DNA-binding proteins search for specific targets via facilitated diffusion along a crowded genome. factors search for DNA lesions in the context of chromatin. DNA-binding proteins must rapidly locate specific sites amidst a vast pool of non-specific 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 via single-molecule and ensemble biochemistry approaches and have also been inferred via single-molecule imaging in live cells2 3 4 5 6 Indeed 1 diffusion is usually a common feature of nearly all proteins that scan both BMS-747158-02 DNA1 2 3 and RNA7 8 for specific 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 specific DNA sequences or structures. To experimentally address this question we investigated facilitated diffusion by yeast Msh2-Msh3 and Msh2-Msh6 two heterodimeric MutS homologue (Msh) complexes that participate in the first 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 lesions13 14 15 Once a lesion is found the Msh complex binds and recruits downstream protein factors to initiate repair. studies have established that Msh2-Msh6 can scan naked DNA for lesions via 1D facilitated diffusion along the DNA track14 15 16 However both yeast and human Msh2-Msh6 diffusion is usually blocked by nucleosomes interactions EP between Msh2-Msh3 and the replication fork are less clear. Msh2-Msh3 is also implicated in other genome maintenance BMS-747158-02 pathways that occur outside of replication-coupled MMR suggesting that it must scan DNA in the context of nucleosomes21 23 24 25 26 Thus Msh2-Msh3 may employ a unique strategy for navigating protein-bound DNA. Here we use single-molecule fluorescence microscopy to BMS-747158-02 reveal that Msh2-Msh3 scans DNA via a facilitated diffusion mechanism comprised of both 1D sliding and microscopic hopping. Msh2-Msh3’s DNA interactions are sufficiently dynamic to allow the bypass of nucleosomes and other protein obstacles while still allowing the complex to recognize a single DNA lesion. In contrast Msh2-Msh6 does not hop on DNA and is largely blocked by nucleosomes. Remarkably a chimeric version of Msh2-Msh6 that encodes the Msh3 mispair-binding domain name (MBD) imparts roadblock bypass activity to Msh2-Msh6. Thus the Msh3 MBD is sufficient to license Msh complex hopping. Our studies contrast how Msh2-Msh3 and Msh2-Msh6 navigate a BMS-747158-02 crowded genome and suggest how Msh2-Msh3 functions outside of replication-coupled repair. More broadly we provide a model for how dynamic fluctuations within DNA-encircling protein domains may facilitate bypass of other protein roadblocks during 1D-facilitated diffusion. Results Visualizing Msh2-Msh3 sliding on DNA curtains We investigated how Msh2-Msh3 slides on DNA by directly monitoring the protein’s movement via total internal reflection fluorescence microscopy of fluorescently labelled Msh2-Msh3. Yeast Msh2-Msh3 with a hemagglutinin (HA) epitope tag around the Msh2 subunit was overexpressed and purified from yeast cells (Supplementary Fig. 1). To fluorescently label Msh2-Msh3 we conjugated the protein with anti-HA antibody-coupled quantum dots (QDs). Gel shift and ATPase assays indicated that this QD-tagged Msh2-Msh3 retained biochemical activities similar to wild-type protein and remained responsive to specific DNA templates (Supplementary Fig. 1). These data indicate that this QD does not compromise communication between the DNA-binding and ATPase domains of Msh2-Msh3. This epitope-labelling strategy has also been used successfully with yeast Msh2-Msh6 (refs 17 27 We used a high-throughput DNA.