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Structure and dynamics in site-specific recombination

Wagner, Nicole
http://orcid.org/0000-0002-1568-6936
http://rave.ohiolink.edu/etdc/view?acc_num=osu1669910785900193

Abstract Details

2022, Doctor of Philosophy, Ohio State University, Biochemistry Program, Ohio State.
The field of gene editing, which offers opportunities to treat genetic mutations and diseases at the level of the gene itself, has garnered a great deal of interest in recent years, especially with the advent of the CRISPR-Cas9 system. However, the safety of gene editing agents must be maximized, and their off-target effects minimized, if they are to be deployed therapeutically in patients. Many well-characterized gene editing systems, such as CRISPR-Cas9, TALENs, and zinc finger nucleases, are programmable to target specific DNA sequences but rely on nuclease activity, which generates double-stranded DNA breaks. The presence of double-strand breaks in a living cell activates error-prone cellular repair pathways, which can cause serious off-target effects on the genome. The tyrosine recombinase Cre presents an alternative approach to gene editing. It recombines loxP DNA sites without requirements for external host proteins or energy sources, and without generating double-strand breaks. These features bode well for the simplicity and safety of Cre as a gene editing tool. However, one limitation is its substrate scope; its activity is fairly specific for its loxP substrate, and it cannot easily be programmed to recognize alternate DNA sequences. Furthermore, there are gaps in our understanding of how it controls its activity and the determinants of DNA selectivity and recombination efficiency. A deeper understanding of the structural and dynamic features underlying these processes will enhance the potential to target Cre toward therapeutically relevant DNA sequences. NMR spectroscopy, our tool of choice, offers the advantage of insight into both structural and dynamic features of biomolecules in solution. We begin with dual introductory chapters that contextualize the subsequent studies. Chapter 1 is a literature review of Cre recombinase structural biology – what has been learned in the past 25 years and where the field stands now. Chapter 2 is a literature review of applications of NMR spectroscopy to study DNA structure and dynamics. Chapter 3 describes our work to identify a tractable oligonucleotide for NMR studies of the loxP spacer. The spacer, the only asymmetric region of the loxP site, impacts recombination efficiency and guides strand exchange order despite making few base-specific contacts with Cre. However, resonance overlap makes it difficult to study the spacer in the context of the 34-bp loxP site. Therefore, we seek to identify an oligonucleotide that yields simpler spectra while retaining the chemical environment experienced by the spacer in longer oligonucleotides. Using chemical shifts as reporters of DNA structure, we describe the identification of a 16-bp loxP construct well-suited for further structural and dynamics studies. We also discuss comparisons with chemical shifts of the lox4 spacer, which inverts strand exchange order, and predicted B-form chemical shifts. Our findings prompt preliminary speculation about the role of the spacer sequence in modulating intrinsic bending and strand exchange order. Building on the work in Chapter 3, Chapter 4 describes studies of Mg2+ effects on structural features of the loxP and lox4 spacers. We begin with 1D 1H NMR titrations to demonstrate that Mg2+ concentration has an effect on chemical shifts of the spacer. We then carry out plasmid-based recombination assays at a range of Mg2+ concentrations in order to select optimal conditions for more detailed NMR experiments. Finally, we quantify the effects of 10 mM Mg2+ on the chemical shifts of aromatic and anomeric protons in the 16-bp loxP and lox4 spacer oligonucleotides. Chapter 5 focuses on characterizing interactions of active site residues in the recently discovered autoinhibited conformation of apo Cre. In particular, we seek to investigate underlying causes of the unique and sensitive chemical shift of the active site W315 sidechain. We demonstrate that the Y324F mutation perturbs the 1H-15N chemical shift of the W315 indole in a manner similar to the effects of C-terminal truncation or loxP DNA binding. In the process, we also identify an unexpected pattern of additional chemical shift perturbations induced by the Y324F mutation throughout the NMR spectrum of the catalytic domain, CreCat. We report progress toward investigating the impact of the Y324F mutation not only on the chemical environment of the W315 sidechain, but also on the conformation of Cre’s C-terminus. In Chapter 6, we design Cre mutants incorporating the S. aureus sortase A recognition motif via substitutions and insertions. Previous NMR studies of Cre have focused on isolated domains, but we also seek to study its structure and dynamics in its full-length context while avoiding resonance overlap. Sortase-mediated ligation of isotopically labeled and unlabeled domains enables simpler spectra of large proteins, but leaves behind its “LPXTG” recognition motif in the final product. Therefore, before generating ligated Cre constructs, we aimed to verify a means of incorporating the LPXTG motif into Cre without perturbing its structure or function.
Mark Foster (Advisor)
Charles Bell (Committee Member)
Rafael Brüschweiler (Committee Member)
Michael Poirier (Committee Member)
277 p.
Biochemistry
Cre; recombinase; Cre recombinase; DNA; NMR; structure; dynamics; structural biology

Recommended Citations

Citations

  • Wagner, N. (2022). Structure and dynamics in site-specific recombination [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1669910785900193

    APA Style (7th edition)

  • Wagner, Nicole. Structure and dynamics in site-specific recombination. 2022. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1669910785900193.

    MLA Style (8th edition)

  • Wagner, Nicole. "Structure and dynamics in site-specific recombination." Doctoral dissertation, Ohio State University, 2022. http://rave.ohiolink.edu/etdc/view?acc_num=osu1669910785900193

    Chicago Manual of Style (17th edition)

osu1669910785900193
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