Mechanisms of target search by DNA-binding proteins

All genetic information is preserved within the DNA duplex. For successful propagation of this genetic code, DNA must be accurately replicated, repaired, and transcribed. These fundamental processes require DNA-binding proteins that recognize specific DNA sequences or structures. These proteins must quickly find a specific target amidst a vast excess of non-specific, yet structurally similar, DNA. Moreover, target search must proceed in the context of nucleosomes, transcription factors, and other protein roadblocks. Uncovering how proteins locate their targets is important for understanding key regulatory mechanisms involved in nearly all genomic transactions. My research aims to understand the mechanisms and structural motifs that regulate target search. Our approach uses single-molecule microscopy to visualize this multi-step and dynamic process in real-time. My first project studied the heterodimeric DNA MisMatch Repair (MMR) complex Msh2-Msh3. Msh2-Msh3 recognizes DNA lesions implicated in MMR, Single-Strand DNA Anealing (SSA), and TriNucleotide Repeat (TNR) expansion disorders. My work provided the first comprehensive characterization of how Msh2-Msh3 can ‘hop’ over nucleosomes via the Msh3 Mispair Binding Domain (MBD) as it searches for DNA lesions (Chapter 2). I then focused on the type I CRISPR-Cas system, which confers bacteria and archaea with immunity against foreign DNA. This process is initiated by the sequence-specific targeting of foreign DNA by Cascade; a multi-subunit, RNA-guided, target recognition complex. I discovered that sequence and structural conservation of basic residues in Cascade regulates the diffusion of the complex on non-specific DNA. I then observed the Cascade mediated recruitment of downstream proteins Cas3 and Cas1-Cas2, culminating in the formation of a Primed Acquisition Complex (PAC) which I conjecture is required for cells to respond to mutated DNA targets (Chapter 4). Furthermore, I developed new methods relating to single-molecule microscopy, including a high-throughput method to make DNA curtains, a fluorescent labeling strategy for conjugating dyes to proteins, and a high-throughput force-based assay (Chapter 3). In addition to providing protocols and tools that will be useful for future single-molecule studies, my research has made key contributions to the MMR and CRISPR fields by shedding light on mechanisms that contribute to target search and roadblock removal/avoidance on DNA.Biochemistr