Mismatched nucleotides arise from polymerase misincorporation errors, recombination between heteroallelic parents and chemical or physical DNA damage1. Highly conserved MutS (MSH) and MutL (MLH/PMS) homologues initiate mismatch repair and, in higher eukaryotes, act as DNA damage sensors that can trigger apoptosis2. Defects in human mismatch repair genes cause Lynch syndrome or hereditary non-polyposis colorectal cancer and 10–40% of related sporadic tumours3. However, the collaborative mechanics of MSH and MLH/PMS proteins have not been resolved in any organism. We visualized Escherichia coli (Ec) ensemble mismatch repair and confirmed that EcMutS mismatch recognition results in the formation of stable ATP-bound sliding clamps that randomly diffuse along the DNA with intermittent backbone contact. The EcMutS sliding clamps act as a platform to recruit EcMutL onto the mismatched DNA, forming an EcMutS–EcMutL search complex that then closely follows the DNA backbone. ATP binding by EcMutL establishes a second long-lived DNA clamp that oscillates between the principal EcMutS–EcMutL search complex and unrestricted EcMutS and EcMutL sliding clamps. The EcMutH endonuclease that targets mismatch repair excision only binds clamped EcMutL, increasing its DNA association kinetics by more than 1,000-fold. The assembly of an EcMutS–EcMutL–EcMutH search complex illustrates how sequential stable sliding clamps can modulate one-dimensional diffusion mechanics along the DNA to direct mismatch repair.

SUMMARY The mismatch repair (MMR) initiation protein MutS forms at least two types of sliding clamps on DNA: a transient mismatch searching clamp (~1 s) and an unusually stable (~600 s) ATP-bound clamp that recruits downstream MMR components. Remarkably, direct visualization of single MutS particles on mismatched DNA has not been reported. We have combined real-time particle tracking with fluorescence resonance energy transfer (FRET) to image MutS diffusion dynamics on DNA containing a single mismatch. We show searching MutS rotates during diffusion independent of ionic strength or flow rate, suggesting continuous contact with the DNA backbone. In contrast, ATP-bound MutS clamps that are visually and successively released from the mismatch spin freely around the DNA, and their diffusion is affected by ionic strength and flow rate. These observations show that ATP binding alters the MutS diffusion mechanics on DNA, which has a number of implications for the mechanism of MMR.

Mismatch repair (MMR) is activated by evolutionarily conserved MutS homologs (MSH) and MutL homologs (MLH/PMS). MSH recognizes mismatched nucleotides and form extremely stable sliding clamps that may be bound by MLH/PMS to ultimately authorize strand-specific excision starting at a distant 3′-or 5′-DNA scission. The mechanical processes associated with a complete MMR reaction remain enigmatic. The purified human (Homo sapien or Hs) 5′-MMR excision reaction requires the HsMSH2-HsMSH6 heterodimer, the 5′ → 3′ exonuclease HsEXOI, and the single-stranded binding heterotrimer HsRPA. The HsMLH1-HsPMS2 heterodimer substantially influences 5′-MMR excision in cell extracts but is not required in the purified system. Using real-time single-molecule imaging, we show that HsRPA or Escherichia coli EcSSB restricts HsEXOI excision activity on nicked or gapped DNA. HsMSH2-HsMSH6 activates HsEXOI by overcoming HsRPA/EcSSB inhibition and exploits multiple dynamic sliding clamps to increase tract length. Conversely, HsMLH1-HsPMS2 regulates tract length by controlling the number of excision complexes, providing a link to 5′ MMR.is a highly conserved strand-specific excision-resynthesis process that corrects nucleotide misincorporation errors during replication and nucleotide mismatches arising from recombination between heteroallelic parents or physical damage to the DNA (for review see ref. 1). Mutation of core MMR components results in elevated mutation rates and susceptibility to a variety of cancers (2).MMR has been reconstituted with purified Escherichia coli, Saccharomyces cerevisae, and human proteins (3-6). The core MutS homologs (MSH) and MutL homologs (MLH/PMS) components direct a strand-specific excision reaction, whereas resynthesis appears to be uniquely performed by the replicative polymerase complex (1). In all organisms the excision process is initiated at a single-strand DNA scission (ssDNA/S) that may be located either 3′ or 5′ and hundreds to thousands of base pairs distant from the mismatch (4, 7). An ssDNA/S positioned on the newly replicated strand ensures accurate correction of replication misincorporation errors (1).Excision directionality in γ-proteobacteria (E. coli) is linked to the choice of 3′ or 5′ exonucleases that specifically degrade ssDNA generated by the EcUvrD helicase in concert with EcMutS and EcMutL (1). The lack of a helicase distinguishes yeast and human MMR from γ-proteobacteria. Moreover, the eukaryotic 3′-and 5′-excision reactions require different core MMR components and likely occur by different mechanisms (1). For example, the 3′-MMR excision requires the replicative processivity factor PCNA to activate a cryptic MLH/PMS endonuclease activity (8), whereas 5′ MMR uses the only known MMR exonuclease EXOI (3, 5, 6). Unlike the E. coli ssDNA exonucleases, EXOI will initiate 5′ excision from a ssDNA/S in the absence of a helicase (9). Whereas the purified 5′-MMR reaction does not require MLH/PMS or PCNA, complementation studies with cellular extracts displayed a substantial requirement for...

Base-pair mismatches that occur during DNA replication or recombination can reduce genetic stability or conversely increase genetic diversity. The genetics and biophysical mechanism of mismatch repair (MMR) has been extensively studied since its discovery nearly 50 years ago. MMR is a strand-specific excision-resynthesis reaction that is initiated by MutS homolog (MSH) binding to the mismatched nucleotides. The MSH mismatch-binding signal is then transmitted to the immediate downstream MutL homolog (MLH/PMS) MMR components and ultimately to a distant strand scission site where excision begins. The mechanism of signal transmission has been controversial for decades. We have utilized single molecule Forster Resonance Energy Transfer (smFRET), Fluorescence Tracking (smFT) and Polarization Total Internal Reflection Fluorescence (smP-TIRF) to examine the interactions and dynamic behaviors of single Thermus aquaticus MutS (TaqMutS) particles on mismatched DNA. We determined that Taq-MutS forms an incipient clamp to search for a mismatch in ∼1 s intervals by 1-dimensional (1D) thermal fluctuation-driven rotational diffusion while in continuous contact with the helical duplex DNA. When MutS encounters a mismatch it lingers for ∼3 s to exchange bound ADP for ATP (ADP → ATP exchange). ATP binding by TaqMutS induces an extremely stable clamp conformation (∼10 min) that slides off the mismatch and moves along the adjacent duplex DNA driven simply by 1D thermal diffusion. The ATP-bound sliding clamps rotate freely while in discontinuous contact with the DNA. The visualization of a train of MSH proteins suggests that dissociation of ATP-bound sliding clamps from the mismatch permits multiple mismatch-dependent loading events. These direct observations have provided critical clues into understanding the molecular mechanism of MSH proteins during MMR.

Post-translational modifications (PTMs) of receptor tyrosine kinases (RTKs) at the plasma membrane (PM) determine the signal transduction efficacy alone and in combination. However, current approaches to identify PTMs provide ensemble results, inherently overlooking combinatorial PTMs in a single polypeptide molecule. Here, we describe a single-molecule blotting (SiMBlot) assay that combines biotinylation of cell surface receptors with single-molecule fluorescence microscopy. This method enables quantitative measurement of the phosphorylation status of individual membrane receptor molecules and colocalization analysis of multiple immunofluorescence signals to directly visualize pairwise site-specific phosphorylation patterns at the single-molecule level. Strikingly, application of SiMBlot to study ligand-dependent epidermal growth factor receptor (EGFR) phosphorylation, which is widely thought to be multi-phosphorylated, reveals that EGFR on cell membranes is hardly multi-phosphorylated, unlike in vitro autophosphorylated EGFR. Therefore, we expect SiMBlot to aid understanding of vast combinatorial PTM patterns, which are concealed in ensemble methods, and to broaden knowledge of RTK signaling.