The goal of this project is to attain detailed mechanistic, structural and functional information on protein and G-Quadruplex (GQ) interactions at the single molecule level on three systems. Genome-wide computational studies have identified ~300,000 potentially GQ forming sequences (PQS) in the human genome. PQS are concentrated in telomeres and promoter sites of certain oncogenes. In addition to the well-known connection between telomeres and cancer, the concentration of PQS in promoter sites suggests a potential role in transcription level gene expression regulation. GQs were recently visualized in human cells at both telomeric and non-telomeric sites. Mutations in proteins that destabilize GQs resulted in DNA breaks, slowing of replication machinery, and genomic instability. Therefore, a better understanding of GQ-protein interactions is of critical importance We propose to study the following three systems to achieve this goal: 1) Understanding the function of GQ in telomere protection and elongation: Binding of RPA to single-stranded telomeric DNA (ssTEL) is a potential source of genomic instability as it activates ATR checkpoint. POT1/TPP1, a component of shelterin, is involved in protection of ssTEL against this pathway. However POT1/TPP1's lower concentration and lower affinity to ssTEL compared to RPA suggest the involvement of other factors in preventing RPA's access to ssTEL. On the other hand, RPA binding to ssTEL is required during telomere elongation by telomerase, as it likely removes GQ type secondary structures. How the cell manages to transition between these different modes remains unclear. We hypothesize that synergistic activity of POT1/TPP1 and GQ enhances protection of ssTEL against RPA while the dynamic nature of GQ folding/unfolding facilitates the transition to telomere elongation mode. Finally, we propose to study the influence of hnRNPA, TERRA, GQ-GQ interactions, GQ stabilizing drugs on this competition. 2) Understanding the structural and functional implications of GQ destabilization due to protein binding in its vicinity. We observed Bloom (BLM)-mediated telomeric GQ unfolding in the absence of ATP. The efficiency of this activity correlates with the binding stability of BLM to the overhang ssDNA in the vicinity of GQ. We hypothesize that binding of proteins in the vicinity of GQs significantly reduces GQ stability, facilitating efficient removal f these structures in cellular context. Such a mechanism would provide a new perspective on removal of GQs in cellular context. We will study the generality of this observation with other RecQ family helicases and establish the factors that influence it. 3) Unfolding of non-telomeric GQs by RPA: RPA, the major ssDNA binding protein in eukaryotes, is highly efficient at destabilizing GQs. We hypothesize that ssDNA binding affinity and structural arrangement of RPA's DNA binding domains (DBD) determine its efficiency in unfolding different GQs. We propose to perform systematic studies on a broad class of GQs to establish guidelines for their physiological viability.
Elevated telomerase activity is a common feature of cancer cells while G-quadruplex (GQ) formation is known to slow down this activity. In addition, GQ formation in promoter sites of certain genes, particularly oncogenes, is known to regulate transcription level gene expression. These observations combined with the unique layered structure of GQ have recently motivated studies to develop specific drugs that target GQ to regulate telomerase activity or gene expression, which makes the proposed studies very significant in terms of their potential impact on public health.
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