Expanding the Functional Landscape of GIY-YIG Nuclease Domains: Mechanism, Modularity, Evolution, and Engineering

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2025-10-28

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Abstract

GIY-YIG homing endonucleases are compact, site-specific nucleases that promote the mobility of introns and inteins through DNA cleavage. Despite their broad phylogenetic distribution and potential utility as programmable nucleases, the structural basis for their sequence preferences, cleavage modalities, and evolutionary adaptability has remained poorly understood. The overarching goal of this thesis was to define the modular determinants that control GIY-YIG nuclease function and to establish frameworks for their systematic characterization and reprogramming. To this end, I combined biochemical reconstitution, domain and subdomain shuffling, directed evolution, bioprospecting, and phylogenetic analysis. A buffer-tuning approach was used to decouple nuclease activity from DNA-binding scaffolds, while fluorescent capillary electrophoresis and Oxford Nanopore sequencing provided scalable and sensitive methods to profile cleavage preferences without complex libraries or Illumina sequencing. Using recombination between diverse nucleases (I-TevI, I-BmoI, I-BamI, and F-SP01I), I identified discrete structural elements controlling sequence preference and strand bias. The first α-helix and adjacent loop emerged as a portable “preference switch region,” while the second α-helix encoded cleavage modality. Substitution of only two residues (R47E + S48E) was sufficient to convert a double-strand cleavase into a strand-selective nickase. Bioprospecting uncovered I-BamI as the first nickase GIY-YIG homing endonuclease, and revealed scaffolds such as F-SP01I that tolerate extensive recombination, supporting an evolutionary model in which nicking represents the ancestral state of the domain. These findings demonstrate that GIY-YIG nuclease domains are defined by modular elements that can be reprogrammed to alter both sequence preference and cleavage modality. This modularity provides the mechanistic basis for their evolutionary diversification and establishes practical routes for their engineering. By expanding the functional landscape of GIY-YIG nuclease domains, this thesis positions them as accessible, evolvable, and engineerable platforms with broad relevance for molecular biology, synthetic biology, and genome editing.

Summary for Lay Audience

Our DNA is constantly being cut, copied, and repaired by small proteins called nucleases. One family, the GIY-YIG homing endonucleases, has a deep evolutionary history spanning hundreds of millions of years, helping pieces of DNA move around in the genomes of microorganisms. Despite knowing their role in nature, almost nothing was understood about how these enzymes decide where to cut DNA or how they cut it — whether they cleave through both strands or just nick a single strand. In this thesis, I explored these questions by studying the nuclease domains of several GIY-YIG enzymes, including I-TevI, I-BmoI, I-BamI, and F-SP01I. I developed new methods that allowed me to test these enzymes quickly and precisely, without the need for expensive, large-scale DNA sequencing. By swapping pieces between different enzymes and simulating evolution in test tubes and bacteria, I found that very small segments act like “switches.” One switch determines the DNA sequences an enzyme prefers to cut, while another switch controls whether it functions as a double-strand cutter (cleavase) or a single-strand cutter (nickase). Remarkably, changing only two amino acids was enough to flip an enzyme from a cleavase into a nickase. I also discovered the first natural nickase in this family, I-BamI, which confirmed that nicking likely represents the ancestral state of GIY-YIG enzymes. Together, these results show that GIY-YIG nucleases are made of small, interchangeable pieces and are extremely adaptable, explaining how they evolved new functions over evolutionary time. More importantly, these insights provide a roadmap for using them as customizable tools in research, biotechnology, and editing DNA in human cells.

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Keywords

GIY-YIG nuclease domain, Homing endonuclease, Modularity, Cleavage preference, Cleavage modality, Nickase, Cleavase, Block shuffling, DNA shuffling, Directed evolu tion, Bioprospecting, Phylogenetic analysis, Capillary electrophoresis, Oxford Nanopore sequencing, I-TevI, I-BmoI, I-BamI, F-SP01I, Genome editing, Gene editors, Synthetic biology, Protein engineering.

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https://hdl.handle.net/20.500.14721/39185

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