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, 2012www.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 onflow offflow on
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
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