The topology and spatial organization of DNA in cells is essential to its function and is tightly regulated by proteins. Two prominent features of protei-mediated structure are DNA loops and the formation of condensed DNA. Looping allows protein-protein and/or protein-DNA interactions over large genomic distances. An example of protein-mediated DNA looping is the telomere structure. Telomeres are nucleoprotein structures that cap the ends of linear chromosomes. Human telomeric DNA can be arranged into DNA T-loops as large as thousands of base pairs. Only static snapshots of T-loop formation are known. TRF1 and TRF2 are essential proteins associated with the loop formation, but their precise role and function in the dynamics of T-loop formation remains unclear. Nuclear DNA exists in an intrinsically crowded environment containing a very high volume fraction of DNA and proteins. Generally there is a hierarchy of domains of order, and thus the traditional crowding model may not fully capture the influence of the environment. To improve understanding of the looping process, we propose to study time-resolved DNA looping at the single-molecule level by confining DNA and proteins to nanofluidic volume. We argue that this confinement mimics certain aspects of the nuclear environment that are not reproduced by crowding in small molecules. We will use nanochannel-confined telomeric DNA to observe the dynamic actions of TRF1 and TRF2, and form an operational model of how the telomeric loop forms. The same will be performed for the model enzyme T4 ligase. All experiments use dynamic single molecules in real time. To establish the role of confinement in loop formation, we will apply high-resolution AFM imaging to derive the static conformations of T4 ligase, TRF1 and TRF2 mediated DNA loops formed inside nanochannels and that formed in free solution. Comparison with DNA loops formed by the model system, T4 DNA ligase, will aid in the interpretation of looping mechanisms mediated through TRF1/TRF2. In the last study, we will align independent DNA strands parallel or perpendicular to each other in nanochannel junctions, and follow the actions in real time using fluorescence imaging to measure the capture rate by ligase and TRF1/TRF2 as DNA molecules are scanned past each other. We anticipate that moving molecules in parallel past each other increases DNA-DNA capture when compared to mere point contacts. The results are essential for advancing our understanding of telomere biology, the influence of geometric constraints on the utilization of nuclear DNA, and physics of confined stiff biopolymers in general.
Protein-mediated DNA looping is an important process underlying many biological pathways. We propose an innovative study to use nanochannel-confined DNA to observe dynamic DNA looping actions of DNA ligase, TRF1 and TRF2, which are important for telomere T-loop formation. The results from the proposed studies will considerably expand our understanding of telomere maintenance, which plays important roles in aging and carcinogenesis.
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