Identification of the New Type of G-Quadruplex with Multiple Vacant Sites in Human Telomeric DNA

To date,few studies have reported on the folding mechanism of tandem G-quadruplexes in human telomeric DNA.Hence,the control of the biofunctions of G-quadruplex,which requires a thorough understanding of its dynamic behavior,is limited.Here,we investigated the folding/unfolding behavior of human telomeric sequences with lengths over 10 kilonucleotide(knt)by circular dichroism(CD)spectroscopy,UV melting assay,and atomic force microscopy(AFM)-based single-molecule force spectroscopy.

Enhanced radiosensitivity by 6-thio-dG via increasing telomere dysfunction and ataxia telangiectasia mutated inhibition in non-small cell lung cancer

Objective:To investigate the radiosensitivity of 6-thio-dG and its underlying molecular mechanisms in non-small cell lung cancer(NSCLC).Methods:H1299 and A549 NSCLC cells were pretreated with 6-thio-dG for one week and then exposed toγ-irradiation.Cell proliferation and survival were quantified using clonogenic assays.DNA damage was assessed using immunofluorescence forγH2AX.Telomere dysfunction-induced foci analysis was performed by the colocalization of telomere signals(FISH)andγH2AX.Telomere fusion was defined as two telomere signals merged into one at the chromosome by immuno-FISH in metaphase spreads.Proteins related to the DNA damage response were detected using Western blot analysis.Apoptosis wasanalyzed using flow cytometry and Western blot.Results:The presence of 6-thio-dG increased the radio sensitivity of H1299 and A549 cells(P<0.05),but had no effect on the normal human lung fibroblast line,MRC5.6-thio-dG pretreatment significantly reduced the clonogenic potential induced byγ-ray irradiation and aggravated genomic DNA and telomeric DNA damage(P<0.05).In addition,6-thio-dG pretreatment effectively increasedγ-ray irradiation induced telomere dysfunction(P<0.05),resulting in disruption of chromosome stability and inhibition of the ATM pathway,thereby impairing genomic DNA and telomeric DNA repair,which was closely associated with enhanced drug-mediated radiation-induced apoptosis.Conclusions:6-thio-dG increases the radiosensitivity of NSCLC by inhibiting ATM and inducing telomere dysfunction,which can potentially be used as a strategy for radiotherapy for NSCLC.

ILF3 safeguards telomeres from aberrant homologous recombination as a telomeric R-loop reader

Telomeres are specialized structures at the ends of linear chromosomes that protect genome stability.The telomeric repeat-containing RNA(TERRA)that is transcribed from subtelomeric regions can invade into double-stranded DNA regions and form RNA:DNA hybrid-containing structure called R-loop.In tumor cells,R-loop formation is closely linked to gene expression and the alternative lengthening of telomeres(ALT)pathway.Dysregulated R-loops can cause stalled replication forks and telomere instability.However,how R-loops are recognized and regulated,particularly at telomeres,is not well understood.We discovered that ILF3 selectively associates with telomeric R-loops and safeguards telomeres from abnormal homologous recombination.Knocking out ILF3 results in excessive R-loops at telomeres and triggers telomeric DNA damage responses.In addition,ILF3 deficiency disrupts telomere homeostasis and causes abnormalities in the ALT pathway.Using the proximity-dependent biotin identification(BioID)technology,we mapped the ILF3 interactome and discovered that ILF3 could interact with several DNA/RNA helicases,including DHX9.Importantly,ILF3 may aid in the resolution of telomeric R-loops through its interaction with DHX9.Our findings suggest that ILF3 may function as a reader of telomeric R-loops,helping to prevent abnormal homologous recombination and maintain telomere homeostasis.

NDI-induced Topological Conversion of Human Telomeric G-Quadruplexes from Hybrid-2 to Parallel Form

G-Quadruplexes(GQs),which are formed by G-rich DNA sequences in human telomeres,have become an attractive target for cancer treatment.The ligands to stabilize the conformation of human telomeric GQs in vivo are particularly important for structure-based ligand design and drug development targeting the noncanonical DNA structure.Here we report the conformational conversion of Tel26 induced by a naphthalene diimide(NDI)ligand in K^(+)buffer,even at cellular physiological temperature(37℃)and under mimetic cellular crowding conditions created by Ficoll 70.We provide an insight into the dynamic conversion from initial hybrid-2 GQ topology to final parallel GQ topology.These results are helpful for the design of ligands with GQ conformation regulation.