Single molecule investigation of transcription activator-like effector search dynamics

Recent advances in genetic engineering hold great potential to profoundly change the treatment of human disease. Precise manipulation of genetic material allows for the creation of new disease models and the rapid translation of new therapies into the clinic. Several classes of programmable nucleases allowing for precise and targeted genomic edits are central to the rising popularity, flexibility, and accessibility of gene engineering. Transcription activator-like effectors (TALEs) are one such class that form a powerful gene-editing platform when fused to nuclease domains, thereby yielding TALEN systems. Despite pervasive use of TALENs for editing crops, small animals, eukaryotic stem cells, and human T-cells, remarkably little is known about the molecular mechanisms used to locate and bind their DNA target sites. This work describes the application of single molecule fluorescence imaging to the study the TALE search process along specific and non-specific DNA templates. Our work provides a molecular-level picture of the dynamics of TALE-DNA interactions, and our results have revealed an apparently unique search mechanism for DNA binding proteins. We directly observe TALEs diffusing along non-specific DNA in one-dimension. Our results show that TALE diffusion occurs in a directionally unbiased and thermally driven manner along double surface tethered and extended DNA templates (Chapter 2). Interestingly, we observe significant intra-trajectory heterogeneity for diffusion of full-length TALE proteins. We further isolate and study the single molecule dynamics of TALE truncation mutants containing only the N-terminal region (NTR), and these results reveal the importance of the NTR for nucleating non-specific binding. We find that the TALE NTR alone is capable of short, rapid non-specific search. Furthermore, we study the diffusion of a series of TALEs with variable size central repeat domains (CRDs). Taken together with insights from NTR dynamics and the heterogeneity of full-length TALE diffusion, we propose a two-state search mechanism for TALEs that is comprised of rapid search and interspersed periods of local sequence checking along DNA templates. We further expand our characterization of TALE search by determining the impact of solution conditions, ionic strength, probe size, and the role of hydrodynamic flow on TALE dynamics (Chapter 3). Using this combination of single molecule experiments, we find that TALE diffusion does not fit the traditional definitions of binary classification of DNA-binding protein search, which have been characterized as protein hopping or sliding along DNA templates. Instead, our results suggest a mechanism wherein TALEs encircle DNA templates during search, but form only transient contacts with the DNA backbone. Furthermore, the non-specific search trajectory of TALEs is rotationally decoupled, in contrast to a broad class of other DNA binding proteins including DNA repair proteins and transcription factors. We further utilize a combination of bulk fluorescence anisotropy measurements and single molecule experiments to characterize the effects of divalent cations on TALE binding (Chapter 4). Our results show that TALE specificity is significantly enhanced in the presence of certain divalent cations, which can be attributed to a decrease in non-specific binding affinity. Finally, we generate long DNA templates with TALE target binding arrays at precise locations, and we directly visualize specific binding and localization of TALEs to their respective target sites following 1-D search. Taken together, our results have elucidated the fundamental search mechanism of TALE proteins along DNA templates

Single-molecule visualization reveals the damage search mechanism for the human NER protein XPC-RAD23B

DNA repair is critical for maintaining genomic integrity. Finding DNA lesions initiates the entire repair process. In human nucleotide excision repair (NER), XPC-RAD23B recognizes DNA lesions and recruits downstream factors. Although previous studies revealed the molecular features of damage identification by the yeast orthologs Rad4-Rad23, the dynamic mechanisms by which human XPC-RAD23B recognizes DNA defects have remained elusive. Here, we directly visualized the motion of XPC-RAD23B on undamaged and lesion-containing DNA using high-throughput single-molecule imaging. We observed three types of one-dimensional motion of XPC-RAD23B along DNA: diffusive, immobile and constrained. We found that consecutive AT-tracks led to increase in proteins with constrained motion. The diffusion coefficient dramatically increased according to ionic strength, suggesting that XPC-RAD23B diffuses along DNA via hopping, allowing XPC-RAD23B to bypass protein obstacles during the search for DNA damage. We also examined how XPC-RAD23B identifies cyclobutane pyrimidine dimers (CPDs) during diffusion. XPC-RAD23B makes futile attempts to bind to CPDs, consistent with low CPD recognition efficiency. Moreover, XPC-RAD23B binds CPDs in biphasic states, stable for lesion recognition and transient for lesion interrogation. Taken together, our results provide new insight into how XPC-RAD23B searches for DNA lesions in billions of base pairs in human genome

Don\u27t Bring a Gun to a Fist Fight: Deconstructing Hegemonic Masculinity Through the Gun in Lina Wertmuller\u27s \u27Pasqualino Settebellezze\u27

Book Summary: Feminism in Italy has left a great area of investigation uncovered, buried under the pressure of a backlash that runs over women\u27s identity, their expectations, and their artistic expression. Featuring essays by top scholars and interviews with acclaimed directors, this book examines Italian women\u27s authorship in film and their visions of reality. This anthology unites artists such as Lina Wertmuller, Liliana Cavani, Alina Marazzi, Ilaria Borrelli, and Francesca Comencini with intellectuals such as Dacia Maraini and Patrizia Carrano, two groundbreaking Italian writers whose work has been dedicated to the tireless defense of women\u27s literature and cinema, from a perspective of continuity with past issues and openness towards the more complex gender challenges of today. [From the publisher] Chapter Summary: This chapter examines Lina Wert’s use of the gun in Pasqualino Settebellezze as a means for ridiculing and ‘emasculating’ representations of hegemonic masculinity. A phallic extension of the male body, the gun is traditionally perceived as a glorified symbol of patriarchal authority and order in contemporary western culture; however, in Pasqualino Settebellezze, the phallic gun is both disempowered and disempowering through an often comedic yet dark caricature