Graduation Date

Summer 8-11-2023

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Pharmaceutical Sciences

First Advisor

Yuri L. Lyubchenko

Abstract

Genome dynamics and integrity are the two crucial features defining the successful functioning of cells and their maintenance and evolution. The genetic processes in the cell require distant communications between the regulatory regions controlled by specific proteins. Mistakes in this interaction process will lead to termination of the genetic process may lead to the cell damage, disease development or the cell death. Similar distant regulatory process is required for numerous genome integration systems such as Variable Diversity Joining (V(D)J) recombination system resulting in the specificity of the immunoresponse, a defining property of the adaptive immune system. A common feature of these genetic processes is a transient formation of the complex between the two DNA segments termed synaptosome. However, a molecular model explaining how the recognition of specific regions on DNA by specialized proteins is taking place remains uncertain. This dissertation combines experimental studies with theoretical ones (collaboration with Prof. A. Kolomeisky, Rice University) enabling us to build a novel framework, which will also explain how the search process of distant DNA segments in a long DNA molecule occurs. We used restriction enzyme SfiI as our experimental system for developing a model for specific recognition of two sites on DNA. SfiI requires binding to two specific sites on DNA to cleave. This catalytic reaction required Mg2+ cations, but no cleavage occurs if Mg2+ is replaced with Ca2+ cations, although the specific complexes assemble. AFM studies of interaction of SfiI with DNA with sites separated with different revealed the assembly of lopped complexes with sizes of the loops coinciding with the distances between the specific sites on DNA. Theoretical model explaining the variability of yield of loops with the size was build and validated with the experiments. We applied a high-speed time-lapse AFM (HS-AFM) to directly visualize the interaction of SfiI with DNA. The use of this cutting-edge nanoimaging approach allowed us to visualize various pathways of the site search process including the formation and dynamics of looped complexes. Identification of pathways at various steps of the site-search process led us to the model of the search process of two sites on DNA, which start with the search of the first site using sliding, jumping and site-transfer pathways followed by the assembly specific looped complexes via of the formation of transient loops of various sizes using novel threading and segment transfer pathways. Transient states of the synaptosome assembly were qualitatively characterized with the use of single molecule fluorescence measurements. Lifetimes of all types of transient synaptic complexes were measured and a theoretical model was built to explain the variability of lifetimes depending on the complex type. Importantly, the model explains the efficiency of the threading pathway. The experimental approaches were applied to studies of interaction with DNA for apurinic/apyrimidinic endonuclease 1 (APE1). This is a multifunctional protein, which in addition to involvement in DNA repair, including base excision repair (BER) and nucleotide incision repair (NIR), critically involved in gene activity regulation interacting with DNA along with transcription factors. Specificity of APE1 binding to G-rich promoter regions led to a number of hypotheses explaining its interaction with DNA. We applied AFM to characterize the interaction of APE1 with DNA, using a DNA substrate containing two well-separated G-rich segments. We demonstrated a high affinity of APE1 to G-specific motifs. The formation of loops was also demonstrated, but in addition to specific loops between the G-rich segments, non-specific loops are formed as well. No G-quadruplex structures were identified on the DNA substrate alone, suggesting that their formation is not required for APE1 specific binding, rather such structures can be stabilized by APE1 binding. Finally, loops are formed by the monomeric APE1, suggesting that the protein has two DNA binding sites. These findings led us to the model on the site search process for monomeric APE1 in which both DNA-binding site of the protein are involved. These experiments laid a foundation for future studies will test this model and other functional properties of APE1 that can also explain its role in the cell death and development of various diseases, specifically cancer. The discovery on the importance of the threading pathways in the site-search process of DNA looping system is supported by recent studies of chromosomes, in which the formation and dynamics of topologically associated domains (TAD) is explained by the threading mechanism mediated by cohesin in complexes with other proteins regulating the TAD dynamics.

Comments

2023 Copyright, the authors

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