle and are not always coupled to DNA replication. In addition to potential direct recruitment via proteinprotein interactions, Msh2Msh3 may also need to recognize lesions via diffusion on a nucleosome-coated track. Further studies will be required to dene how lesion binding and post-recognition complexes alter Msh2Msh3 diffusion on a nucleosome-coated DNA.
In conclusion, our results demonstrate that Msh2Msh3 diffuses on DNA via a combination of 1D sliding and hopping. Hopping is facilitated by transient opening of the DNA-binding clamp, which is in turn modulated by the Msh3 MBD. Msh2Msh3 can hop over protein obstacles to recognize lesions that are on a nucleosome-coated DNA substrate. To our knowledge, this is the rst direct demonstration that dynamic opening of a clamp-like DNA-binding domain can facilitate roadblock bypass during 1D-facilitated diffusion. These results provide insight into how a eukaryotic DNA repair factor bypasses roadblocks to function outside of replication-coupled MMR. More broadly, this study provides a paradigm for how uctuations within DNA-binding domains may facilitate bypass of protein roadblocks during 1D-facilitated diffusion.
Methods
Buffers. Our typical imaging buffer conditions contained 40 mM Tris-HCl pH 8, 25150 mM NaCl, 2 mM MgCl2, 2 mM DTT, 0.2 mg ml 1 BSA. The total ionic strength I was calculated using:
I
1
2 X
N
i1
ciz2i 1
where c is the molar concentration of ion i, z is the charge number of that ion, and the sum is taken over all ions N. The pKa of Tris base is 8.1 at 25 C, therefore 45% of the 40 mM Tris in our solution will be charged, contributing 9 mM to the total ionic strength. The hydrochloric acid used to titrate the Tris base down to a pH of 8.0 contributes 11 mM to the total ionic strength. Adding 2 mM MgCl2 contributes 6 mM to the total ionic strength (for the divalent magnesium ions,z2 4). Addition of 25150 mM NaCl adds an additional 25150 mM of ionic
strength. Thus, we varied the total ionic strength (I) from 51 to 176 mM.
Purication of Msh23 and Msh26. Saccharomyces cerevisiae Msh2HAMsh3 and Msh2HAMsh6 were puried by sequential ion exchange chromatography and ssDNA-afnity chromatography27,34. Msh2HAMsh6 bound less tightly to Q-Sepharose fast ow (QFF, GE Life Sciences) than to PBE94 and therefore the starting NaCl concentration for the QFF column was reduced to 250 mM. Msh2HAMsh6 eluted from QFF at B300 mM NaCl. E. coli-expressed
Msh2HAMsh6 and Msh2HAMsh6(3MBD) were puried over QFF, ssDNA cellulose and QFF columns in the same manner as S. cerevisiae-expressed Msh2HAMsh6 (refs 20,68). Triple-FLAG epitope-tagged EcoRI(E111Q) was puried from E. coli using an inteinchitin-binding domain fusion construct (NEB IMPACT Kit)30.
Purication of wild-type and 3 FLAG hH2A. The wild-type or 3 FLAG H2A
plasmid was transformed into BL21(DE3) codon plus RIL cells (Agilent). A colony was inoculated into 50 ml LB broth with 50 mg ml 1 carbenicillin and 34 mg ml 1 chloramphenicol, and grown at 37 C overnight. Fifteen millilitres of the overnight culture was seeded into 1.5 l LB broth and grown in the presence of both antibiotics. When the culture reached an OD600 of 0.6, 0.2 mM IPTG was added and the induction continued at 37 C for 3.5 h. Cells were harvested at 5,000g for15 min, and resuspended in 150 ml lysis buffer (100 mM NaPO4 pH 8.0, 8 M urea, 10 mM DTT, 15 mg benzamidine). Urea was deionized (501-X8 resin, Bio-Rad) immediately before use. Cells were lysed by sonication on ice, and centrifuged at 12 C and 100,000g for 30 min. A 100 ml column was packed with 25 ml of SP-Sepharose Fast Flow resin (GE Healthcare), washed with 10 column volumes (CV) of water, and equilibrated with 10 CV of wash buffer (100 mM NaPO4pH 8.0, 7 M urea, 10 mM DTT, 0.3 mM benzamidine). The 150 ml supernatant was added to the column and rotated for 1 h at room temperature (RT). The supernatant was washed with 5 CV of wash buffer, and eluted with ve fractions of 5 ml elution buffer (100 mM NaPO4, pH 8.0, 1 M NaCl, 7 M urea, 10 mM DTT,4 mM benzamidine). The resulting 25 ml eluent was loaded onto a Superdex-200 column (GE Healthcare) equilibrated in SAU-100 buffer (20 mM NaAcetatepH 5.2, 7 M urea, 100 mM NaCl, 1 mM EDTA, 5 mM b-mercaptoethanol). Gel ltration was performed using 120 ml SAU-100 buffer and the histone-containing fractions were loaded onto a tandem Q/SP column (10 ml each). After loading the histones, the tandem column was washed with 5 CV of SAU-100. The Q column was removed and 3 FLAG H2A was eluted with a gradient from 0 to 100%
SAU-600 (20 mM NaAcetate pH 5.2, 7 M urea, 600 mM NaCl, 1 mM EDTA, 5 mM b-mercaptoethanol) over 20 CV. The eluate was fractionated in 1.2 ml fractions and the histone-containing fractions were conrmed by SDSPAGE. Protein concentration was determined by running an SDSPAGE gel with BSA standards (Pierce Biotechnologies). Puried protein was lyophilized and stored in 80 C.
Both wild-type and 3 FLAG H2A proteins puried with similar elution proles
and nal yields.
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Inclusion body purication of histones. Each of the three histones (H2B, H3 and H4) was puried from inclusion bodies as previously described, with minor modications69. Briey, each histone was overexpressed in BL21(DE3) codon plus RIL cells. Cells were grown at 37 C and 0.2 mM IPTG was added at OD600 0.6,
followed by additional 3 h of induction at 37 C. Cells were harvested by centrifugation at 5,000g for 20 min at RT. Cell pellets were suspended in 25 ml TW buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM EDTA), and stored at
80 C until use. Each pellet was thawed and diluted up to 35 ml total volume using TW2 buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM EDTA, 5 mM b-mercaptoethanol, 1 mM benzamidine, and 1% (w/v) Triton X-100). Cells were lysed by sonication on ice for 2 min (10 s on50 s off). To harvest the inclusion bodies, the lysate was centrifuged at 20,000g for 20 min at 4 C. The pellets were rinsed with TW2 buffer by suspending, and centrifuged at 20,000g for 20 min at 4 C. The pellet was washed in same way twice using TW2 without Triton X-100, and the nal pellet was stored at 80 C.
Purication of histones. Each inclusion body pellet was mixed with 200 ml DMSO and 6.5 ml unfolding buffer (20 mM Tris-HCl, pH 7.5, 7 M guanidinium-HCl, and 10 mM DTT) by gently agitating for 1 h at RT, and centrifuged at 20,000g for20 min at 4 C. This was repeated two more times, and the supernatant from each centrifugation was dialyzed against 1 l urea buffer (10 mM Tris-HCl pH 8.0, 7 M urea, 1 mM EDTA, 5 mM b-mercaptoethanol, and 100 mM NaCl for H2B, 200 mM
NaCl for H3 and H4) using 3,500 or 7,000 MWCO dialysis tubing (SnakeSkin, Pierce Biotechnologies). A tandem Q/SP column was equilibrated with 10% buffer B (10 mM Tris-HCl pH 8.0, 7 M urea, 1 mM EDTA, 1 mM DTT, 1 M NaCl) and 90% buffer A without 1 M NaCl. The dialyzed histones were loaded and washed with 10% buffer B. H2B was eluted from 10 to 40% buffer B over 20 CV in 200 min and H3 and H4 were eluted from 20 to 50% buffer B over 20 CV. The puried histones were checked by SDSPAGE, lyophilized and stored at 20 C until use.
DNA substrates for total internal reection uorescence microscopy.
DNA substrates for single-molecule experiments were prepared by annealing oligonucleotides IF003 and IF004 to l-phage DNA (Supplementary Table 4;
New England Biolabs). Briey, B15 nM l-phage DNA was heated to 65 C, combined with 1 mM IF003 and IF004, and allowed to slowly cool to RT. After cooling, the reaction was supplemented with ATP to 1 mM, T4 DNA ligase (2,000 units; New England Biolabs) and incubated overnight at RT. The ligase was heat inactivated and the reaction was passed over an S-1000 gel ltration column (GE) to remove excess proteins and oligonucleotides. The DNA was stored at 4 C or immediately isopropanol precipitated for nucleosome reconstitution.
Histone octamer assembly. Each of the four histone was dissolved in unfolding buffer (20 mM Tris-HCl pH 7.5, 7 M guanidinium-HCl, and 10 mM DTT), and gently agitated for 1 h at RT. The histones were mixed in equimolar ratios of H3/H4, and a 10% higher molar ratio of H2A/H2B relative to H3/H4. The mixture was adjusted to a nal concentration of 1 mg ml 1 and dialyzed against refolding buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 5 mM b-mercaptoethanol, 2 M NaCl) using 3,500 MWCO dialysis tubing with several buffer exchanges over 48 h. The dialyzed mixture was centrifuged to remove aggregates, and concentrated using spin-concentrators (Amicon Ultra-15; Millipore) to a nal volume of about 1 ml. Gel ltration over a Superdex-200 (GE Healthcare) using SAU-200 was performed to resolve histone octamers from dimers and tetramers in the refolding buffer. The octamer peak fractions were combined, concentrated using a 10,000 MWCO spin-concentrator (Amicon Ultra-4, Millipore), and ash frozen using liquid N2. The resulting histone octamers were stored in 80 C until use.
Nucleosome reconstitution. To reconstitute human nucleosomes on the l-phage DNA substrate, the DNA was rst ligated to biotinylated and DIG-terminated oligonucleotides (IF003 and IF004, respectively) and gel-ltered through an S-1000 column (GE). The DNA was concentrated using isopropanol precipitation, and dissolved to a nal concentration of 70 ng ml 1 in TE with high salt (10 mM
Tris-HCl pH 8.0, 1 mM EDTA, 2 M NaCl). For reconstitution, 30 ml of the DNA (nal concentration of B20 ng 1 was used in total volume of 100 ml. The octamer was diluted 10-fold in dilution buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 2 M
NaCl) right before use. The 100 ml mixture was dialyzed using a mini dialysis button (10 K MWCO, Bio-Rad) against 400 ml storage buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 1 mM DTT) that contained gradually decreasing concentrations of NaCl. Dialysis was performed in a cold room at 4 C for at least 90 min for each 1.5 M, 1 M, 0.8 M, 0.6 M, 0.4 M NaCl containing storage buffer.0.2 M NaCl buffer was used for overnight dialysis. At a nominal input ratio of 1:75 (DNA:octamer), we counted approximately 15 nucleosomes per DNA molecules. The large nominal DNA:octamer ratio probably stems from octamer loss due to aggregation onto the dialysis membrane and polypropylene tubing during the extended dialysis procedure70. The nucleosome-coated DNA was stored at 4 C for up to two weeks.
Msh2Msh3 DNA-binding and ATPase activities. DNA substrates for the gel mobility shift and ATPase assay were radio-labelled (where appropriate) using
homoduplex (LS1/LS2), 1 loop (LS2/LS6T), 8 loop (LS2/LS8) and 30-ssDNA
ap (LS1/LS3/LS16) (Supplementary Table 4)34. Next, we performed titrations of Msh2HAMsh6, Msh2HAMsh3 and Msh2HAMsh6(3MBD) according to standard protocols34,35. Briey, each protein was incubated at the indicated concentration with 1 nM labelled DNA substrate, 20 mM HEPES (pH 7.5), 100 mM NaCl. 1 mM DTT, 40 mg ml 1 BSA, 2 mM MgCl2 in a total of 10 ml. The reactions were assembled on ice and then incubated at RT for 5 min following the addition of the DNA substrate. The reactions were electrophoresed through a 4%, X TBE polyacrylamide gel at 130 V for 45 min in the cold. The gels were dried, exposed to a PhosphorImager screen (Molecular Dynamics) and quantied with ImageQuant (GE Life Sciences).
For gel mobility shift assays in the presence of antibody and/or QDs, Msh2HAMsh3 (200 ( ) or 400 ( ) nM) was incubated with stoichiometric
concentrations of aHA antibody, aFLAG antibody or aHA-coupled QDs on ice for 15 min before the addition of the labelled DNA substrate. In these reactions, 20 nM DNA substrate was included. The reactions were then incubated at RT for 10 min and electrophoresed through a 3%, X TBE gel at 145 V for 90 min. The gels were dried, exposed to a PhosphorImager screen (Molecular Dynamics) and quantied with ImageQuant (GE Life Sciences).
The ATPase assays were performed in 5 ml reactions at 100 nM protein complex, 25 mM Tris (pH 7.5), 2 mM MgCl2, 1 mM DTT and 40 mg ml 1 BSA.
When present, aHA-QDs were incubated with the protein on ice at stoichiometric concentrations for 15 min before addition to the reaction. DNA template was added to 2 mM nal concentration and was incubated with protein for 5 min at RT.
ATP spiked with g32P-ATP was added last at increasing concentrations (0, 20, 33, 50, 67, 100 and 250 mM ATP). The reaction was incubated at 30 C for 30 min and then quenched by the addition of EDTA. The fraction hydrolyzed was determined by PEI-cellulose TLC with 0.6 M potassium phosphate buffer pH 3.4. The data were analysed in Prism (GraphPad).
Single-molecule microscopy. Images were collected with a Nikon Ti-E microscope in a prism-TIRF conguration. The inverted microscope setup allowed for the sample to be illuminated by a 488 nm laser light (Coherent) through a quartz prism (Tower Optical, Co.). To minimize spatial drift, experiments were conducted on a oating TMC optical table. A 60 water immersion objective lens
(1.2 NA, Nikon), two EMCCD cameras (Andor iXon DU897, 80 C) and
NIS-Elements software (Nikon) were used to collect the data with a 200 ms exposure time. Two-color imaging was conducted using a 638 nm dichroic beam splitter (Chroma). Frames were saved as TIFF les without compression for further image analysis in ImageJ (NIH). All single-molecule results were the product of at least two independent experiments.
Quantum dots. QDs were conjugated to Rabbit anti-HA tag antibodies (ICL labs #RHGT-45A-Z) or Mouse anti-FLAG tag antibodies (Sigma #F3165) using SiteClick antibody labelling kits (Life Technologies) according to the manufacturers instruction. The unconjugated antibodies were removed using a HiPrep Sephacryl S-300 HR gel ltration column (GE). QDs were stored in PBS (137 mM NaCl, 2.7 mM KCl, 9.1 mM K2HPO4 and 2.8 mM KH2PO4) at 4 C.
Fluorescent labelling of MMR complexes. The Msh2 subunit encodes an HA epitope tag between amino acids 644 and 645. HA epitope-tagged Msh2Msh3, Msh2Msh6(3MBD), and Msh2Msh6 were labelled with anti-HA conjugated QDs27. Briey QDs and protein were co-incubated at a 1:1 molar ratio (150 nM protein and QDs) in BSA buffer (40 mM Tris-HCl pH 8.0, 1 mM DTT, 2 mM MgCl2, 0.2 mg ml 1 BSA) for 15 min on ice followed by dilution to a nal concentration of 510 nM in BSA buffer containing the experimentally indicated concentration of NaCl (25150 mM) and nucleotide (1 mM ADP, ATP, or AMPPNP; Roche). The diluted proteinQD mixture was injected into the owcell, and allowed to incubate with DNA curtains for 510 min. After incubation, excess QDs and all non-DNA-bound proteins were ushed out of the owcells, buffer ow was terminated, and data acquisition was initiated. For experiments where the protein lifetime on DNA was o30 s (Msh2Msh3 and
Msh2Msh6(3MBD) at NaCl concentrations above 75 mM), free protein and QDs were retained in the owcell. This allowed for repeated rounds of protein binding and rapid dissociation. The short lifetimes guaranteed that individual DNA molecules accumulated fewer than three protein complexes.
Fluorescent labelling of EcoRI(E111Q) and nucleosomes. Triple FLAG epitope-tagged EcoRI(E111Q) was incubated with DNA at 0.5 nM concentration in buffer containing 10 mM Tris pH 7.8, 1 mM EDTA, and 150 mM NaCl. The reaction was then diluted 10-fold with BSA buffer and injected into the owcell. 3 FLAG-EcoRI(E111Q) was labelled in situ by injecting 10 nM of anti-FLAG
conjugated QDs directly into the owcell at a rate of 100 ml min 1. Before injection, anti-FLAG conjugated QDs were diluted to 5 nM in BSA buffer containing the experimentally indicated concentration of NaCl and nucleotide. Nucleosomes were reconstituted with 3 FLAG H2A containing octamers.
The nucleosomes were labelled in situ with a strategy identical to EcoRI(E111Q).
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Data analysis of particle tracking. Fluorescent particles were tracked in ImageJ with a custom-written particle tracking script (available on request). The resulting trajectories were analysed in Matlab (Mathworks). For each image frame the uorescent particle was t to a two-dimensional Gaussian function to obtain its position with sub-pixel resolution. The series of positions of a given particle were used to obtain trajectories. To ensure that trajectories corresponded to proteins on DNA, only molecules that responded to buffer ow controls and diffused fora minimum of 10 s were analysed. Only DNA-bound QDs were counted for statistical analysis.
Data analysis of measuring diffusion coefcients. For all diffusion experiments, we used double-tethered DNA curtains, which extend DNA molecules between two nano-fabricated chromium features in the absence of buffer ow28. The average separation between the two chromium features was 13 mm (B80% extension relative to B-form DNA). Trajectories of individual molecules were used to calculate the 1D MSD as a function of time interval using:
MSD nDt
1N n
XN n
i1
yin yi
2 2
where N is the total number of frames in the trajectory, n is the number of frames for a given time interval, Dt is the time between frames, and yi is the position of
Msh2Msh3 at frame i. To minimize systematic errors associated with estimating diffusion coefcients for very short trajectories, we only considered particles that diffused for more than 10 s (50 frames). The MSD was calculated for the rst10 time intervals (Dt 0.2 s-2 s) and plotted as a function of Dt to generate the line:
MSD Dt
2DDt 3 where D is the diffusion coefcient. Plots were t to a line and the t was used to calculate diffusion coefcients of individual molecules. Diffusion coefcients were calculated for Z45 molecules per condition, and are reported as a means.d.
Statistical analysis was performed on the Logarithm of the data using a two tailed students t-test, with a 95% condence interval.
Characterizing DNA transfer events. We scored the frequency of protein transfer between two DNA molecules that were tethered to adjacent pedestals (separated by 1 mm). Msh2Msh3, Msh2Msh6(3MBD) or Msh2Msh6 was injected into the owcell and allowed to diffuse on DNA for at least 10 min. After the diffusion traces were collected, the DNA molecules were visualized via uorescent staining. Our criteria for identifying Msh-protein transfer between two adjacent DNA molecules were: (i) the protein rst diffused in register with one DNA for greater than 4 s, (ii) the protein shifted positions to the neighbouring DNA in less than one frame (200 ms) and (iii) the same protein continued to diffuse in register with an adjacent DNA for at least 4 additional seconds. All DNA transfer experiments were performed in imaging buffer with 1 mM ADP and 100 mM NaCl.
Characterizing roadblock bypass events. Roadblock bypass was observed using the following DNA roadblocks on double-tethered DNA: Msh2Msh3, EcoRI(E111Q) and nucleosomes. For Msh2Msh3 roadblocks, Msh2Msh3 was divided into two 100 nM reactions individually labelled with QDs that uorescently emit at either 605 or 705 nm. The reactions were incubated for 15 min on ice and diluted 10-fold in BSA buffer supplemented with 10 mM NaCl and 1 mM ADP. The reactions were then mixed and injected into the owcell. After DNA incubation, the buffer was switched from 10 to 100 mM NaCl using a six port, two position manual valve (Valco Instruments, Co., Inc.). After buffer exchange, the proteins were visualized on DNA for B10 min
For EcoRI(E111Q), the hydrolytically dead restriction enzyme was rst incubated with DNA and labelled with QDs in situ30. Next, 10 nM of QD-labelled Msh2Msh3 was injected into the owcell. Buffer exchange and imaging was done under identical conditions as for Msh2Msh3 on naked DNA. Nucleosome bypass experiments were also done using the same buffer exchange strategy. We checked whether the Msh2Msh3 nucleotide state altered nucleosome bypass probabilities by repeating these experiments with 1 mM ADP, AMPPNP or ATPMg 2, respectively. Nucleosomes were labelled in situ as described for EcoRI(E111Q).
To test the effect that QDs had on roadblock bypass, nucleosomes were labelled either before Msh2Msh3 injection or after Msh2Msh3 image acquisition.
We considered a protein to pass a roadblock if it satised the following criteria:(a) the protein was initially diffusing on one side of the roadblock, (b) the protein transiently co-localized with the roadblock within a collision zone of 750 bp (200 nm) and (c) the diffusing protein clearly continued diffusing on the opposite side of the roadblock. The collision zone is dened as 3 the s.d. of the random
motion exhibited by a stationary protein on double-tethered DNA curtains over 4100 frames. This random motion is the result of thermal uctuations experienced by the DNA.
Lysogen DNA purication. The E. coli lysogen used to generate ap-containing l-DNA was a generous gift from the Greene lab. The lysogen was created by replacing the region between the NgoMIV and the XbaI restriction enzyme cutsites (20,041 bp and 24,374 bp, respectively) with a 151 bp DNA segment containing
three BspQI sites and a unique NcoI cut site. To purify the DNA, a single E. coli colony was grown to conuency in 50 ml of LB broth overnight at 30 C and used to inoculate 500 ml of LB broth. When OD600 0.6, the asks were rapidly heated
to 42 C followed by a 15 min heat shock at 45 C. The cells were then grown at 37 C for 2 h. Cells were pelleted at 3,000g for 30 min, and suspended in SM buffer (50 mM Tris-Cl pH 7.5, 100 mM NaCl and 8 mM MgSO4). Cells were lysedwith 2% chloroform, and the genomic nucleic acids degraded with 50 ng ml 1of DNAseI and 30 ng ml 1 RNAse A (Sigma). The lysate was claried by centrifugation at 6,000g for 15 min. Phage heads were precipitated using cold buffer
L2 (30% PEG 6000, 3 M NaCl) for 30 min at 4 C. Phage heads were pelleted by centrifuging at 10,000g for 10 min. The phage pellet was suspended in buffer L3 (100 mM Tris pH 7.5, 100 mM NaCl and 25 mM EDTA). Phage heads were degraded by buffer L4 (4% SDS) and 100 mg ml 1 of Proteinase K (NEB). SDS was precipitated by adding buffer L5 (3 M potassium acetate pH 5.5) followed by centrifugation at 15,000 g for 30 min. The supernatant was passed over a pre-equilibrated QIAGEN Genomic-tip 500 DNA purication column (QIAGEN). DNA was puried from the columns according to the manufacturers instruction, and dissolved in Te buffer (10 mM Tris pH 7.5, 0.1 mM EDTA). DNA was stored in 4 C for up to 6 months.
Flap incorporation. 50 mg of puried l-DNA (see above) was diluted in NEB buffer 3 and digested with 50,000 units of Nt.BspQI (NEB) for 1 h at 55 C. Oligonucleotides IF003 and IF004, along with oligonucleotides incorporating either a 30-ssDNA ap (MB34, MB35), a digylated 30-ssDNA ap (MB35,MB36) or a complementary sequence (MB32) were added in 500 molar excess to the nicked
l-DNA (Supplementary Table 4). The reaction was heated to 70 C and cooled at a rate of 0.5 C min 1 to promote oligonucleotide incorporation. The reaction was supplemented with 1 mM of ATP and 3,000 units of T4 DNA ligase (NEB) and incubated overnight at RT. Reactions were quenched with 50 mM EDTA. An aliquot of the reaction was taken for agarose gel insertion diagnostics, while the remainder was incubated with ve units of Proteinase K (NEB) at 50 for 30 min to degrade Nt.BspQI and T4 DNA ligase. The DNA was passed over an S-1000 gel ltration column (GE) and stored at 4 C.
Characterizing ap insertion efciency. Insertion of the ap oligonucleotide abolishes an NcoI site within the l-DNA. NcoI cleavage can thus be used to monitor the oligo insertion efciency. After the ap oligonucleotide was ligated into the nicked l-DNA, a Bio-Spin size exclusion column (Bio-Rad) was used to exchange the buffer into NEB Buffer 4. 1.5 mg of the l-DNA was digested with 20 units of NcoI-HF (NEB) at 37 C for 1 h. Digests were run on a 0.8% agarose gel containing 0.5 mg ml 1 of ethidium bromide (Apex) for three hours at 100 V.
Gels were imaged with a Typhoon gel imager (GE).
Alkaline agarose gels were used to monitor complete re-ligation of the nicked l-DNA. 1.5 mg of proteinase K treated DNA was loaded into a 0.6% alkaline agarose gel using 6 alkaline gel-loading buffer (300 mM NaOH, 6 mM EDTA,
18% (w/v) glycerol and 0.15% (w/v) Orange G (NEB)). Gels were run in 1x alkaline electrophoresis buffer (50 mM EDTA pH 8.0, 1 M NaOH) for 24 h at 20 V and4 C. Gels were incubated in neutralization buffer (1 M Tris-Cl pH 7.6, 1.5 M NaCl) for 45 min at RT, stained in a solution of 1 TAE and 0.5 mg ml 1 ethidium
bromide for 30 min, and de-stained by soaking in ddH20 for 20 min. Gels were imaged with a Typhoon gel imager.
Visualizing via single-molecule uorescence imaging. To directly visualize the ap oligonucleotide within DNA curtains, oligonucleotides MB35 and MB36 were inserted into the DNA to generate a substrate with a 30-ssDNA ap terminated with a DIG label. The oligonucleotide was incorporated into the DNA substrate, puried and assembled into DNA curtains. The digylated DNA was labelled in situ with 12.5 ng ml 1 of DIG monoclonal antibody (Life Technologies #700772)
followed by 10 nM of Goat anti-Rabbit QD605 conjugate (Life Technologies #Q-11401MP). The average signal from 150 frames was used to calculate the QD position.
References
1. Tafvizi, A., Mirny, L. A. & van Oijen, A. M. Dancing on DNA: kinetic aspects of search processes on DNA. Chem. Phys. Chem. 12, 14811489 (2011).
2. Halford, S. E. & Marko, J. F. How do site-specic DNA-binding proteins nd their targets? Nucleic Acids Res. 32, 30403052 (2004).
3. Blainey, P. C. et al. Nonspecically bound proteins spin while diffusing along DNA. Nat. Struct. Mol. Biol. 16, 12241229 (2009).
4. Cravens, S. L. et al. Molecular crowding enhances facilitated diffusion of two human DNA glycosylases. Nucleic Acids Res. 43, 40874097 (2015).
5. Schonhoft, J. D. & Stivers, J. T. Timing facilitated site transfer of an enzyme on DNA. Nat. Chem. Biol. 8, 205210 (2012).
6. Elf, J., Li, G.-W. & Xie, X. S. Probing transcription factor dynamics at the single-molecule level in a living cell. Science 316, 11911194 (2007).7. Koh, H. R., Kidwell, M. A., Ragunathan, K., Doudna, J. A. & Myong, S. ATP-independent diffusion of double-stranded RNA binding proteins. Proc. Natl Acad. Sci 110, 151156 (2013).
10 NATURE COMMUNICATIONS | 7:10607 | DOI: 10.1038/ncomms10607 | http://www.nature.com/naturecommunications
Web End =www.nature.com/naturecommunications
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10607 ARTICLE
8. Chandradoss, S. D., Schirle, N. T., Szczepaniak, M., MacRae, I. J. & Joo, C.
A dynamic search process underlies microRNA targeting. Cell 162, 96107 (2015).
9. Marcovitz, A. & Levy, Y. Obstacles may facilitate and direct DNA search by proteins. Biophys. J. 104, 20422050 (2013).
10. Brackley, C. A., Cates, M. E. & Marenduzzo, D. Intracellular facilitated diffusion: searchers, crowders, and blockers. Phys. Rev. Lett. 111, 108101 (2013).
11. Jiricny, J. Postreplicative mismatch repair. Cold Spring Harb. Perspect. Biol. 5, a012633 (2013).
12. Kunkel, T. A. & Erie, D. A. DNA mismatch repair. Annu. Rev. Biochem. 74, 681710 (2005).
13. Gradia, S. et al. hMSH2-hMSH6 forms a hydrolysis-independent sliding clamp on mismatched DNA. Mol. Cell 3, 255261 (1999).
14. Lee, J.-B. et al. Single-molecule views of MutS on mismatched DNA. DNA Repair 20, 8293 (2014).
15. Erie, D. A. & Weninger, K. R. Single molecule studies of DNA mismatch repair. DNA Repair 20, 7181 (2014).
16. Pluciennik, A. & Modrich, P. Protein roadblocks and helix discontinuities are barriers to the initiation of mismatch repair. Proc. Natl Acad. Sci. USA 104, 1270912713 (2007).
17. Gorman, J., Plys, A. J., Visnapuu, M.-L., Alani, E. & Greene, E. C. Visualizing one-dimensional diffusion of eukaryotic DNA repair factors along a chromatin lattice. Nat. Struct. Mol. Biol. 17, 932938 (2010).
18. Li, F., Tian, L., Gu, L. & Li, G.-M. Evidence that nucleosomes inhibit mismatch repair in eukaryotic cells. J. Biol. Chem. 284, 3305633061 (2009).
19. Sogo, J. M., Stahl, H., Koller, T. & Knippers, R. Structure of replicating simian virus 40 minichromosomes: the replication fork, core histone segregation and terminal structures. J. Mol. Biol. 189, 189204 (1986).
20. Shell, S. S., Putnam, C. D. & Kolodner, R. D. Chimeric Saccharomyces cerevisiae Msh6 protein with an Msh3 mispair-binding domain combines properties of both proteins. Proc. Natl Acad. Sci. USA 104, 1095610961 (2007).
21. Hombauer, H., Srivatsan, A., Putnam, C. D. & Kolodner, R. D. Mismatch repair, but not heteroduplex rejection, is temporally coupled to DNA replication. Science 334, 17131716 (2011).
22. Goellner, E. M., Putnam, C. D. & Kolodner, R. D. Exonuclease 1-dependent and independent mismatch repair. DNA Repair 32, 2432 (2015).
23. Sugawara, N., Pques, F., Colaicovo, M. & Haber, J. E. Role of Saccharomyces cerevisiae Msh2 and Msh3 repair proteins in double-strand break-induced recombination. Proc. Natl Acad. Sci. USA 94, 92149219 (1997).
24. Li, F. et al. Role of Saw1 in Rad1/Rad10 complex assembly at recombination intermediates in budding yeast. EMBO J. 32, 461472 (2013).
25. Iyer, R. R., Pluciennik, A., Napierala, M. & Wells, R. D. DNA triplet repeat expansion and mismatch repair. Annu. Rev. Biochem. 84, 199226 (2015).26. Spies, M. & Fishel, R. Mismatch repair during homologous and homeologous recombination. Cold Spring Harb. Perspect. Biol. 7, a022657 (2015).
27. Gorman, J. et al. Dynamic basis for one-dimensional DNA scanning by the mismatch repair complex Msh2-Msh6. Mol. Cell 28, 359370 (2007).
28. Gallardo, I. F. et al. High-throughput universal DNA curtain arrays for single-molecule uorescence imaging. Langmuir 31, 1031010317 (2015).
29. Robison, A. D. & Finkelstein, I. J. Rapid prototyping of multichannel microuidic devices for single-molecule DNA curtain imaging. Anal. Chem. 86, 41574163 (2014).
30. Finkelstein, I. J., Visnapuu, M.-L. & Greene, E. C. Single-molecule imaging reveals mechanisms of protein disruption by a DNA translocase. Nature 468, 983987 (2010).
31. Hohng, S. & Ha, T. Near-complete suppression of quantum dot blinking in ambient conditions. J. Am. Chem. Soc. 126, 13241325 (2004).
32. Srivatsan, A., Bowen, N. & Kolodner, R. D. Mispair-specic recruitment of the Mlh1-Pms1 complex identies repair substrates of the Saccharomyces cerevisiae Msh2-Msh3 complex. J. Biol. Chem. 289, 93529364 (2014).
33. Harrington, J. M. & Kolodner, R. D. Saccharomyces cerevisiae Msh2-Msh3 acts in repair of base-base mispairs. Mol. Cell. Biol. 27, 65466554 (2007).
34. Surtees, J. A. & Alani, E. Mismatch repair factor MSH2-MSH3 binds and alters the conformation of branched DNA structures predicted to form during genetic recombination. J. Mol. Biol. 360, 523536 (2006).
35. Kumar, C. et al. ATP binding and hydrolysis by Saccharomyces cerevisiae Msh2-Msh3 are differentially modulated by mismatch and double-strand break repair DNA substrates. DNA Repair 18, 1830 (2014).
36. Thompson, R. E., Larson, D. R. & Webb, W. W. Precise nanometer localization analysis for individual uorescent probes. Biophys. J. 82, 27752783 (2002).
37. Kochaniak, A. B. et al. Proliferating cell nuclear antigen uses two distinct modes to move along DNA. J. Biol. Chem. 284, 1770017710 (2009).
38. Bonnet, I. & Desbiolles, P. The diffusion constant of a labeled protein sliding along DNA. Eur. Phys. J. E 34, 110 (2011).
39. Diki, J. et al. The rotation-coupled sliding of EcoRV. Nucleic Acids Res. 40,
40644070 (2012).
40. Lin, J. et al. TRF1 and TRF2 use different mechanisms to nd telomeric DNA but share a novel mechanism to search for protein partners at telomeres. Nucleic Acids Res. 42, 24932504 (2014).
41. Junop, M. S., Obmolova, G., Rausch, K., Hsieh, P. & Yang, W. Composite active site of an ABC ATPase: MutS uses ATP to verify mismatch recognition and authorize DNA repair. Mol. Cell 7, 112 (2001).
42. Owen, B. A. L., Lang, W. H. & McMurray, C. T. The nucleotide binding dynamics of human MSH2-MSH3 are lesion dependent. Nat. Struct. Mol. Biol. 16, 550557 (2009).
43. Warren, J. J. et al. Structure of the human MutSa DNA lesion recognition complex. Mol. Cell 26, 579592 (2007).
44. Obmolova, G., Ban, C., Hsieh, P. & Yang, W. Crystal structures of mismatch repair protein MutS and its complex with a substrate DNA. Nature 407, 703710 (2000).
45. Cho, W.-K. et al. ATP alters the diffusion mechanics of MutS on mismatched DNA. Structure 20, 12641274 (2012).
46. Snowden, T., Acharya, S., Butz, C., Berardini, M. & Fishel, R. hMSH4-hMSH5 recognizes Holliday junctions and forms a meiosis-specic sliding clamp that embraces homologous chromosomes. Mol. Cell 15, 437451 (2004).
47. Wilson, T., Guerrette, S. & Fishel, R. Dissociation of mismatch recognition and ATPase activity by hMSH2-hMSH3. J. Biol. Chem. 274, 2165921664 (1999).
48. Blainey, P. C., van Oijen, A. M., Banerjee, A., Verdine, G. L. & Xie, X. S.
A base-excision DNA-repair protein nds intrahelical lesion bases byfast sliding in contact with DNA. Proc. Natl Acad. Sci. USA 103, 57525757 (2006).
49. Etson, C. M., Hamdan, S. M., Richardson, C. C. & van Oijen, A. M. Thioredoxin suppresses microscopic hopping of T7 DNA polymerase on duplex DNA. Proc. Natl Acad. Sci. USA 107, 19001905 (2010).
50. Record, Jr M. T., Lohman, T. M. & deHaseth, P. Ion effects on ligand-nucleic acid interactions. J. Mol. Biol. 107, 145158 (1976).
51. Qiu, R. et al. Large conformational changes in MutS during DNA scanning, mismatch recognition and repair signalling. EMBO J. 31, 25282540 (2012).
52. Gupta, S., Gellert, M. & Yang, W. Mechanism of mismatch recognition revealed by human MutSb bound to unpaired DNA loops. Nat. Struct. Mol. Biol. 19, 7278 (2012).
53. Lamers, M. H. et al. The crystal structure of DNA mismatch repair protein MutS binding to a G T mismatch. Nature 407, 711717 (2000).
54. Pathak, S., Davidson, M. C. & Silva, G. A. Characterization of the functional binding properties of antibody conjugated quantum dots. Nano Lett. 7, 18391845 (2007).
55. Wright, D. J., King, K. & Modrich, P. The negative charge of Glu-111 is required to activate the cleavage center of EcoRI endonuclease. J. Biol. Chem. 264, 1181611821 (1989).
56. Lang, W. H. et al. Conformational trapping of mismatch recognition complex MSH2/MSH3 on repair-resistant DNA loops. Proc. Natl Acad. Sci. USA 108, E837E844 (2011).
57. Lee, S. D., Surtees, J. A. & Alani, E. Saccharomyces cerevisiae MSH2-MSH3 and MSH2-MSH6 complexes display distinct requirements for DNA binding domain I in mismatch recognition. J. Mol. Biol. 366, 5366 (2007).
58. Jeong, C. et al. MutS switches between two fundamentally distinct clamps during mismatch repair. Nat. Struct. Mol. Biol. 18, 379385 (2011).
59. Sharma, M., Predeus, A. V., Mukherjee, S. & Feig, M. DNA bending propensity in the presence of base mismatches: implications for DNA repair. J. Phys. Chem. B 117, 61946205 (2013).
60. Rossetti, G. et al. The structural impact of DNA mismatches. Nucleic Acids Res. 43, 43094321 (2015).
61. Wang, H. et al. DNA bending and unbending by MutS govern mismatch recognition and specicity. Proc. Natl Acad. Sci. USA 100, 1482214827 (2003).
62. Li, F. et al. The Histone mark H3K36me3 regulates human DNA mismatch repair through its interaction with MutSa. Cell 153, 590600 (2013).
63. Schpf, B. et al. Interplay between mismatch repair and chromatin assembly. Proc. Natl Acad. Sci. USA 109, 18951900 (2012).
64. Kadyrova, L. Y., Blanko, E. R. & Kadyrov, F. A. CAF-I-dependent control of degradation of the discontinuous strands during mismatch repair. Proc. Natl Acad. Sci. USA 108, 27532758 (2011).
65. Javaid, S. et al. Nucleosome remodeling by hMSH2-hMSH6. Mol. Cell 36, 10861094 (2009).
66. Forties, R. A. et al. A quantitative model of nucleosome dynamics. Nucleic Acids Res. 39, 83068313 (2011).
67. Honda, M. et al. Mismatch repair protein hMSH2-hMSH6 recognizes mismatches and forms sliding clamps within a D-loop recombination intermediate. Proc. Natl Acad. Sci. USA 111, E316E325 (2014).
68. Antony, E. & Hingorani, M. M. Mismatch recognition-coupled stabilization of Msh2-Msh6 in an ATP-bound state at the initiation of DNA repairw.
Biochemistry 42, 76827693 (2003).69. Thstrm, A., Lowary, P. T. & Widom, J. Measurement of histone-DNA interaction free energy in nucleosomes. Methods 33, 3344 (2004).
70. Lee, J. Y. & Greene, E. C. Assembly of recombinant nucleosomes on nanofabricated DNA curtains for single-molecule imaging. Methods Mol. Biol. 778, 243258 (2011).
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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10607
Acknowledgements
We are grateful to members of the Finkelstein and Surtees laboratories for carefully reading the manuscript. This work was supported by the National Institute of General Medical Sciences of the National Institutes of Health (GM097177 to I.J.F. and GM087459 to J.A.S.), the American Cancer Society (RSG-14-235-01 to J.A.S.), CPRIT (R1214 to I.J.F.) and the Welch Foundation (F-l808 to I.J.F.). J.A.S. is an ACS Research Scholar. I.J.F. is a CPRIT Scholar in Cancer Research. The content is solely the responsibility of the authors and does not necessarily represent the ofcial views of the National Institutes of Health. We are indebted to our colleagues Richard Kolodner, Eric Greene, Eric Alani and Manju Hingorani for valuable reagents and helpful discussions.
Author contributions
M.W.B., Y.K., G.M.W. and J.A.S. prepared proteins and DNA samples. M.W.B. and Y.K. conducted the single-molecule experiments. G.M.W., J.D.H. and J.A.S. performed gel shift and ATPase experiments. J.A.S. and I.J.F. directed the project. M.W.B., Y.D.K., J.A.S. and I.J.F. co-wrote the paper. All authors commented on the manuscript.
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How to cite this article: Brown, M. W. et al. Dynamic DNA binding licences a repair factor to bypass roadblocks in search of DNA lesions. Nat. Commun. 7:10607doi: 10.1038/ncomms10607 (2016).
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Abstract
DNA-binding proteins search for specific targets via facilitated diffusion along a crowded genome. However, little is known about how crowded DNA modulates facilitated diffusion and target recognition. Here we use DNA curtains and single-molecule fluorescence imaging to investigate how Msh2-Msh3, a eukaryotic mismatch repair complex, navigates on crowded DNA. Msh2-Msh3 hops over nucleosomes and other protein roadblocks, but maintains sufficient contact with DNA to recognize a single lesion. In contrast, Msh2-Msh6 slides without hopping and is largely blocked by protein roadblocks. Remarkably, the Msh3-specific mispair-binding domain (MBD) licences a chimeric Msh2-Msh6(3MBD) to bypass nucleosomes. Our studies contrast how Msh2-Msh3 and Msh2-Msh6 navigate on a crowded genome and suggest how Msh2-Msh3 locates DNA lesions outside of replication-coupled repair. These results also provide insights into how DNA repair factors search for DNA lesions in the context of chromatin.
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