My goal in obtaining this K99 award is to acquire the additional training to become an independent investigator in the field of single-molecule studies of telomere protein-DNA and protein-protein interactions. This study will be under the guidance of Dr. Van Houten and sponsored by Drs. Erie and Opresko. The expertise and resources from these three labs provide an excellent environment for me to advance my skills in AFM and single-molecule fluorescence imaging, telomere protein biochemistry, and QPCR assays. We hypothesize that, in addition to disrupting TRF1, TRF2 and POT1 proteins binding to DMA, environmentally-induced DMA damage (such as UV light or oxidative stress) at telomeres can cause stochastically unstable assemblies of telomere binding proteins on DMA. This in turn progressively favors the disruption of the T-loop structure and the exposure of the 3'overhang.
The specific aims of this study are two-fold.
The first aim i s to evaluate the effects of environmentally-induced DMA damage on G-quadruplex formation, protein binding, protein assemblies, and T-loop formation. We will use AFM to examine the effects of DMA damage on G-quadruplex assembly. The impact of bulky DMA lesions on the binding of POT1, TRF1 and TRF2 to short telomeric DMA substrates will be evaluated using electrophoresis mobility shift assays (EMSAs), and the T-loop formation will be examined using AFM. In AFM studies, loading of POT1 (marked by quantum dots, QDs) onto duplex telomeric DMA will be used as a reporter of shelterin assembly.
The second aim i s to evaluate the effects of DNA damage on dynamics of protein-DNA interaction and protein assembly on telomeric DNA. We will characterize the functionality of TRF1, TRF2, and POT1-QD conjugates using AFM imaging and EMSA. These protein-QD conjugates will be used in single-molecule fluorescence studies to evaluate how UV-induced DNA damage affects the dynamics of TRF1-, TRF2-DNA interactions and shelterin assembly. It is known that certain environmental DNA damaging agents cause increased telomere shortening. Short telomeres are characteristic of various human diseases. This study will greatly advance our understanding of how exposure to environmental stressors. such as UV light and oxidative stress, is associated with telomere dysfunction in the etiology of several human disorders including age-associated degenerative diseases and cancer.
|Wu, Dong; Kaur, Parminder; Li, Zimeng M et al. (2016) Visualizing the Path of DNA through Proteins Using DREEM Imaging. Mol Cell 61:315-23|
|Kaur, Parminder; Wu, Dong; Lin, Jiangguo et al. (2016) Enhanced electrostatic force microscopy reveals higher-order DNA looping mediated by the telomeric protein TRF2. Sci Rep 6:20513|
|Benarroch-Popivker, Delphine; Pisano, Sabrina; Mendez-Bermudez, Aaron et al. (2016) TRF2-Mediated Control of Telomere DNA Topology as a Mechanism for Chromosome-End Protection. Mol Cell 61:274-86|
|Roushan, Maedeh; Azad, Zubair; Lim, Shuang Fang et al. (2015) Interference of ATP with the fluorescent probes YOYO-1 andYOYO-3 modifies the mechanical properties of intercalator-stained DNA confined in nanochannels. Mikrochim Acta 182:1561-1565|
|Parikh, Dhvani; Fouquerel, Elise; Murphy, Connor T et al. (2015) Telomeres are partly shielded from ultraviolet-induced damage and proficient for nucleotide excision repair of photoproducts. Nat Commun 6:8214|
|Lin, Jiangguo; Countryman, Preston; Buncher, Noah et al. (2014) TRF1 and TRF2 use different mechanisms to find telomeric DNA but share a novel mechanism to search for protein partners at telomeres. Nucleic Acids Res 42:2493-504|
|Lin, Jiangguo; Kaur, Parminder; Countryman, Preston et al. (2014) Unraveling secrets of telomeres: one molecule at a time. DNA Repair (Amst) 20:142-53|
|Tessmer, Ingrid; Kaur, Parminder; Lin, Jiangguo et al. (2013) Investigating bioconjugation by atomic force microscopy. J Nanobiotechnology 11:25|
|Hughes, Craig D; Wang, Hong; Ghodke, Harshad et al. (2013) Real-time single-molecule imaging reveals a direct interaction between UvrC and UvrB on DNA tightropes. Nucleic Acids Res 41:4901-12|
|Lormand, Justin D; Buncher, Noah; Murphy, Connor T et al. (2013) DNA polymerase Î´ stalls on telomeric lagging strand templates independently from G-quadruplex formation. Nucleic Acids Res 41:10323-33|
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