Hong Wang

Three closely related Kinetoplastid parasites including Trypanosoma brucei, Trypanosoma cruzi, and Leishmania infect more than ten million people worldwide. T. brucei causes fatal human African trypanosomiasis that threatens ~60 million people 2. The animal trypanosomiasis caused by T. brucei also has a great negative impact on local economy. However, few drugs are available to treat these diseases safely and effectively with easy administering, and drug resistance cases have been observed. Our recent observations suggest that TRF helps maintain the Trypanosoma brucei telomere integrity through multiple means. In this project, we propose to examine the underlying mechanisms of how TRF suppresses the telomeric R-loop (TRL) level in Aim 1, test whether TRF facilitates the telomere DNA replication in Aim 2, and investigate how TRF suppresses telomere and subtelomere recombination in Aim 3.

The importance of cohesin in multiple genome maintenance pathways makes it a highly desirable target for anticancer therapies. Uncovering interactions between cohesin and DNA, as well as shelterin proteins at telomeres will provide additional opportunities to reduce cancer cell survival after chemotherapies.

In both prokaryotic and eukaryotic cells, structural maintenance of chromosomes (SMC) complexes play critical roles in 3-D genome organization and maintenance. Each SMC complex consists of a pair of SMC subunits and a varying number of additional regulatory subunits subunits. While recent years have seen significant advances in our understanding of cohesin (SMC1/3) and condesin (SMC2/4) in mediating 3-D genome organization, the molecular mechanism of SMC5/6 complex in regulating DNA replication and repair is still not well understood. This proposed study aims to use High-speed AFM to explore the structure and dynamics of DNA binding by the SMC5/6 complex.

In nanoplasmonic upconverting nanoparticles, the collective oscillation of conduction electrons, known as plasmons, lead to strong light absorption, as well as local field enhancements, which is dependent on the orientation of the nanoparticle with respect to the incident excitation. This upconversion fluorescence anisotropy of the nanoplasmonic upconverting nanoparticles renders them as excellent orientation probes in both 2D and 3D. The upconverting labels could allow the continuous tracking of single-molecules in a variety of settings, which would increase our understanding of cellular function at the molecular level. To further advance our understanding of the biophysical mechanism underlying protein-DNA interactions, this project will use single particle orientation and rotation tracking of nanoplasmonic upconverting-labeled proteins to directly observe the curvilinear movement of proteins involved the telomere maintenance and cohesion pathway.

We hypothesize that the nuclear environment modifies the pathways available for loop formation by enzymes. We will study the mechanism of DNA looping by telomeric proteins and T4 ligase using AFM and fluorescence imaging under confinement to nanochannels resembling reptation tubes.

Defects in cohesin, the mediator of sister chromatid cohesion, drive aneuploidy which can result in many types of cancer. Cohesin is a multi-subunit protein complex that is important for accurate segregation of chromosomes, DNA replication, and gene expression. Structurally, cohesin is a ring-shaped complex that consists of four subunits, one of which can be one of two isoforms, SA1 or SA2. A large gap in our knowledge exists regarding how the cohesin complex bridges two DNA strands together and the different roles that SA1 and SA2 play during the cohesion process. We propose using multi-dimensional single-molecule approaches using fluorescence imaging, atomic force microscopy (AFM) imaging and nano-channel confined DNA to investigate the dynamics and high-resolution structures of SA1 and shelterin protein mediated DNA-DNA pairing.

Telomeres are nucleoprotein structures that cap the ends of linear chromosomes. Telomere dysfunctions are important contributing factors in aging and tumorigenesis. A specialized protein complex called shelterin binds and protects the human chromosome ends. In humans, this complex consists of six core proteins: TRF1, TRF2, POT1, TIN2, TPP1, and RAP1. Electron microscopy studies indicate that human telomeres can form a lasso-like structure called T-loop, in which the single-stranded telomeric tail is folded back and invades the duplex DNA. Telomeric DNA sequences show a higher susceptibility to certain DNA damaging agents than random DNA sequences. It is known that certain environmental DNA damaging agents cause increased telomere shortening. Short telomeres are characteristic of several human diseases. Telomere maintenance involves dynamic actions of multiple proteins on a long complex DNA structure. Given the heterogeneity and complexity of telomeres, single-molecule imaging approaches are essential to fully understand the structure-function relationships that govern the maintenance of telomeres. The goal of my research proposal is to use two highly innovative and complementary single-molecule imaging techniques (Atomic force microscopy and fluorescence imaging) together with fluorescent quantum dot (Qdot) nanocrystal labeled proteins to investigate the effects of DNA damage on the conformational and dynamic properties of telomeric DNA structure and shelterin proteins. AFM generates an image of a surface by scanning with a sharp sensor tip attached to a cantilever. It is a versatile imaging tool for studying the conformation and dynamics of biological systems at nanometer resolution both in air and in solution. Accumulating evidence indicates that dynamic movements on DNA, such as 1-dimensional (1-D) diffusion, jumping, and hopping, are essential for a protein to achieve its function in vivo where nonspecific DNA is in vast excess and bound by other proteins. Consequently, the elucidation of dynamic properties of proteins on DNA has become a central question in biology. To study dynamic protein-DNA interactions in real time at the single-molecule level, we developed a holistic approach to overcome the three key imaging limitations: (1) fluorescence signal intensity and prevention of fluorophore photobleaching, (2) isolation of the DNA from the surface, and (3) reduction of background signals. The cutting-edge single-molecule fluorescence imaging techniques we developed enables the direct observation of movements of individual proteins with up to 17 nanometer positional accuracy and 50 millisecond temporal resolution. We hypothesize that, in addition to disrupting TRF1, TRF2 and POT1 proteins binding to DNA, environmentally-induced DNA damage (such as UV light or oxidative stress) at telomeres can cause stochastically unstable assemblies of telomere binding proteins on DNA. This in turn progressively favors the disruption of the T-loop structure and the exposure of the single-stranded overhang. The specific aims of this study are two-fold. The first aim is to evaluate the effects of environmentally-induced DNA damage on G-quadruplex formation, protein binding, protein assemblies, and T-loop formation. The second aim is to evaluate the effects of DNA damage on dynamics of protein-DNA interaction and protein assembly on telomeric DNA. 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.

Telomere dysfunction and associated chromosomal abnormalities have been strongly associated with cancer and age-related degenerative diseases. Telomeres are highly susceptible to environmental DNA damaging agents due to its unique sequences. Thus, disruption of telomere structure (T-loop) and function due to an accumulation of UV lesions can lead to to skin photoaging by triggering senescence or skin carcin-ogenesis. Consequently, it is essential to understand the mechanism underlying UV-induced telomere dys-functions. Meanwhile, it is known that UV can induce dsDNA breaks and telomere binding proteins TRF1 and TRF2 are known to be recruited to dsDNA breaks. However, the role of TRF1 and TRF2 in double-strand DNA repair (DDR) is still controversial. Single-molecule approaches are essential to fully understand the structure-function relationships that underlying UV-induced telomere dysfunctions and the roles of TRF1 and TRF2 in DDR. To address these questions, in collaboration with Dr. Riehn’s lab, we developed multi-dimensional sin-gle-molecule imaging approaches. We will use single-molecule fluorescence imaging to examine the impact of UV-induced DNA lesions on TRF2-mediated T-loop formation inside nanochannels. To directly investigate whether TRF1 and TRF2 can carry out essential steps required for dsDNA repair, we will directly monitor DNA-DNA pairing by TRF1 and TRF2 inside nanochannels using fluorescence imaging. A new electrostatic force microscopy technique will be used to monitor the high-resolution structure of TRF2-mediated strand invasions. Results obtained from this pilot study will be used to apply for grants from NIEHS and/or ACS.Please add Abstract

The aggregation of biomaterials on graphene started to be investigated relatively recently. Katoch et al. have deposited peptides from buffered aqueous solution onto graphene and have observed a uniform, nanoporous surface coating. Infrared spectroscopy indicates that the peptide remains intact upon adsorption and Raman spectroscopy shows that the graphene is undamaged after the peptide deposition. This experiment represents the proof-of-principle behind non-covalent self-assembly of biologically-relevant materials on a graphene surface. Our proposed work will take the transformative steps of identifying the microscopic molecular-scale mechanisms driving self ?assembly and searching for the impact of surface assembly on functionality of biomolecules with complex meso-scale structure.